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The discovery of nephrin in 1998 has launched a new era in glomerular diseases research, emphasizing its crucial role in the structure and function of the glomerular filtration barrier. In the past 20 years, substantial advances have been made in understanding podocyte structure and function as well as the discovery of several podocyte-related proteins including nephrin. The glomerular filtration barrier is comprised of podocytes, the glomerular basement membrane and endothelial cells. Podocytes, with their specialized slit diaphragm, form the essential backbone of the glomerular filtration barrier. Nephrin is a crucial structural and functional feature of the slit diaphragm that prevents plasma protein, blood cell and macromolecule leakage into the urine. Podocyte damage results in nephrin release. Podocytopathies are kidney diseases in which podocyte damage drives proteinuria, i.e., nephrotic syndrome. Many kidney diseases involve podocytopathy including congenital nephrotic syndrome of Finnish type, diffuse mesangial sclerosis, minimal change disease, focal segmental glomerulosclerosis, collapsing glomerulonephropathy, diabetic nephropathy, lupus nephropathy, hypertensive nephropathy and preeclampsia. Recently, urinary nephrin measurement has become important in the early detection of podocytopathies. In this chapter, we elaborate the main structural and functional features of nephrin as a podocyte-specific protein, pathomechanisms of podocytopathies which result in nephrinuria, highlight the most commonly used methods for detecting urinary nephrin and investigate the diagnostic, prognostic and potential therapeutic relevance of urinary nephrin in primary and secondary proteinuric kidney diseases.

Introduction. Podocyte injury has been reported as an early feature of DN therefore, the assessment of podocyte injury can be accomplished by estimation of podocalyxin in urine. This study aimed to estimate the urinary podocalyxin levels and to determine the sensitivity and specificity of this biomarker for early detection of DN. Materials and methods. A total of 90 patients with type 2 diabetes mellitus (T2DM) were included in this cross-sectional study. Sixty of them were without diagnosed DN, and 30 with diagnosed DN. A control group consisted of 30 healthy subjects. All patients with T2DM were divided into three subgroups according to urinary microalbumin/creatinine ratio (UM/CR): normoalbuminuric, microalbuminuric and macroalbuminuric patients. Urine samples, were used for measurement of podocalyxin by ELISA, creatinine and microalbumin. Fasting venous blood samples was collected for biochemical analyses. Results. The levels of urinary podocalyxin (u-PDX) were higher in patients with T2DM compared to control subjects and a statistically significant difference among studied subgroups regarding u-PDX was found (p < 0.05). Levels of u-PDX are increasing gradually with the degree of DN (p < 0.029). u-PDX levels were positively correlated with UM/CR (r = 0.227, p = 0.002). A cut-off level of 43.8 ng/ml u-PDX showed 73.3% sensitivity and 93.3% specificity to detect DN in early stage. A cut-off level of 30 mg/g UM/CR showed 41.5% sensitivity and 90% specificity in predicting DN. u-PDX was elevated in 48,2% of normoalbuminuric patients. Conclusion. Urinary podocalyxin be useful and more sensitive and specific marker in early detection of DN than microalbuminuria.

Introduction: In December 2019, a highly infectious disease i.e. pneumonia caused by the coronavirus disease SARS-CoV-2, occurred in Wuhan, China, and the World Health Organization has declared its ongoing outbreak a pandemic. The clinical laboratory plays a key role in supporting the monitoring and management of SARS-CoV-2 disease. Material and methods: Our study describes the laboratory characteristics of 103, SARS-CoV-2 patients hospitalized in the public hospital Trifun Panovski in the period April - June 2020. Only two patients died as a result of the SARS-CoV-2. The most common laboratory abnormalities were: elevated lactate dehydrogenase (LDH) and C-reactive protein (CRP). Results: Leukocytosis was found in 23.3%, and leucopenia in 3% of patients with SARS – CoV-2. SARS-CoV-2 infection can be associated with myocardial injury, heart failure, vascular inflammation, myocarditis, cardiac arrhythmias, and hypoxic encephalopathy. We found increased values of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ –glutamyl transferase (γ –GT) in 35 – 50% of patients. We detected affected renal function in 10% of our patients with SARS – CoV 2. Conclusion:We identified potential biomarkers of disease severity. Our results provide guidance in risk assessment, clinical monitoring and therapy decision-making processes

An EC50 database was established to assess the acute toxicity of 16 PESTANAL pesticide standards and of seven pesticide commercial formulations using a Vibrio fischeri bioluminescence method. Half maximal effective concentration (EC50 ) is defined as the concentration of pollutant (in this case, pesticide) destroying 50% of the bacteria population and causing 50% bioluminescence inhibition, after a specified exposure time. Linear curves of bioluminescence inhibition versus pesticide concentration and EC50 values were obtained for exposure times (t) of 5 or 15 min for these pesticides. The EC50 values ranged from 6.90 × 10-4 to 0.83 mg/ml (t = 5 min), and from 9.00 × 10-4 to 0.37 mg/ml (t = 15 min) for pesticide standards, plus from 0.0077 to 0.74 mg/ml (t = 5 min), and from 0.0076 and 0.57 mg/ml (t = 15 min) for pesticide commercial formulations. The EC50 database allowed classification of the pesticides under study into three categories according to their toxicity: very toxic, toxic and moderately toxic. These results demonstrated that the establishment of an EC50 database and of linear curves of bioluminescence inhibition versus the pesticide concentration resulted in very important and irreplaceable tools to estimate the global and individual toxicity of pesticides present in environmental samples.

Lipoprotein(a) – Lp(a) – is an independent risk factor for cardiovascular disease (CVD). Indeed, individuals with plasma concentrations of Lp(a) > 200 mg/l carry an increased risk of developing CVD. Circulating levels of Lp(a) are remarkably resistant to common lipid lowering therapies, currently available treatment for reduction of Lp(a) is plasma apheresis, which is costly and labour intensive. The Lp(a) molecule is composed of two parts: LDL/apoB-100 core and glycoprotein, apolipoprotein(a) – Apo(a), both of them can interact with components of the coagulation cascade, inflammatory pathways and blood vessel cells (smooth muscle cells and endothelial cells). Therefore, it is very important to determine the molecular pathways by which Lp(a) affect the vascular system in order to design therapeutics for targeting the Lp(a) cellular effects. This paper summarises the cellular effects and molecular mechanisms by which Lp(a) participate in atherogenesis, thrombogenesis, inflammation and development of cardiovascular diseases.

Urinary neutrophil gelatinase-associated lipocalin (uNGAL), urinary N-acetyl-bd-glucosaminidase (NAG), urinary α1-microglobulin/creatinine ratio and cystatin C have been suggested as potential early markers of delayed graft function (DGF) following kidney transplantation. We conducted a prospective study in 50 consecutive kidney transplant recipients to evaluate serial changes of these biomarkers within the first week after transplantation and assess their performance in predicting DGF (dialysis requirement during initial post-transplant week) and graft function throughout the first year. Urine samples were collected on post-transplantation days 0, 1, 2, 4, and 7. Statistical analysis: Linear mixed and multivariable regression models, receiver-operating characteristic (ROC), and areas under ROC curves were used. At all-time points, mean urinary NGAL levels were significantly higher in patients developing DGF. Shortly after transplantation (3-6 h), uNGAL and uNAG values were higher in DGF recipients (on average +242 ng/mL; NAG – 6.8 U/mmol creatinine, considering mean dialysis time of 4.1 years) and rose further in the following days, contrasting with prompt function recipients. On Day-1 uNGAL levels accurately predicted DGF (AUC-ROC = 0.93), with a performance higher than serum creatinine (AUC-ROC = 0.76), and similar to cystatin C (AUC-ROC = 0.95). Multivariable analyses revealed that uNGAL levels at days 4 and 7 were strongly associated with one-year serum creatinine level. Urinary NGAL, serum cystatin C is an early marker of graft injury and is independently associated with dialysis requirement within one week after transplantation and one-year graft function.

Fournier’s gangrene (FG) is a necrotizing fasciitis of the genital, perianal and perineal regions, caused by multiple anaerobic/aerobic infection. It is a rare but very serious condition with multiple long-term complications and high mortality rate. Early diagnosis and multidisciplinary approach in treatment of complicated cases of FG are crucial to the successful outcome. We report a case of an extensive FG in a 59-years-old female patient with multiple risk factors such as obesity, type 2 diabetes and hypertension. She was hospitalized as an emergency case with diabetic ketoacidosis, sepsis and extensive necrotic lesions located perineal, perianal, genital and spread to inguinal, hypogastric, gluteal and sacrococcygeal region. Fournier’s gangrene was diagnosed, and after prompt resuscitation, intravenous fluids, broad-spectrum antibiotics, insulin infusion, emergency aggressive surgical debridement was performed. Several aerobic and anaerobic bacteria were isolated from wound culture and hemoculture. Patient has second debridement after four days. After second debridement was applied metabolic control, broad-spectrum antibiotics coverage, dressing the wound and negative pressure wound therapy (NPWT). Patient was discharged home five weeks after a second debridement in good condition. One month later she underwent reconstructive surgical treatment. Besides extensive FG and multiple comorbidity she was successfully managed with good outcome. Fournier’s gangrene remains a life-threatening and fulminant disease which need urgent diagnosis and aggressive medical and surgical treatment, to achieve a reduction in long term complications and mortality rate.

BACKGROUND: Proteinuria, in addition to haematuria, is the most important laboratory parameter in patients with nephro-urological diseases. Low molecular weight proteinuria (LMWP) is of particular importance because some diseases genetic and tubulointerstitial are diagnosed based on its presence.AIM: The purpose of this study is to describe the clinical features, the course and outcome of pediatric patients with a renal disease associated with LMWP.MATERIAL AND METHODS: This retrospective observational study included 250 pediatric patients with various kidney diseases in which the type of proteinuria was defined by 4-20% gradient gel sodium dodecyl sulphate polyacrylamide gel (SDS-PAG) electrophoresis.RESULTS: Isolated LMWP was detected in 12% of patients, while mixed glomerulotubular proteinuria was detected in 18% of patients. It was detected in all patients with the Dent-1/2 disease, Lowe's syndrome and secondary Fanconi syndrome. Transient LMWP was also detected in a series of 12 patients with distal renal tubular acidosis. In patients with nephrotic syndrome, it was associated with corticoresistence and unfavourable clinical course.CONCLUSION: This study contributes to the understanding of the clinical spectrum of various kidney diseases associated with LMWP, their natural course, and the effect of therapy.

Objective: Febrile proteinuria is functional proteinuria and is seen as a transitory phenomenon during acute febrile illness, mainly viral infections. It is a benign phenomenon and clears promptly with resolution of the infection. Clinical Presentation and Intervention: In this report, we present a patient who was thought to have febrile proteinuria. Persistence of significant proteinuria after resolution of the infection prompted biochemical and genetic workup which led to the diagnosis of Dent-2 disease. Conclusion: We recommend the use of SDS-PAGE (sodium dodecyl sulfate electropheresis) for the detection of low molecular weight proteinuria.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. Progressive damage and decline in the number of podocytes often occur in the early stages of DN. Thus, nephrin as a podocyte-specific protein may be regarded as a potential biomarker of early detection of DN. The aim of this study is to determine whether urinary nephrin is an earlier marker in DN than microalbuminuria and to test the significance of urinary nephrin as a marker for early detection of DN.Our cross-sectional study included 90 patients with type 2 diabetes mellitus (T2DM), 30 patients with diagnosed DN and 60 patients without diagnosed DN. As a control group, we used 30 healthy subjects. All patients with T2DM were classified into three subgroups according to urinary microalbumin/creatinine ratio (UMCR): normoalbuminuric, microalbuminuric and macroalbuminuric patients. Nephrin in urine was measured by immunoenzyme assay, microalbumin with turbidimetric and creatinine with the photometric method. In blood sera, we measured a few standard biochemical parameters.Nephrinuria was found to be present in 100% of patients with T2DM and macroalbuminuria, in 88% with microalbuminuria, as well as 82% of patients with T2DM and normoalbuminuria. A concentration of urinary nephrin was significantly increased in all groups of subjects with T2DM compared to the control group (p0.05). Nephrinuria correlated statistically negative with eGFR (r=-0.54). ROC analysis showed that nephrin has a total predicted probability of 96% in patients with DN.Urinary nephrin is earlier, more specific and sensitive marker than microalbumin in early detection of DN.Dijabetesna nefropatija (DN) je vodeći uzrok završnog stadijuma bubrežne bolesti. Progresivno oštećenje i opadanje broja podocita često se javlja u ranim fazama DN, tako da se nefrin kao specifični protein podocita može smatrati potencijalnim biomarkerom ranog otkrivanja DN. Cilj ove studije je da utvrdi da li je urinarni nefrin raniji marker DN u odnosu na mikroalbuminuriju i da testira značaj urinarnog nefrina kao markera za rano otkrivanje DN.Naša studija preseka je obuhvatila 90 pacijenata sa dijabetesom tipa 2 (T2DM), 30 bolesnika sa dijagnozom DN i 60 pacijenata bez dijagnoze DN. Za kontrolnu grupu smo koristili 30 zdravih ispitanika. Svi bolesnici sa T2DM kla sifikovani su u tri podgrupe prema odnosu urinarnog mikro albumina/kreatinina (UMCR): normoalbuminurni, mikro albuminurni i makroalbuminurni pacijenti. Nefrin u urinu je meren imunoenzimskim testom, mikroalbumin uz korišćenje turbidimetrijske i kreatinin koristeći foto metrijsku metodu. U krvnim serumima, merili smo nekoliko standardnih biohemijskih parametara.Ustanovljeno je da je nefrinurija prisutna u 100% pacijenata sa T2DM i makroalbuminurijom, u 88% sa mikro albuminurijom, kao i 82% pacijenata sa T2DM i normoalbuminurijom. Koncentracija urinarnog nefrina značajno je povećana u svim grupama ispitanika sa T2DM u poređenju sa kontrolnom grupom (p0,05). Nefrinurija je korelirala statistički negativno sa eGFR (r = -0,54). ROC analiza je pokazala da je kod pacijenata sa DN ukupna predviđena verovatnoća nefrina 96%.Urinarni nefrin je raniji, specifičniji i osjetljiviji marker od mikroalbumina u ranom otkrivanju DN.

In the last two decades a great progress was observed in understanding of podocytes, their specific structure and function identifying many specific podocyte proteins, such as nephrin and podocalyxin. Podocytes form the final barrier to plasma proteins leakage. Nephrin as a main component of the filtration diaphragm forms a physical barrier while podocalyxin as sialoglycoprotein forms an electrostatic barrier. Podocyte damage, i.e. podocytopathies and their loss through urine-podocyturia, are crucial in pathogenesis and progression of nephropathies with proteinuria as main clinical manifestation. In podocytopathies, nephrin and podocalyxin appear in the urine before proteinuria and microalbuminuria which were previously considered as earliest markers of nephropathies. Nephrinuria and podocalyxuria indicate damage of the podocytes on glomerular level and/or presence of apoptotic and necrotic podocytes in urine. These urinary markers are also important in early diagnosis of secondary nephropathies such as diabetic, lupus and hypertensive nephropathy as the most common causes of end-stage renal failure (ESRF). These markers are also important in the prediction of preeclampsia, which is the most common complication in pregnancy. In this review we elaborate in dept the main structural and functional features of podocytes and their specific proteins, nephrin and podocalyxin, summarizing the recent literature data on their importance in the early diagnosis of the most common secondary nephropathies.

Early diagnosis of renal allograft dysfunction is crucial for the management and long-term survival of the transplanted kidney. Early after transplantation, acute tubular necrosis (ATN) manifesting as delay graft function (DGF) or slow graft function, acute rejection (AR) or drug toxicity (e.g. calcineurin inhibitor) are the leading causes of Acute kidney injury (AKI). Acute rejection, ATN, and calcineurin inhibitor toxicity continue to be major causes of renal allograft dysfunction along with other causes like infections (e.g. BK and CMV viruses, pyelonephritis), obstruction, and recurrence of the original disease. Clinicians have been searching for non-invasive tools that would allow the diagnosis of acute renal allograft dysfunction early and accurately without performing a kidney biopsy. The following comprehensive review will describe the latest findings on urinary biomarkers in acute allograft dysfunction.

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Distal renal tubular acidosis (dRTA) (MIM #267300, #602722 and #179800) is a rare inherited tubulopathy characterized by the inability of the distal tubule to acidify the urine with consecutive systemic acidosis. The clinical features include polyuria, polydipsia, poor appetite, failure to thrive, short stature and rickets. Prominent biochemical features are hypokalemia, hypercalciuria and hypocitraturia. There are reports on patients who presented with unusual biochemical features such as low molecular proteinuria, hypophosphatemia, hypouricemia, generalized hyperaminioaciduria, hyperoxaluria and other making diagnostic confusion to the clinicians. In this work, we report on a series of 8 children with clinically, biochemically and genetically proven dRTA who present with low molecular proteinuria at the disease onset. With metabolic compensation of the disease, there was complete resolution of the low molecular weight protenuria and other proximal tubular abnormalities in all children. Late recognition of the disease with long standing hypokalemia and acidosis may result in abnormal expression and function of the transporters in the proximal tubules. Sodium dodecyl sulphate polyacrylamide gel electrophoeresis is an accurate method for detection and follow up of patients with low molecular weight proteinuria.

A key to effective treatment of cardiovascular disease is to understand the body’s complex lipoprotein transport system. Reverse cholesterol transport (RCT) is the process of cholesterol movement from the extrahepatic tissues back to the liver. Lipoproteins containing apoA-I [high-density lipoprotein (HDL)] are key mediators in RCT, whereas non-high-density lipoproteins (non-HDL, lipoproteins containing apoB) are involved in the lipid delivery pathway. HDL particles are heterogeneous; they differ in proportion of proteins and lipids, size, shape, and charge. HDL heterogeneity is the result of the activity of several factors that assemble and remodel HDL particles in plasma: ATP-binding cassette transporter A1 (ABCA1), lecithin cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), hepatic lipase (HL), phospholipid transfer protein (PLTP), endothelial lipase (EL), and scavenger receptor class B type I (SR-BI). The RCT pathway consists of the following steps: 1. Cholesterol efflux from peripheral tissues to plasma, 2. LCAT-mediated esterification of cholesterol and remodeling of HDL particles, 3. direct pathway of HDL cholesterol delivery to the liver, and 4. indirect pathway of HDL cholesterol delivery to the liver via CETP-mediated transfer There are several established strategies for raising HDL cholesterol in humans, such as lifestyle changes; use of drugs including fibrates, statins, and niacin; and new therapeutic approaches. The therapeutic approaches include CETP inhibition, peroxisome proliferator-activated receptor (PPAR) agonists, synthetic farnesoid X receptor agonists, and gene therapy. Results of clinical trials should be awaited before further clinical management of atherosclerotic cardiovascular disease.

Introduction. End-stage renal disease (ESRD) patients undergoing hemodialysis are at an increased risk of arteriosclerotic vascular disease (ASVD). The increased risk is commonly attributed to the traditional risk factors related to ESRD. However, interest for more recent risk factors for ASVD, such as the level of lipoprotein(a) and its specific apoprotein(a) has been promoted. The aim of this paper is to determine whether apo(a) phenotype is a risk factor for arteriosclerotic vascular disease in ESRD patients who are on hemodialysis. Methods. Apo(a) phenotypisation was performed by using the Western Blot Technique of blood samples from 96 end-stage renal disease patients who were undergoing hemodialysis, and from 100 healthy individuals. Results. Frequency distribution of the basic apo(a) isoforms calculated by means of the χ2-test has shown that there was no significant statistical difference in distribution among patients on hemodialysis, and healthy carriers (χ2-0.36, p2-0.10, p Conclusion. Based on the results obtained, it can be concluded that the apo(a) phenotype is not a risk factor for arteriosclerotic vascular disease in patients undergoing hemodialysis.

Objective: Acute kidney injury is common condition in the neonatal intensive care unit and it is associated with poor outcome. The incidence of neonatal AKI is the highest one followed by adults and children, depending on different factors such as the gestational age, birth weight, contributing conditions and the facilities of the neonatal intensive care unit. The aim of the study was to determine the incidence, risk factors and the outcome of the neonatal acute kidney injury. Subjects and Methods: This was a clinical, prospective study that was performed in a referent NICU at the University Children′s Hospital in Skopje. All neonates admitted from January 2012 to December 20014 with documented acute kidney injury were included. The medical data records of the admitted neonates with AKI were analyzed. The material was statistically processed using methods of the descriptive statistics. Results: During the study period 770 newborn infants were admitted to the NICU and 50 (6.5%) infants developed acute kidney injury. The male to female ratio was 2.1:1. Most of the neonates involved in the study were neonates born at term (62%). Oliguric AKI was found in 28 cases (56%) and no oliguric in 22 cases (44%). The prevalence of prerenal, renal and post renal AKI were 78.5%, 19.5% and 2.0% respectively. Perinatal asphyxia was the most common predisposing factor for AKI and was evaluated in 38% of the cases with predominance of term infants and male. The mortality rate was 32% and was significantly higher in the group of patients with congenital heart diseases. Conclusion: AKI is a life threatening condition with still high mortality rate. Early recognition of the risk factors and the rapid effective treatment of the contributing conditions will reduce AKI in the neonatal period.

Background: Chronic infections in CHD are due to one or both of the organisms Chlamydia pneumoniae and Helicobacter pylori. Aim: To examine the association between serum markers of Chlamydia pneumoniae and Helicobacter pylori infection and markers of myocardial damage. in patients with acute coronary syndrome (ACS), with chronic coronary artery disease (CAD) and in–control group. Material and methods: Sera were taken from a total of 153 subjects. Subjects were divided in three groups: 64 patients with ACS; 53 patients with CAD and a group of 35 conditionally healthy individuals. Analysis of patients’ sera for IgG antibodies to H. pylori and markers for myocardial damage was done on the Immulite system.. The presence of specific IgG and IgA antibodies to C. pneumoniae was determined with MIF, Sero FIA (Savyon Diagnostics, Israel). Statistical analysis of data was done using the statistical program SPSS (Statistical Package for Social Sciences), version 13. Results and discussion: There was a high significant difference in troponin levels between the three groups of subjects (p=0.0000). Levels of creatine kinase isoenzyme (CK-MB) were highest in the ACS group (500.0 ng/mL). There was a statistically significant difference between CG subjects and ACS patients due to more frequent detection of antichlamydial IgA antibodies in patients with acute coronary syndrome. Positive serum immune response for Helicobacter pylori was 17 (53.1%) and 29 (80.6%), respectively. Conclusion: Increased IgA antibody titers for C. Pneumoniae, increased CRP values as well as classic markers of myocardial damage are risk factors for coronary events.

Apoprotein(A) Isoforms and Plasma LP(A) Concentration in Members of Four FamiliesApoprotein(a) is a multikringle protein which shows a genetically inherited size polymorphism. The APO(a) gene is located at the telomeric region of chromosome 6q2.6-q 2.7. Apo(a) size polymorphism is a major determinant of Lp(a) levels. The aim of this study is to describe the influence of apo(a) size polymorphism on the plasma Lp(a) levels in the members of four families. K3EDTA plasma was obtained from every subject after over-night fast. Apo(a) isoforms were determined by 3-15% SDS-PAGE followed by Western immunoblot technique. Plasma Lp(a) level was de - termined with immunonephelometric method. Every child inherited one isoform from its mother and the other from its father. The children from the first family had Lp(a) levels similar to those measured in their parents. The daughters from the second and fourth family inherited the dominant S3 apo(a) isoform from their mothers and also mother's high Lp(a) levels (0.365 g/L - daughter from the second, and 0.465 g/L and 0.446 g/L - daughter from the fourth family respectively). The elder daughter from the third family, carrier of double banded S4S1 apo(a) isoform, had the highest Lp(a) level among the children from all four families. We found out a generation decrease of the Lp(a) level in two families. On the basis of our findings we concluded that the inheritance of the apo(a) isoforms in the members of all four families is in accordance with the simple Mendelian's model and that the apo(a) size polymorphism influences the Lp(a) level in the blood of the examined subjects.

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.

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.

The disorders of serum concentration of Apolipoproteins A-1 and B (Apo A-1 and Apo B), as well as of Apolipoprotein B/Apoliporotein A-1 ratio (Apo B/ Apo A-1) are considered to be independent risk factors for coronary heart disease. It is recommended that each laboratory should asign its own reference intervals for Apo A-1 and Apo B, as well as for Apo B/Apo A-1 ratio, as these depend on the geographical location. For this purpose we determined the serum concentration of Apo A-1 and Apo B and we calculated the Apo B/Apo A-1 ratio in 122 healthy individuals from Macedonian population (70 women and 52 men), aged 18-60 years. We used commercial, standardized manual immunoturbidimetric assay (Randox). The obtained concentrations (x ± SD) were: Apo A-1 = 1.39 ± 0.28 g/l in the female population and 1.42 ± 0.27 g/l in the male population, Apo B = 0.95 ± 0.24 g/l in the female and 1.00 ± 0.23 g/l in the male population. The calculated Apo B/Apo A-1 ratio was 0.70 ± 0.17 in the female subgroup and 0.72 ± 0.18 in the male subgroup. Student's t-test detected no significant difference (p>0.05) in mean values for Apolipoproteins A-1 and B and for Apo B/Apo A-1 ratio between female and male subgroups, which lead to the conclusion that division according to sex is not necessary from diagnostic point of view. The Apo A-1 and Apo B concentrations estimated for the mixed group (N=122) were 1.40 ± 0.28 g/l and 0.97 ± 0.23 g/l. The Apo B/Apo A-1 ratio estimated for whole group was 0.71 ± 0.17. The type of distribution of values for Apo A-1, Apo B and Apo B/A-1 ratio was tested by calculating the values of coefficient of asymetry (Skewness), coefficient of flatness (Kurtosis) and standard deviations of these coefficients. The distribution of values was according to Gaussian curve for all three parameters. Therefore, according to the normal distribution reference intervals for Apo A-1, Apo B and Apo B/Apo A-1 ratio was estimated parametrically (mean ± 2 standard deviations). Reference intervals for Apo A-1 were 0.84 - 1.96 g/l, 0.51 - 1.43 g/l for Apo B and 0.37- 1.05 for Apo B/Apo A-1 ratio.

OBJECTIVE With increasing interest in the role of non-traditional lipid risk factors in coronary artery disease, we undertook this study to relate LDL subclass size and carotid intima-media thickness of the common carotid artery in coronary artery disease patients. METHODS The study was conducted in 106 patients during their first visit (study group I) and after 12 months (study group II). Intima-media thickness of the common carotid artery was determined using B-mode ultrasound. Separation of LDL subclasses was preformed by 3-31 % polyacrylamide gradient gel electrophoresis. RESULTS LDL3 was the dominant subclass in both study groups, but there was statistically significant difference in the distribution of dominant LDL subclasses (p

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

Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop-p-ethyl (I), diclofop-methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol-water solutions of each pesticide were then added at concentrations of 2.4 x 10(-4)-2.4 x 10(-8) mol/L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 x 10(-4) mol/L and was lowered by about 15-26% for pesticide I at concentrations of 2.4 x 10(-5)-2.4 x 10(-8) mol/L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3-20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide.

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.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.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.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.