Vladmila Bojanić

Identification and validation of six proteins as marker for endemic nephropathy

Endemic nephropathy (EN) is defined as a slow progressive renal tubulointestitial disease that mainly occurs in the restricted areas of the Balkan Peninsula. The complexity of the pathogenesis of EN makes its earlier diagnosis very difficult. Urine samples from healthy volunteers from EN regions, EN patients with proteinuria less than 150 mg/L and EN patients with proteinuria more than 150 mg/L, patients with acute kidney injury, patients with diabetic nephropathy and healthy volunteers from Germany were collected. The urinary proteome analyses were performed using 2-D DIGE and mass spectrometry. The validation of biomarkers was investigated by two approaches (Western blot (WB) and dot blot) in successively increasing size - and partially overlapping - sample sets. Comparative and statistical analyses of the proteomics data from the different patient groups allowed the identification of six proteins (alpha-1-microglobulin, alpha-2-glycoprotein-1, beta-2-microglobulin, mannose-binding-lectin-2, protection-of-telomeres-protein-1, and superoxide-dismutase [Cu-Zn]), which were able to discriminate EN with low and high proteinuria from the other groups with high significance (p<0.05). The reliability of the identified proteins as EN marker was underlined with high statistical significance using WB analyses (sensitivity 66.7-98% and specificity 70-100%), whereas the dot blot analyses revealed a decrease in the sensitivity and specificity of these biomarkers.

HPLC method development for determination of doxycycline in human seminal fluid.

The present paper reports the development and validation of an analytical method for doxycycline quantification in human seminal fluid by HPLC with UV detection. The separation of doxycycline was achieved at 40°C on a reversed-phase C18 column using isocratic elution. The mobile phase consisted of acetonitrile (A) and water buffered at pH 2.5 with a concentrated orthophosphoric acid (B) in the volume ratio of 20:80 (v/v), respectively. The detection was performed at 350nm. As an internal standard (IS), tetracycline was used. The proposed method involves the extraction of doxycycline from seminal fluid based on acidic precipitation of the proteins using perchloric acid. The method showed good intra- and inter-day precisions (RSD<7.0%), good accuracy (recovery for doxycycline>80%), and high correlation coefficient (r=0.998) for standards subjected to the entire procedure. The detection and quantification limits were 0.087μg/ml and 0.264μg/ml. The developed method was used to analyze doxycycline in the seminal fluids obtained from male subjects who were treated with doxycycline-hyclate. The mean doxycycline concentrations of 0.89±0.07μg/ml and 0.45±0.26μg/ml were detected in seminal fluid after 6h and 12h, respectively. This is the first study reporting extraction and HPLC determination of doxycycline in this complex sample and can be very useful in support of clinical and pharmacokinetic studies on this antibiotic.

Crosstalk of inflammatory mediators and lipid parameters as early markers of renal dysfunction in stable renal transplant recipients with regard to immunosuppression

BACKGROUND Kidney transplantation is still the treatment of choice for end-stage renal disease, therefore it is important to establish all modifiable risk factors for initiation of renal dysfunction. MATERIAL/METHODS We enrolled 73 renal transplant recipients, who were more than 12 months post-renal transplant surgery, had a stable graft function, had no clinically present cardiovascular disease, and were on standard immunosuppressive therapy. The concentrations of intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), CRP, lipids, and lipoproteins were measured. We used logistic regression to calculate non-adjusted, age, and multivariable-adjusted ORs and 95% confidence intervals for glomerular filtration rate, GFR <60 ml/min/1.73 m(2). RESULTS Non-adjusted OR showed that there was a significant risk of reduced GFR in patients with total cholesterol higher than 5.19 mmol/L, LDL cholesterol ≥ 4.1 mmol/L, non- HDL ≥ 4.2 mmol/L, and higher VCAM-1 concentration. After adjustment for age and in multivariable model, OR showed a significant risk for reduced GFR in patients with total cholesterol ≥ 5.2 mmol/L, LDL ≥ 4.1 mmol/L, non-HDL ≥ 4.2 mmol/L, and higher VCAM-1 concentration. HDL, triglycerides, CRP, and lipoprotein ratios did not have any significance as predictors of renal dysfunction. There were no differences in all evaluated parameters between groups in regard to immunosuppressive therapy. CONCLUSIONS Total cholesterol, LDL, non-HDL, and VCAM-1 are strong and independent predictors of renal dysfunction in stable renal transplant recipients. In contrast, HDL, CRP, triglycerides, and ICAM-1 did not seem to have any impact on renal dysfunction.

Drug interactions with grapefruit

INTRODUCTION The concentration of many orally given medications may be affected by grapefruit or grapefruit juice consumption. It may result in numerous harmful effects. INTERACTION OF GRAPEFRUIT WITH DRUGS Taking only one cup of juice may induce interactions with different drugs even during the period of a few days. The effect is induced by suppression of cytochrome P450 isoenzyme CYP3A4 in the intestinal wall. The Latin name of grapefruit, Citrus paradisi, is quite opposite to the effects which could be induced by taking grapefruit and some medications at the same time. It is necessary to avoid taking grapefruit with the drugs whose pharmacokinetics could be altered by the active principles found in that fruit. DISCUSSION The coloured grapefruit contains less furanocoumarins, but there is no difference in induction and intensity of pharmacokinetic interaction with drugs related to its colour. Other citrus fruits (orange, lemon) do not have such effects, but some other fruits (pomegranate, stella fruit, banpeiyu, hassaku, takaoka-buntan and kinkan) exert inhibitory effects on the activity of cytochrome P450 isoenzyme.

LIPID PEROXIDATION AND OXIDATIVE PROTEIN PRODUCTS IN CHILDREN WITH EPISODIC FEVER OF UNKNOWN ORIGIN / LIPIDNA PEROKSIDACIJA I OKSIDATIVNI PROTEINSKI PRODUKTI KOD DECE SA EPIZODINOM GROZNICOM NEPOZNATOG UZROKA

Summary Background: Episodic fever syndromes are commonly seen in pediatric practice. Episodic fever of unknown origin (FUO) lasts for a few days or weeks and is followed by a fever-free period with a sense of well-being. In this condition, activated neutrophils and monocytes intensively generate reactive oxidative species that may further damage various mole- cules. The aim of the study was to evaluate oxidative stress biomarkers, lipid peroxidation in erythrocytes and plasma, and advanced oxidation protein products (AOPP) in children with episodic FUO. Methods: The study enrolled 25 children with episodic FUO in afebrile phase and 25 healthy children as controls. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) production with the thiobarbituric-acid-reactive sub- stances (TBARS) assay in erythrocytes and plasma. Oxidative modification of proteins was measured spectrophotometri- cally by the determination of AOPP in plasma. Results: Mean duration of episodic fevers was 3.96±2.8 years. Erythrocyte MDA levels were higher in children with FUO than in controls (86.26± 10.75 vs. 78.0±3.21 nmol/g hemoglobin), although not significantly (p=0.202). The MDA plasma concentrations were similar (2.42±0.35 vs. 2.41 ±0.39 (xmol/L) between the groups (p=0.732). Unexpectedly, levels of AOPP were significantly lower in chil- dren with FUO than in healthy controls (18.8±5.04 vs. 25.1 ±3.35 nmol/L, p=0.047). Conclusions: Episodic fevers of unknown origin with an aver- age duration of 3.96±2.8 years do not cause significant oxidative modifications of lipids and proteins in children. Kratak sadržaj Uvod: Sindromi epizodične ili rekurentne groznice često se sreću u pedijatrijskoj praksi. Epizodična groznica nepoznatog uzroka (FUO) traje nekoliko dana do nekoliko nedelja, nakon čega sledi miran period bez povišene temperature uz osećaj potpunog zdravlja. U ovim stanjima, aktivirani neutrofili i monociti intenzivno produkuju reaktivne kiseonične vrste koje naknadno mogu oštetiti različite molekule. Cilj našeg rada bio je oceniti biomarkere oksidativnog stresa, odnosno lipidnu peroksidaciju u eritrocitima i plazmi, kao i uznapredovale oksidativne proteinske produkte (AOPP) kod dece sa epizo- dičnom FUO. Metode: U istraživanje je uključeno 25 dece sa epizodičnom groznicom u afebrilnoj fazi i 25 zdrave dece. Lipidna peroksi- dacija je utvrđena merenjem produkcije malondialdehida (MDA) korišćenjem testa tiobarbiturnih reagujućih supstanci (TBARS) u eritrocitima i plazmi. Oksidativna modifikacija proteina je određivana spektrofotometrijski, merenjem AOPP-a u plazmi. Rezultati: Srednje vreme trajanja epizodičnih groznica bilo je 3,96±2,8 godina. Vrednosti MDA u eritrocitima su bile vise kod dece sa epizodičnom FUO nego kod zdrave dece (86,26 ±0,75 vs. 78,0±3,21 nmol/g hemoglobina), iako ne sta- tistički značajno (p=0,202). Koncentracije MDA u plazmi su bile slične kod ove dve grupe dece (2,42±0,35 vs. 2,41 ± 0,39(jmol/L, p=0,732). Neočekivano, nivoi AOPP-a su zna- čajno bili manji kod dece sa FUO nego kod zdravih kontrol- nih subjekata (18,8±5,04 vs. 25,1 ±3,35 (jmol/L, p=0,047). Zaključak: Epizodične groznice nepoznatog uzroka u trajanju od 3,96 ±2,8 godina ne izazivaju značajnu oksidativnu mo- difikaciju lipida i proteina kod dece.

Adipoparacrinology of Atherosclerosis: Evidence Updated

Abstract: Recently, the secretory - endocrine, paracrine and autocrine - phenotype of adipose tissue, consisting of adipo-cytes, stromovascular cells and immune cells, has increasingly been recognized. In humans, adipose tissue is partitioned into two large depots (subcutaneous and visceral) and many small depots associated with heart, blood vessels, major lymph nodes, pancreas, prostate gland, ovaries. Accordingly, two major subfields of adipobiology have emerged, adi-poendocrinology (studying the endocrine activity of adipose tissue) and adipoparacrinology (studying the paracrine activ-ity of adipose tissue). Traditional concept of the pathogenesis of atherosclerosis focuses on intimal surface, where endo-thelial dysfunction expressed by an “inside-out” inflammatory process triggers the formation of atherosclerotic plaque. The present short review highlights evidence for the possible role of dysfunctional paracrine activity of epicardial adipose tissue and of periadventitial adipose tissue in an “outside-in” pathway in the development of coronary and peripheral athe-rosclerosis, respectively. Such a paradigm may have various therapeutic applications including in coronary artery bypass surgery.