Alpha-1-Antitrypsin in Pathogenesis of Hepatocellular Carcinoma

Context: Alpha-1-antitrypsin (A1AT) is the most abundant liver-derived, highly polymorphic, glycoprotein in plasma. Hereditary deficiency of alpha-1-antitrypsin in plasma (A1ATD) is a consequence of accumulation of polymers of A1AT mutants in endoplasmic reticulum of hepatocytes and other A1AT-producing cells. One of the clinical manifestations of A1ATD is liver disease in childhood and cirrhosis and/or hepatocellular carcinoma (HCC) in adulthood. Epidemiology and pathophysiology of liver failure in early childhood caused by A1ATD are well known, but the association with hepatocellular carcinoma is not clarified. The aim of this article is to review different aspects of association between A1AT variants and hepatocellular carcinoma, with emphasis on the epidemiology and molecular pathogenesis. The significance of A1AT as a biomarker in the diagnosis of HCC is also discussed. Evidence Acquisitions: Search for relevant articles were performed through Pub Med, HighWire, and Science Direct using the keywords "alpha-1-antitrypsin", "liver diseases", "hepatocellular carcinoma", "SERPINA1". Articles published until 2011 were reviewed. Results: Epidemiology studies revealed that severe A1ATD is a significant risk factor for cirrhosis and HCC unrelated to the presence of HBV or HCV infections. However, predisposition to HCC in moderate A1ATD is rare, and probably happens in combination with HBV and/or HCV infections or other unknown risk factors. It is assumed that accumulation of polymers of A1ATD variants in endoplasmic reticulum of hepatocytes leads to damage of hepatocytes by gain-of-function mechanism. Also, increased level of A1AT was recognized as diagnostic and prognostic marker of HCC. Conclusions: Clarification of a carcinogenic role for A1ATD and identification of pro-inflammatory or some still unknown factors that lead to increased susceptibility to HCC associated with A1ATD may contribute to a better understanding of hepatic carcinogenesis and to the development of new drugs. Published by Kowsar Corp, 2012. cc 3.0

Hemiluminiscencija - teorijski princip, reakcije i primena u kliničkoj laboratorijskoj praksi i istraživanjima

This article reviews the fundamental and applied aspects of chemiluminescence and its applications in both routine clinical analysis and in the diverse applications in clinical research. Chemiluminiscence reactions are basic of luminometric assays, induce ultra sensitive detection limits (attomole-zeptomole), rapid assays, and a broad range of analytical applications. In the routine clinical laboratory, chemiluminescence is now commonly used for immunoassay and DNA probe assays, in the form of a chemiluminescent labels (acridinium ester, acridinium sulfonamide) and detection reactions for peroxidase and alkaline phosphatase enzyme labels (luminol and adamantyl 1,2-dioxetane-based reactions, respectively). Chemiluminescent immunoassay test kits and automated immunoassay analyzers have been developed. There are broad range of chemiluminescent immunoassay tests which are routinely used for the measurement of the biochemistry, immunology, toxicology, virology, and endocrinology analytes in the assessment of thyroid function, fertility, myocardial damage, anemia, therapeutic drug levels, and diagnosing and monitoring drug abuse, cancer and infectious diseases (e.g. hepatitis). In clinical research, the sensitivity, dynamic range and diversity of chemiluminescent assays has led to a vast range of applications, notably in protein and nucleic acid blotting, microarray-based assays, monitoring reactive oxygen species, and as detection reactions for substances separated by HPLC and capillary electrophoresis. Nowadays, in clinical research, chemiluminescent detection techniques are used to measure enzymes expressed by reporter genes, cellular luminescence, blotted proteins (Western blotting) and nucleic acids (Northern and Southern blotting).U ovom radu, dato je teorijsko objašnjenje nastanka hemiluminiscencije i prikazane su najznačajnije hemiluminiscentne i bioluminiscentne rekcije. Na bazi hemiluminiscencije razvijena je moćna, visoko osetljiva analitička tehnika - luminometrija, sa detekcionionim limitom do atomola (10-18) i čak zeptomola (10-21), koja je našla široku primenu u imunohemijskim određivanjima i DNK analizama. U tu svrhu, ispitivana jedinjenja se vezuju za hemiluminiscentne obeleživače (akridinijum estar, akridinijum sulfonamid), ili se obeležavaju enzimima (peroksidazom ili alkalnom fosfatazom), koji pojačavaju signal hemiluminiscentnih reakcija. U rutinskoj praksi, primenjuje se veliki broj hemiluminiscentnih imuno-testova za merenje biohemijskih, imunoloških, toksikoloških, virusoloških i endokrinoloških analita u cilju ispitivanja funkcije tiroideje, fertiliteta, oštećenja miokarda, anemije, praćenja koncentracije leka, dijagnoze i praćenja zloupotrebe lekova, otkrivanja i praćenja terapije kancera, kao i infektivnih bolesti (npr. hepatitis). U kliničkim istraživanjima, zahvaljujući osetljivosti, brzini i raznolikosti izvođenja, došlo je do široke primene hemiluminiscencije, posebno u metodama ispitivanja proteina i nukleinskih kiselina pomoću blotting metoda (Western, Northern i Southern blotting), microarray matoda, praćenja reaktivnih kiseoničnih radikala, kao i u reakcijama detekcije supstanci, koje su prethodno razdvojene tečnom hromatografijom i kapilarnom elektroforezom. Danas se tehnike hemiluminiscentne detekcije koriste i za merenje aktivnosti enzima za čiju sintezu su odgovorni određeni geni (reporters genes)

Deep Eutectic Solvent Based Reversed-Phase Dispersive Liquid–Liquid Microextraction and High-Performance Liquid Chromatography for the Determination of Free Tryptophan in Cold-Pressed Oils

A fast and straightforward reversed-phase dispersive liquid–liquid microextraction (RP-DLLME) using a deep eutectic solvent (DES) procedure to determine free tryptophan in vegetable oils was developed. The influence of eight variables affecting the RP-DLLME efficiency has been studied by a multivariate approach. A Plackett–Burman design for screening the most influential variables followed by a central composite response surface methodology led to an optimum RP-DLLME setup for a 1 g oil sample: 9 mL hexane as the diluting solvent, vortex extraction with 0.45 mL of DES (choline chloride–urea) at 40 °C, without addition of salt, and centrifugation at 6000 rpm for 4.0 min. The reconstituted extract was directly injected into a high-performance liquid chromatography (HPLC) system working in the diode array mode. At the studied concentration levels, the obtained method detection limits (MDL) was 11 mg/kg, linearity in matrix-matched standards was R2 ≥ 0.997, relative standard deviations (RSD) was 7.8%, and average recovery was 93%. The combined use of the recently developed DES -based RP-DLLME and HPLC provides an innovative, efficient, cost-effective, and more sustainable method for the extraction and quantification of free tryptophan in oily food matrices. The method was employed to analyze cold-pressed oils from nine vegetables (Brazil nut, almond, cashew, hazelnut, peanut, pumpkin, sesame, sunflower, and walnut) for the first time. The results showed that free tryptophan was present in the range of 11–38 mg/100 g. This article is important for its contributions to the field of food analysis, and for its development of a new and efficient method for the determination of free tryptophan in complex matrices, which has the potential to be applied to other analytes and sample types

Cytotoxicity and antimicrobial activity of Teucrium scordium L. (Lamiaceae) extracts

Cytotoxicity and antimicrobial activity of cyclohexane, dichlormethane and methanol extracts of Teucrium scordium subspec. scordioides was studied. Cyclohexane and dichlormethane extracts of T. scordium possessed high citotoxicity against MDA-MB-361 cells (IC(50)=130.33+/-0.1 mu g/ml and IC(50)=189.89+/-3.99 mu g/ml, respectively). Dichlormethane extract was more effective against MDA-MB-453 cell line (IC(50)=130.33 +/- 0.1 mu g/ml). The methanol extract of T. scordium possessed no cytotoxicity against breast cancer cell lines, MDA-MB-361 and MDA-MB-453. Herb extracts of T. scordium have shown weak antibacterial activity on Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Bacillus subtilis with no activity against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Enterococcus faecalis, and Candida albicans

Alpha-1-Antitrypsin Deficiency in Serbian Adults with Lung Diseases

Aim: Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population. Methods: The study included the adults with chronic obstructive pulmonary disease (COPD) (n = 348), asthma (n = 71), and bronchiectasis (n = 35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis. Results: PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1: 5519, 1: 38, and 1:5519). Conclusion: The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population

Insulin mimetički efekat niskih koncentracija amonijum dekavanadata na izolovane adipocite pacova

In this study, the insulin mimetic activity of ammonium decavanadate (DV) (in concentration 0.1 and 1.0 mM) dissolved in saline (0.9 % w/v of NaCl) or dimethyl sulfoxide, DMSO (2% v/v) was evaluated by means of in vitro measurements of the glucose uptake (GU) and inhibition of free fatty acids release (IFFAR) using isolated epinephrine-pretreated white adipocytes from Wistar rats. Our data showed strong insulin mimetic effect of DV (> 80 %) in both concentration and independently of the solvent were used. Having in mind satisfactory pharmacological effect of DMSO as well as its capability of penetration enhancer it would be reasonable to continue in vivo testing of dose-dependent insulin mimetic effect of decavanadate in DMSO. It is also necessary to examine toxicity of decavanadate in order to estimate its therapeutic index as potential antidiabetic agent.Ispitana je insulin mimetička aktivnost amonijum dekavanadata (DV), in vitro merenjem preuzimanja glukoze (GU) i merenjem inhibicije oslobađanja slobodnih masnih kiselina (IFFAR) na modelu izolovanih belih adipocita Wistar pacova. Ovaj biološki efekat dekavanadata (u koncentracijama od 0,1 i 1,0 mM) koji je rastvoren u fiziološkom rastvoru (0.9 % NaCl) ili u dimetil sulfoksidu (2% DMSO) ispitan je na adipocitima koji su prethodno tretirani adrenalinom. Prema dobijenim rezultatima dekavanadat pokazuje snažan insulin mimetički efekat (> 80 %) i to u obe koncentracije i nezavisno od vrste rastvarača. Na osnovu ovog in vitro eksperimenta može se zaključiti da bi bilo opravdano dozno-zavisno ispitivanje insulin mimetičkog efekta dekavanadata u DMSO-u kao rastvaraču u in vivo uslovima. Takođe, neophodno je ispitati i toksičnost ovog jedinjenja kako bi se što bezbednije iskoristio njegov insulin mimetički efekat

Rizik razvoja multiple skleroze povezan sa oksidativnim stresom

Background: Multiple sclerosis (MS) is characterized by inflammation, demyelination and axonal degeneration. Oxidative stress (OS) plays a significant role in the pathogenesis of the disease. The aim of the study was to examine the association between OS and smoking on the MS development. Methods: The study included 175 patients with relapsing- remitting multiple sclerosis (RRMS) (76 males, 99 females) and 254 healthy subjects (81 males and 173 females). Oxidative stress biomarkers in serum, Total Antioxidant Status (TAS) and Total Oxidative Status (TOS) were determined spectrophotometrically. Oxidative Stress Index (OSI) was calculated as the ratio of TOS and TAS. Urinary 8-oxo- 7,8-dihydro-2’-deoxyguanosine were determined by HPLC-MS/MS and expressed as 8-oxodG/creatinine. Results: In females with RRMS were higher TOS, OSI and 8-oxodG/creatinine than in females in control group. The group of males with RRMS had lower level of TAS than the males in control group. Higher levels of 8-oxodG/creatinine was obtained in active, passive and former smokers with RRMS than in control group with the same exposition to tobacco smoke. Independent predictors of MS are passive smoking, increased OSI and increased levels of urinary 8-oxodG/creatinine. Conclusions: Our results demonstrate that the OS parameters should be included in the assessment of the risk for MS development. Due to the more sensitivity to oxidative stress, females may be at higher risk of MS development. This data indicates the importance of introducing the antioxidant therapy as a complementary treatment in patients with RRMS.Uvod: Multipla skleroza (MS) se karakteriše upalom, demijelinizacijom i degeneracijom aksona. Oksidativni stres (OS) igra značajnu ulogu u patogenezi bolesti. Cilj studije je bio da se ispita povezanost OS i pušenja na razvoj MS. Metode: Studija je obuhvatila 175 pacijenata sa relapsnoremitentnom multiplom sklerozom (RRMS) (76 muškaraca, 99 žena) i 254 zdrava ispitanika (81 muškarac i 173 žene). Biomarkeri oksidativnog stresa u serumu, ukupni antioksidativni status (TAS) i ukupni oksidativni status (TOS) su određeni spektrofotometrijski. Indeks oksidativnog stresa (OSI) je izračunat kao odnos TOS i TAS. Urinarni 8-okso7,8-dihidro-2'-deoksiguanozin je određen HPLC-MS/MS i izražen kao 8-oksoG/kreatinin. Rezultati: Kod žena sa RRMS bili su viši TOS, OSI i 8okodG/kreatinin nego kod žena u kontrolnoj grupi. Grupa muškaraca sa RRMS imala je niži nivo TAS od muškaraca u kontrolnoj grupi. Veći nivoi 8-okodG/kreatinina su dobijeni kod aktivnih, pasivnih i bivših pušača sa RRMS nego u kontrolnoj grupi sa istom izloženošću duvanskom dimu. Nezavisni prediktori MS su pasivno pušenje, povećan OSI i povećani nivoi 8-okodG/kreatinina u urinu. Zaključak: Naši rezultati pokazuju da parametre OS treba uključiti u procenu rizika za razvoj MS. Zbog veće osetljivosti na oksidativni stres, žene mogu biti izložene većem riziku od razvoja MS. Ovi podaci ukazuju na značaj uvođenja antioksidativne terapije kao komplementarnog lečenja kod pacijenata sa RRMS

Pozitivan učinak dimetilsulfoksida na hepatoprotektivnu aktivnost volframata

Tungstates and polyoxotungstates have been known to be bioactive compounds for a long time. Based on our previous work about sodium tungstate (ST) and 12-tungstophosphoric acid (WPA) as hepatoprotective agents, we performed a seven week long experiment in rats, using ST and WPA pretreatments in thioacetamide (TAA) induced acute liver necrosis. The possible influences of dimethyl sulfoxide (DMSO), given orally for 3 consecutive days before inducing hepatic lesions, were also investigated. The effects were evaluated by the activity of serum enzymes, oxidative stress parameters, antioxidative defense markers, and histopathology in Wistar rats. The obtained results suggest that oral pretreatment with tungstates, especially ST, in combination with DMSO, before the TAA inducted liver necrosis, could be useful for the prevention of hepatic injury in rats.Volframati i polioksovolframati su decenijama poznati kao biološki aktivna jedinjenja. Kao nastavak prethodnog istraživanja hepatoprotektivnog efekta natrijum volframata (ST) i 12-volframfosforne kiseline (WPA), izveden je eksperiment na Wistar pacovima, koji su pretretirani sa ST i WPA tokom sedam nedelja a nakon tog perioda im je intraperitonealno ubrizgan tioacetamid (TAA) u cilju indukovanja akutne nekroze jetre. Ispitivan je uticaj dimetil sulfoksida (DMSO), koji je primenjivan per os u toku 3 konsekutativna dana pre indukcije hepatične nekroze. Efekti su evaluirani praćenjem aktivnosti serumskih enzima, parametara oksidativnog stresa, antioksidativnog odbrambenog markera, kao i histopatološkom analizom tkiva jetre. Dobijeni rezultati ukazuju da oralni pretretman pacova sa volframatima, pre svega ST, u kombinaciji sa DMSO, može biti koristan u prevenciji nekroze jetre indukovane tioacetamidom

Alpha-1-Antitrypsin Deficiency in Serbian Adults with Lung Diseases

Aim: Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population. Methods: The study included the adults with chronic obstructive pulmonary disease (COPD) (n = 348), asthma (n = 71), and bronchiectasis (n = 35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis. Results: PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1: 5519, 1: 38, and 1:5519). Conclusion: The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population

Smaller HDL particles are associated with absence of obstructive coronary artery disease in stable angina pectoris patients

Background A research on novel cardiovascular risk factors is mainly focused on patients with clinically verified coronary artery disease (CAD), while less is known about their presence in symptomatic patients, but without angiographically proven occlusion of coronary arteries. The aim of this study was to compare plasma low-density lipoprotein (LDL) and high-density lipoprotein (HDL) size and subclasses in stable angina patients with and without significant obstructive CAD. Methods LDL and HDL subclasses were analysed in 100 stable angina patients with >= 50% of obstruction and 40 patients with less than 50% of luminal narrowing, as assessed by coronary angiography. Results Patients with lt 50% of obstruction had reduced mean HDL size and higher proportion of small HDL particles (P lt 0.05). HDL size and proportion of small HDL particles were significant and independent predictors of obstructive CAD (P lt 0.05, respectively). Conclusions Stable angina patients wit