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.

Oxidative stress-related risk of the multiple sclerosis development.

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-oxo7,8-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.

The Effects of Disease-Modifying Therapies on Oxidative Stress in Patients With Relapsing-Remitting Multiple Sclerosis.

OBJECTIVE: Oxidative stress (OS) has a role in the pathogenesis and progression of multiple sclerosis. The effects of disease-modifying therapies (DMTs) on OS are unclear. We aimed to explore the association between DMTs and OS in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: The study conducted in 167 patients (102 received and 65 not received the DMTs). The DMTs included interferon beta-1a (n = 15), interferon beta-1b (n = 20), glatiramer acetate (n = 10), and sphingosine-1-phosphate receptor modulators (n = 57). Oxidative stress assessed by total antioxidant status (TAS) and total oxidant status (TOS) (determined by spectrophotometric method), oxidative index (OSI was calculated), and urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG/creatinine was determined by high-performance liquid chromatography and tandem mass spectrometry). Patients were classified by Multiple Sclerosis Severity Score (MSSS) to mild/moderate (MSSS, <6.7) and severe (MSSS, >6.7). RESULTS: Disease-modifying therapies are associated with increased TAS, decreased TOS, OSI, and 8-oxodG/creatinine. Regardless of therapy, women had a less favorable redox status (lower TAS, higher TOS and OSI). Patients with MSSS>6.7 and without DMTs had higher OSI than patients who received DMTs. Women with MSSS>6.7 without DMTs had lower TAS than women with DMTs, whereas in the same stage of MS, men without DMTs had higher TOS than patients with DMTs. Women with MSSS<6.7 and with DMTs had lower 8-oxodG/creatinine compared with those without DMT therapy. CONCLUSIONS: The antioxidant effects of DMTs were evidenced in this study. The gender-related effects of DMTs on the OS imply the personalized antioxidant pharmacotherapy, especially for the women. The OS biomarkers have a potential as the prognostic for the assessment of DMTs outcomes in patients with RRMS.

Associations of Common Variants in HFE and TMPRSS6 Genes with Hepcidin-25 and Iron Status Parameters in Patients with End-Stage Renal Disease.

BACKGROUND: Influence of TMPRSS6 A736V and HFE (C282Y and H63D) polymorphisms on serum hepcidin-25 levels and iron status parameters in end-stage renal disease (ESRD) patients stratified according to gender has not been previously investigated. In addition, we aimed to evaluate the diagnostic accuracy of the parameters to separate iron-deficiency anemia (IDA) from anemia of chronic disease. MATERIALS AND METHODS: Iron status parameters and genetic analysis were performed in 126 ESRD patients and in 31 IDA patients as the control group. RESULTS: ESRD patients had significantly higher ferritin and hepcidin-25 (<0.001) relative to IDA patients. Cut-off values with the best diagnostic accuracy were found for hepcidin ≥9.32 ng/mL, ferritin ≥48.2 µg/L, transferrin saturation ≥16.8%, and MCV ≥81 fL. Interaction between gender and HFE haplotypes for the hepcidin-25 and ferritin levels in ESRD patients (p = 0.005, partial eta squared = 0.09; p = 0.027, partial eta squared = 0.06, respectively) was found. Serum transferrin was influenced by the combined effect of gender and TMPRSS6 A736V polymorphism in ESRD patients (p = 0.002, partial eta squared = 0.07). CONCLUSION: Our findings could contribute to the further investigation of mechanisms involved in the pathophysiology and important gender-related involvement of the TMPRSS6 and HFE polymorphisms on anemia in ESRD patients.

Influence of Dental Restorations on Oxidative Stress in Gingival Crevicular Fluid.

Biocompatibility of dental materials (DM) can be evaluated by gingival crevicular fluid (GCF) oxidative stress (OS) status. The goal of the study was to ascertain influence of dental caries degree, teeth position, and type and amount of applied DM on GCF OS profile. For this purpose, we tested six DMs that were sealed in one session: amalgam (Amg), composites: Tetric EvoCeram and Beautifil (BF), phosphate cement-zinc phosphate and polycarboxylate cements-zinc polycarboxylate cements, and glass ionomer cement (GIC). The study included 88 dental outpatients. Follow-up was scheduled at 7th and 30th day. Oxidative stress parameters (malondialdehyde (MDA) and glutathione (GSH) levels and total superoxide dismutase (tSOD) activity) were measured before (0th day) and after the treatment (7th and 30th day) in GCF. Control teeth were mirror-positioned healthy teeth. The DM accomplished the following effects (listed in descending order): increase of GSH in GCF was realized by ZPoC > BF > GIC > Amg; tSOD activity increase by ZPoC > BF > Amg; and MDA decrease by ZPoC > ZPhC > Amg > TEC. Dental caries provokes insignificant rise of OS in GCF. ZPoC and ZPhC showed the highest antioxidant effect, contrary to GIC. Restorations with antioxidant properties may reduce gum diseases initiated by caries lesion, what is of great clinical relevance in dentistry.

The Role of Oxidative Stress in the Clinical Manifestations of Childhood Asthma.

INTRODUCTION: The significance of oxidative stress in pathogenesis of childhood asthma was recognized, but its role in the clinical manifestations of disease is still unclear. MATERIALS AND METHODS: The study was conducted in 96 asthmatic children. The urinary biomarker of oxidative stress, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG/creatinine) was determined by using HPLC-MS/MS. ELISA was performed to measure myeloperoxidase (MPO) and Cu,Zn- superoxide dismutase (Cu,Zn-SOD) in serum. RESULTS: Logistic regression analysis revealed that female gender, tobacco smoke exposure, and increased 8-oxodG/creatinine were associated with risk for intermittent asthma, while the positive allergy test and increased Cu,Zn-SOD were associated with eczema in asthmatic children. Higher MPO (p = 0.033), and percent of granulocytes (p = 0.030) were found in severe persistent asthma in comparison to intermittent or mild persistent asthma. CONCLUSION: The main findings that TSE-induced oxidative stress is a risk for intermittent asthma and eczema may be clinically significant for the disease prevention and therapeutic improvements.

Alpha-1-Antitrypsin Antagonizes Cisplatin-Induced Cytotoxicity in Prostate Cancer (PC3) and Melanoma Cancer (A375) Cell Lines.

Increased circulating alpha-1-antitrypsin (AAT) correlates with cancer stage/aggressiveness, but its role in cancer biology is unclear. We revealed antagonistic effect of AAT to cisplatin-induced cytotoxicity in prostate (PC3) and melanoma (A375) cancer cell lines. Moreover, AAT abrogated cytotoxicity of MEK inhibitor U0126 in PC3 cell line. Weaker antagonistic effect of AAT on cytotoxicity of PI3/Akt and NF-kB inhibitors was also observed. In addition, cisplatin increased AAT gene expression in transfected PC3 cells. However, AAT derived from transfected PC3 cells did not antagonize cisplatin-induced cytotoxicity. In conclusion, these results suggest possible association between high circulating AAT and cisplatin resistance.

Oxidative Stress and Genetic Variants of Xenobiotic-Metabolising Enzymes Associated with COPD Development and Severity in Serbian Adults.

The genetic and non-genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are still poorly understood. We investigated the potential role of genetic variants of xenobiotic-metabolising enzymes (glutathione-S-transferase M1, GSTM1; glutathione-S-transferase T1, GSTT1; microsomal epoxide hydrolase, mEH), oxidative stress (assessed by urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG/creatinine), sex, ageing and smoking habits on susceptibility to development of COPD and its severity in Serbian population. The investigated population consisted of 153 healthy subjects (85 males and 68 females) and 71 patients with COPD (33 males and 38 females). Detection of GSTM1*null, GSTT1*null, mEH Tyr113His and mEH His139Arg gene variants was performed by PCR/RFLP method. Urinary 8-oxodG was determined using HPLC-MS/MS, and expressed as 8-oxodG/creatinine. We revealed that increased urinary 8-oxodG/creatinine and leucocytosis are the strongest independent predictors for COPD development. Increased level of oxidative stress increased the risk for COPD in males [odds ratio (OR), 95% confidence interval (CI): 8.42, 2.26-31.28], more than in females (OR, 95% CI: 3.60, 1.37-9.45). Additionally, independent predictors for COPD were ageing in males (OR, 95% CI: 1.29, 1.12-1.48), while in females they were at least one GSTM1 or GSTT1 gene deletion in combination (OR, 95% CI: 23.67, 2.62-213.46), and increased cumulative cigarette consumption (OR, 95% CI: 1.09, 1.01-1.16). Severity of COPD was associated with the combined effect of low mEH activity phenotype, high level of oxidative stress and heavy smoking. In conclusion, early identification of GSTM1*null or GSTT1*null genotypes in females, low mEH activity phenotype in heavy smokers and monitoring of oxidative stress level can be useful diagnostic and prognostic biomarkers.

Oxidative Stress and Polymorphism of Xenobiotic-Metabolizing Enzymes in Two Patients with Severe Alpha-1-Antitrypsin Deficiency.

Alpha-1-antitrypsin deficiency (AATD) and tobacco smoke play a key role in the pathogenesis of early-onset emphysema. Differences in AATD-related chronic obstructive pulmonary disease stages imply the existence of modifying factors associated with disease severity. We present two male patients with emphysema caused by severe AATD (PiZZ genotype). Both are former smokers and have epoxide hydrolase low-activity phenotype. Extremely high level of oxidative stress (high urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine), increased inflammation (high serum CRP), and GSTP1 105Val mutation were found in patient with a worse lung function and prognosis. These data provide more evidence that oxidative stress-related gene variants and inflammation are associated with worse symptoms of AATD-related emphysema. Therefore, prevention against severe stage of AATD-related emphysema would include early identification of the risk gene variants, cessation or never smoking, and treatment with anti-inflammatory and anti-oxidant drugs. Additionally, urinary 8-oxodG could be a candidate for predictive biomarker for routine assessment of the oxidative stress level in AATD patients.

Gender-related differences in susceptibility to oxidative stress in healthy middle-aged Serbian adults.

Gender-related differences in the association between polymorphism of xenobiotic-metabolising enzymes or non-genetic biomarkers and susceptibility to oxidative stress was assessed in healthy middle-aged Serbian adults, by urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG/creatinine) and total antioxidant status in serum (TAOS). Females were more susceptible to oxidative stress. In both genders, positive predictor of the antioxidative protection was serum triglyceride, while BMI <25 kg/m(2) was associated with oxidative stress. Susceptibility to oxidative stress in males was associated with GSTT1*null allele and increased serum iron, but in females, it was decreased serum bilirubin. Early identification of the risk factors could be important in the prevention of oxidative stress-related diseases.

Oxidative stress, bioelements and androgen status in testes of rats subacutely exposed to cadmium.

The objective of our study was to examine testicular toxicity of cadmium (Cd), focusing on oxidative stress (OS), essential metals and androgenic status and morphological changes. Male Wistar rats [controls and four Cd-subgroups (n = 6) organized according to the exposure (1, 3, 10 and 21 days)] were intraperitoneally (i.p.) treated with 1 mg CdCl2/kg/day. Testicular Cd deposition was noticed from the 1st day. After 10 and 21 days, copper (Cu) and iron (Fe) increased by 60-109% and 43-67%, respectively, while zinc (Zn) decreased by 24-33%. During 1-21 days of the exposure, decrease in testicular total superoxide dismutase (SOD) and total glutathione-s-transferase (GST) activities occurred gradually by 30-78% and 15-84%, respectively, while superoxide anion radical (O2(-)) increased gradually by 114-271%. After 10-21 days, decrease in testicular catalase (CAT) activity appeared by 13-31%. After 21 days, malondialdehyde (MDA) decreased by 44% and the ratio of oxidized glutathione/reduced glutathione (GSSG/GSH) increased by 130% in testes of the rats exposed to Cd. Additionally, decreased testicular testosterone level and the relative testes mass, along with induced microscopic and macroscopic changes were occured, what can be explained as the consequence of instantly developed OS, impaired essential metals status and Cd testicular deposition.

Alpha-1-antitrypsin deficiency in children: clinical characteristics and diagnosis.

INTRODUCTION: Alpha-1-antitrypsin deficiency (AATD) is a relatively rare and clinically very heterogeneous autosomal recessive disorder. OBJECTIVE: Presentation of clinical characteristics of AATD in the first months after birth, as well as the significance of testing brothers and sisters for its presence. METHODS: Objectives of the study were analyzed on a sample of eight children (four male and four female, aged 63 months (mean 14.81 ± 23.96 months; range 1-63 months) with AATD confirmed based on its low serum value and pathological phenotype. RESULTS Of the total of eight patients, six manifested cholestasis syndrome (three male and three female, mean age 2.25 ± 1.37 months; range 1-4.5 months), while two patients, a 3.5-year-old girl and a 5.25-year-old boy, were without symptoms and clinical-laboratory signs of the disease, disclosed during family testing. Serum alpha-1-antitrypsin level rated 0.30-0.66 g/L (0.37 ± 0.12), among which seven were with ZZ phenotype 0.30-0.39 (0.33 ± 0.04), and in a boy with FZ the phenotype was disclosed on family screening, 0.66 g/L. In the group of patients with cholestasis syndrome (serum GTT 444.80 ± 203.15 U/L; range 201-676 U/L), three had mild to moderate hepatomegaly, one had longitudinal growth delay (< P3; -10.50%) and two had icterus with conjugated hyperbilirubinemia (92 and 109 µmol/L) and prolonged prothrombin time (PT 14.8 and 17 sec). All children with cholestasis syndrome also had hypertransaminasemia (ALT 80.83 ± 33 U/L; range 37-124 U/L and AST 116.67 ± 62.82 U/L; range 58-230 U/L). CONCLUSION: Cholestasis syndrome represents a basic manifestation of AATD in the first months after birth, while early testing of brothers and sisters enables early disclosure and adequate treatment of the subclinical forms of the disease.

Polymorphisms of beta2-adrenergic receptor gene in serbian asthmatic adults: effects on response to Beta-agonists.

BACKGROUND AND OBJECTIVES: Polymorphisms of beta2-adrenergic receptor gene (ADRB2) are clinically relevant for several reasons, including as a risk factor for asthma development/severity and predicting the effectiveness of treatment with beta2-agonists in reducing asthma symptoms. The aim of this study was to examine the association between ADRB2 gene polymorphisms and asthma in the Serbian population, and to evaluate the therapeutic response in relation to the ADRB2 genotype. METHODS: The study included 171 patients with asthma and 101 healthy subjects as the control group. Genotyping of Arg16Gly and Gln27Glu polymorphisms was performed by direct sequencing of polymerase chain reaction (PCR) products. RESULTS: In Serbian adults, carriers of the 27Gln allele and 27Gln/Gln genotype were at higher risk of asthma [odds ratio (OR) 2.5, 95% confidence interval (CI) 1.6-3.8, and OR 3.00, 95% CI 1.7-5.3, respectively], while the presence of the 27Glu allele and 27Gln/Glu genotype were found to be protective of asthma (OR 0.4, 95% CI 0.3-0.6, and OR 0.3, 95% CI 0.1-0.7, respectively). Furthermore, we found that the presence of the 27Gln allele in asthmatics younger than 50 years leads to a better response to therapy with long-acting beta2-agonists (LABA) in combination with prevailing low and moderate doses of inhaled corticosteroids (ICS), while carriers of the 27Glu allele over 50 years old are more likely to respond to LABA + ICS therapy. CONCLUSION: We identified that in Serbian adults the 27Gln allele and 27Gln homozygosity are risk factors for asthma, which may be of clinical interest in disease prevention. The finding that younger carriers of the 27Gln allele respond better to LABA + ICS therapy may be utilized in personalized asthma treatment.

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 <50% of obstruction had reduced mean HDL size and higher proportion of small HDL particles (P < 0.05). HDL size and proportion of small HDL particles were significant and independent predictors of obstructive CAD (P < 0.05, respectively). CONCLUSIONS: Stable angina patients with <50% of coronary obstruction have more favourable HDL subclasses distribution than patients with significant coronary stenosis.

Diagnostic characteristics and application of alcohol biomarkers.

Alcohol biomarkers play a significant role in the early diagnosis of alcohol intoxication/abuse, alcohol-related organ damages, assessment of alcoholism therapy outcomes, and in forensic medicine. Laboratory detection of excessive alcohol consumption can be carried out by direct measuring of the ethanol and/or metabolites in biological samples which is of particular importance in the cases of acute ethanol intoxication/controlling and/or monitoring of alcohol consumption, or indirectly, by using biomarkers. Preferred diagnostic characteristics of alcohol biomarkers, specificity and sensitivity dependent on the particular demands such as: prevention and treatment of alcoholism in primary and social care, criminal justice, workplace health and safety screening, trafficking control, etc. Alcohol biomarkers traditionally used in clinical practice [blood alcohol concentration (BAC), gamma-glutamyl transferase (GGT), carbohydrate-deficient transferrin (CDT), the ratio GGT/CDT, alanine aminotransferase (ALT), aspartate aminotransferase (AST), the rati. AST/ALT, mean cbrpuscular volume (MCV), phosphatidylethanol (PEth)] are well validated. They are used as screening/monitoring markers of acute/chronic excessive alcohol intake, alcoholism in pregnancy, and other disorders/conditions related to alcohol abuse. Numerous potential alcohol biomarkers have been discovered, but few are validated. Potential alcohol biomarkers (ethanol and serotonin metabolites, sialic acids, etc.) have good diagnostic characteristics, but their application in clinical practice is limited due to the costly equipment necessary for their measurement. Significant progress has been made in the development of sensitive and practical alcohol transdermal devices that can instantly/continuously measure BAC through human skin. Transdermal sensing of alcohol may become a valuable method for monitoring abstinence. A special aspect of alcoholism is genetic predisposition to alcohol abuse and alcoholism, or alcohol-related organ damage. Recent genome-wide association studies (GWASs) have proposed several susceptibility loci for alcohol dependence.

Gender-related reference intervals of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine determined by liquid chromatography-tandem mass spectrometry in Serbian population.

OBJECTIVES: Although there are many nucleobase modifications, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the dominant form of oxidative modifications of DNA. Urinary 8-oxodG is potentially the best non-invasive biomarker of oxidative stress. Defining reference interval for urinary 8-oxodG is a prerequisite for its clinical use as biomarker. DESIGN AND METHODS: Reference population included 229 healthy Serbian adults (130 males and 99 females). The spot urinary 8-oxodG was determined using high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). Urinary creatinine was measured by the kinetic Jaffé method. RESULTS: Analytical performances of the HPLC-MS/MS: CVs within and between-run variations were 5.6% and 2.6%; LOD and LOQ were 1.65 nmol/L and 3.30 nmol/L; mean recovery and relative accuracy were 96% and 97%. Creatinine level was higher in males than in females, but no gender difference in 8-oxodG level was observed. Upon the adjustment of 8-oxodG to creatinine (8-oxodG/creatinine), higher values were obtained in females (1.38 ± 0.65 nmol/mmol) than in males (1.05 ± 0.48 nmol/mmol). Distribution of 8-oxodG/creatinine in spot urine sample was log-normal and gender-related reference intervals (estimated as the 2.5th-97.5th percentiles) were 0.45-2.22 nmol/mmol for males, and 0.54-3.11 nmol/mmol for females. Body mass index (BMI) affects excretion of the 8-oxodG in males, independently of urinary creatinine, while in females it does not. Therefore, BMI might contribute to the gender-related differences of 8-oxodG/creatinine in spot urine samples. CONCLUSIONS: This is the first established gender-related reference intervals of spot urinary 8-oxodG/creatinine. Our results contribute to the full validation of 8-oxodG as biomarker of oxidative stress.

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 proinflammatory 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.

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.

Intrastriatal pre-treatment with L-NAME protects rats from diquat neurotoxcity.

INTRODUCTION: Contact herbicide diquat (DQ), redox cycling compound, mediates its systemic toxicity throughout the enlarged production of free radicals. Target organs are liver and kidney in humans. To-date, the mechanism of DQ-induced neurotoxicity has not been rationalized. OBJECTIVE: The objectives of the study were to examine the ability of DQ to induce oxidative stress (OS) and/or nitrosative stress (NS) upon intrastriatal (i.s.) administration and to investigate the role of nitric oxide (NO(x)) using NG-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of nitric oxide synthase (NOS) in the pretreatment of DQ i.s. administration. MATERIAL AND METHODS: The experiment was conducted on Wistar rats, randomly divided in experimental groups, receiving different treatments i.s. applied. Parameters of OS/NS such as: superoxide anion radical (O(2)(•-)), superoxide dismutase (SOD), malondialdehyde (MDA) and nitrates (NO(3)(-)) were measured in the cortex (bilaterally), at 30(th) min, 24 hours and 7 days after the treatments. RESULTS: Lethargy and high mortality rate were observed only in the DQ group (within 24 hours and 2-3 hours, respectively) after awakening from anesthesia. Markedly increased production of NO(x) and O(2)(•-) along with elevated lipid peroxidation altogether contributed to DQ neurotoxicity. The most importantly, the L-NAME i.s. pretreatment protected treated animals from dying and diminished OS/NS response against DQ-induced neurotoxicity. CONCLUSION: The i.s. pretreatment with L-NAME resulted in neuroprotection against DQ neurotoxity, based on animal survival and reduced LPO in the cortex.

Alpha-1-antitrypsin deficiency in early childhood.

Alpha-1-antitrypsin deficiency (AATD), which predisposes liver disease in children, is often undiagnosed. Isoelectric focusing in 161 infants with liver dysfunction revealed 14.7% severe and 12.2% moderate AATD. Positive PAS-D and immunohistochemical staining was found in 60% of severe AATD, but in moderate AATD, only immunohistochemistry was positive in 100%. Bilirubinostasis, hepatomegaly, splenomegaly, cholestasis, hepatomegaly associated with cholestasis, acholia, high transaminases, and low birthweight were significantly more frequent in severe than in moderate AATD. Both AATDs showed significant portal inflammation, hepatic fibrosis, and viral infection. Early screening in children with liver dysfunction can contribute to the successful detection of AATD.