Duško Mirković

Oxytocin in corticosterone-induced chronic stress model: Focus on adrenal gland function

Chronic stress conditions can lead to considerable and extensible changes in physiological and psychological performances, and in emergence of risk for various somatic diseases. On the other hand, the neuropeptide oxytocin is reported to increase the resistance of the organism to stress and modulate activity of autonomic nervous system. Chronic corticosterone administration is used as a rat model for a state observed in terms of chronic stress exposure, when negative feedback mechanism of hypothalamus-pituitary-adrenal axis activity is disrupted. In our study, we aimed to investigate whether chronic administration of oxytocin (10 IU/400μL/day for 14days, s.c.) influenced adrenal gland morphology and activity in adult male Wistar rats during long-term corticosterone administration via drinking water (100mg/L for 21days). We examined the influence of treatments on the levels of adrenal gland hormones, corticosterone, adrenaline and noradrenaline, as well as their response to an acute stress challenge evoked by 15-min forced swimming. In addition, the expression of two main monoamine transporters, the noradrenaline transporter (NAT) and vesicular monoamine transporter 2 (VMAT2) in adrenal medulla was measured in the rats exposed to acute stress. Our results showed that oxytocin treatment prevented corticosterone-induced decrease in body weight gain, attenuated adrenal gland atrophy by increasing glandular weight, and the area of the zona fasciculate and reticularis. Chronic corticosterone intake blunted the response of all measured hormones to acute stress, whereas concomitant oxytocin treatment reversed adrenaline and noradrenaline response to acute stress. Furthermore, in adrenal medulla, oxytocin produced significant vasodilatation and stimulated expression of both catecholamine transporters detected both on mRNA and protein level. Our data suggest that oxytocin, by reducing atrophy of adrenal gland, and by increasing catecholamine storage capacity, may be beneficial in conditions accompanied with high glucocorticoid levels, such as chronic stress exposure.

Incidence of Hyperhomocysteinemia and Mthfr C677T Polymorphism Among Young Patients with Acute Myocardial Infarction

Incidence of Hyperhomocysteinemia and Mthfr C677T Polymorphism Among Young Patients with Acute Myocardial Infarction Hyperhomocysteinemia is considered an independent risk factor for premature cardiovascular disease. Mutation MTHFR C677T reduces the activity of methylenetetra-hydrofolatereductase and may cause hyperhomocysteinemia. Incidence of hyperhomocysteinemia (homocysteine above 12 μmol/L), homocysteine level, and distribution of MTHFR C677T genotypes (C/C, C/T and T/T) are compared between young patients with acute myocardial infarction and healthy persons, matched by age. Study involved 86 patients younger than 45 years (77 men and 9 women) and 35 controls. Homocysteine was measured by an HPLC method and the MTHFR C677T genotype determined using PCR amplification and digestion with Hinf I. Statistical analyses included chisquare and Mann-Whitney U tests. Hyperhomocysteinemia was present in 32.6% patients and 14.3% controls, revealing a significant difference (P= 0.038). Median homocysteine levels in patients (10.4 μmol/L) and controls (9.6 μmol/L) were significantly different (P=0.035). Among patients, 50.0% had C/C, 41.9% C/T and 8.1% T/T genotype, and the genotype had no influence on hyperhomocysteinemia incidence and homocysteine level. Genotype distribution in patients was not significantly different from that observed in controls. The conclusion is that young patients with acute myocardial infarction have higher incidence of hyperhomocysteinemia and higher homocysteine levels than healthy young adults, while there is no significant difference in the distribution of MTHFR C677T genotypes. Incidenca Hiperhomocisteinemije I Polimorfizam Mthfr C677T Kod Mladih Bolesnika SA Akutnim Infarktom Miokarda Hiperhomocisteinemija se smatra nezavisnim faktorom rizika za preuranjeni razvoj kardiovaskularnih bolesti. Mutacija MTHFR C677T snižava aktivnost metilentetra-hidrofolatreduktaze i može dovesti do hiperhomocisteinemije. Incidenca hiperhomocisteinemije (homocisteinemija iznad 12 μmol/L), nivo homocisteina i raspodela MTHFR 677 genotipova (C/C, C/T, T/T) upoređeni su između mladih bolesnika sa akutnim infarktom miokarda i zdravih osoba iste dobi. Studija je obuhvatila 86 bolesnika mlađih od 45 godina (77 muškaraca i 9 žena) i kontrolnu grupu od 35 osoba. Homocistein je određivan metodom HPLC, a MTHFR 677 genotip PCR amplifikacijom i digestijom sa Hinf I. Podaci su statistički obrađeni pomoću Chi-square i Mann-Whitney U testa. Hiperhomocisteinemija je bila prisutna kod 32,6% bolesnika i 14,3% zdravih osoba, što predstavlja statistički značajnu razliku (P=0,038). Medijane homocisteinemija bolesnika (10,4 μmol/L) i zdravih osoba (9,6 μmol/L) bile su statistički značajno različite (P= 0,035). Raspodela MTHFR 677 genotipova kod bolesnika (50,0% C/C, 41,9% C/T i 8,1% T/T) nije se statistički značajno razlikovala od raspodele u kontrolnoj grupi. Genotip MTHFR 677 nije uticao na incidencu hiperhomocisteinemije i nivo homocisteina kod bolesnika. Može se zaključiti da mladi bolesnici sa akutnim infarktom miokarda imaju višu incidencu hiperhomocisteinemije i viši nivo homocisteina nego zdrave osobe iste starosti, pri čemu nema značajne razlike u raspodeli genotipova MTHFR.

Is an integrative laboratory algorithm more effective in detecting alpha-1-antitrypsin deficiency in patients with premature chronic obstructive pulmonary disease than AAT concentration based screening approach?

Introduction: Alpha-1-antitrypsin deficiency (AATD), genetic risk factor for premature chronic obstructive pulmonary disease (COPD), often remains undetected. The aim of our study was to analyse the effectiveness of an integrative laboratory algorithm for AATD detection in patients diagnosed with COPD by the age of 45 years, in comparison with the screening approach based on AAT concentration measurement alone. Subjects and methods: 50 unrelated patients (28 males/22 females, age 52 (24–75 years) diagnosed with COPD before the age of 45 years were enrolled. Immunonephelometric assay for alpha-1-antitrypsin (AAT) and PCR-reverse hybridization for Z and S allele were first-line, and isoelectric focusing and DNA sequencing (ABI Prism BigDye) were reflex tests. Results: AATD associated genotypes were detected in 7 patients (5 ZZ, 1 ZMmalton, 1 ZQ0amersfoort), 10 were heterozygous carriers (8 MZ and 2 MS genotypes) and 33 were without AATD (MM genotype). Carriers and patients without AATD had comparable AAT concentrations (P = 0.125). In majority of participants (48) first line tests were sufficient to analyze AATD presence. In two remaining cases reflex tests identified rare alleles, Mmalton and Q0amersfoort, the later one being reported for the first time in Serbian population. Detection rate did not differ between algorithm and screening both for AATD (P = 0.500) and carriers (P = 0.063). Conclusion: There is a high prevalence of AATD affected subjects and carriers in a group of patients with premature COPD. The use of integrative laboratory algorithm does not improve the effectiveness of AATD detection in comparison with the screening based on AAT concentration alone.

The effects of acth, isoproterenol and Dexamethasone on the rat adrenal gland response to ethane dimethanesulphonate (Eds): A stereological study

Ethane dimethanesulphonate (EDS), an alkylating agent, caused marked atrophy of the adrenal cortex of adult male rats, in addition to its toxic effect on testicular Leydig cells. The aim of this work was to examine whether a 9-day treatment with ACTH (40 IU/kg/d), isoproterenol (120 mg/kg/d) or dexamethasone (0.25 mg/kg/d), which started 4 days prior to intraperitoneal administration of a single dose of EDS (75 mg/kg), affected the morphological changes in the adrenal cortex evoked by EDS alone. The animals were killed 15 days after EDS injection. Stereological analysis revealed that both ACTH and isoproterenol almost completely prevented cortical atrophy induced by EDS. They also considerably stimulated corticosterone secretion in EDS-injected animals. By contrast, in dexamethasone-suppressed rats, the deleterious effect of EDS on adrenocortical cells was augmented. The volume and cellularity of all cortical zones were reduced, but the remaining cells of the zona reticularis displayed considerable hypertrophy which was probably responsible for the maintenance of corticosterone secretion. These results clearly demonstrate that both ACTH and b adrenoceptor stimulation have protective action against the toxic effects of EDS on rat adrenal cortex, whereas dexamethasone exerts an opposite influence.

Double positivity of the IgG isotype of both anticardiolipin and anti-β2gpI antibodies is associated with the highest number of vascular impairment parameters in patients with primary antiphospholipid syndrome: preliminary data

Although numerous studies investigated the association between homocysteine (Hcy), tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) and apolipoproteins (apos) with thrombosis and/or recurrent pregnancy losses, studies that analyzed the abovementioned parameters and multiple positivity of antiphospholipid antibodies (aPL Abs) in patients with primary antiphospholipid syndrome (PAPS) are lacking. Therefore, the aim of this study was to analyze the presence of various combinations of the abovementioned parameters and their associations with clinical and/or serological features of PAPS. High-pressure liquid chromatography (HPLC) was used for determination of Hcy, while apoAI, apoB, and lipoprotein (Lp) (a) concentrations were estimated by immunonephelometry. High-sensitivity C-reactive protein (hsCRP) was measured by immunoturbidimetry. Apo (a), TNF-α, and aPL Abs were measured by ELISA. Various combinations of analyzed parameters (Hcy/CRP/TNF/apoAI/apoB/apo (a)/Lp (a)) were not associated with a single presence of either aPL Abs. Double positivity for both isotypes of anticardiolipin (aCL) Abs (IgG + IgM) was associated with the increased apoB levels. The presence of the IgG isotype of both aCL + anti-beta2 glycoprotein I (aβ2gpI) Abs was associated with the highest number of analyzed parameters (i.e., increased levels of hsCRP, Lp (a), and apo (a)). The presence of the IgG isotype of both aCL + aβ2gpI Abs was associated with the highest number of vascular impairment parameters in patients with PAPS, and this combination confers the highest risk for the recurrence of thrombotic episodes. This is the first report that analyzed the association between various combinations of vascular impairment parameters with multiple aPL Ab positivity. Our results provide a rationale for further investigations of therapeutic approaches for PAPS patients.

Isoelectrofocusing and PCR Amplification-Reverse Hybridization Assay in Evaluation of Alpha-1-Antitrypsin Deficiency

Isoelectrofocusing and PCR Amplification-Reverse Hybridization Assay in Evaluation of Alpha-1-Antitrypsin Deficiency Alpha-1-antitrypsin deficiency is a potentially lethal genetic disorder, which has pulmonary and liver manifestations. The standardized biochemical and molecular diagnostic protocol for detection of clinically relevant alleles is needed. The paper summarizes current concepts about AATD, describes the potentials of isoelectric focusing and PCR amplification-reverse allele specific oligonucleotide hybridization assay in the detection of affected individuals and shortly presents our experiences in the evaluation of AATD. We conclude that the systematic clinical laboratory approach to AATD might be based on the combination of mentioned methods, coordinated by alpha-1-antritrypsin quantification. Additionally, its complete medical implementation is achieved through teamwork between clinical chemists, molecular biologists and clinicians. Izoelektrofokusiranje i PCR Amplifikacija-Reverzna Hibridizacija u Proceni Nedostatka Alfa-1-Antitripsina Nedostatak alfa-1-antitripsina je potencijalno smrtonosan genetski poremećaj sa pulmonarnim i hepatičnim manifestacijama. Uočljiva je potreba za standardizovanim protokolom detekcije klinički značajnih alela, koji bi uključivao biohemijske i molekularne metode. Rad prikazuje savremena shvatanja o nedostatku alfa-1-antitripsina, opisuje mogućnosti primene izoelektrofokusiranja i testa zasnovanog na kombinaciji PCR amplifikacije i reverzne hibridizacije sa alel-specifičnim oligonukleotidima i ukratko opisuje naša iskustva u toj oblasti dijagnostike. Može se zaključiti da bi, u kliničkim laboratorijama, kombinacija dveju pomenutih metoda, uz dodatne informacije dobijene kvantitativnom analizom, mogla predstavljati osnovu detekcije genetski uslovljenog nedostatka alfa-1-antitripsina. Neophodno je naglasiti da je za sveobuhvatnu medicinsku primenu takvog laboratorijskog protokola neophodna saradnja medicinskih biohemičara, molekularnih biologa i lekara odgovornih za lečenje bolesnika sa genetski uslovljenim nedostatkom alfa-1-antitripsina.

Acth-induced model of depression resistant to tricyclic antidepressants: Neuroendocrine and behavioral changes and influence of long-term magnesium administration

ABSTRACT Magnesium (Mg), is not only a modulator of the glutamatergic NMDA receptors’ affinity, it also prevents HPA axis hyperactivity, thus possibly being implicated in neurobiological features of mood disorders. Further uncovering of molecular mechanisms underlying magnesiums proposed effects is needed due to the recent shift in research of treatment resistant depression (TRD) towards glutamatergic pathways. Here, we applied Mg via drinking water for 28days (50mg/kg/day), in ACTH‐treated rats, an established animal model of depression resistant to tricyclic antidepressants. Using this model in male rats we measured (1) changes in hippocampal neurogenesis and behavioral alterations, (2) adrenal hormones response to acute stress challenge and (3) levels of biometals involved in regulation of monoamines turnover in rat prefrontal cortex. Our results support beneficial behavioral impact of Mg in TRD model together with increased hippocampal neurogenesis and BDNF expression. Furthermore, Mg prevented ACTH‐induced disruption in HPA axis function, by normalizing the levels of plasma ACTH, corticosterone and interleukin‐6, and by increasing the peripheral release of adrenaline, noradrenaline and serotonin after the acute stress challenge. Finally, the influence on copper/zinc ratio suggested probable magnesiums involvement in monoamine turnover in PFC. Our findings provide further insights into the possible pathways implicated in the behavioral modulation effects of Mg, as well as its central and peripheral effects in ACTH‐induced TRD model. Thus, further investigation of molecular signaling related to the glutamatergic transmission and role of Mg, could reveal prospects to novel treatment strategies that could be of particular importance for patients suffering from TRD. HIGHLIGHTSIn ACTH model of treatment‐resistant depression Mg alleviates behavioral changes.HPA axis hyperactivity induced by ACTH was attenuated in Mg‐administered rats.Study suggests involvement of Mg in central and peripheral turnover of monoamines.In ACTH model of treatment‐resistant depression Mg increases neurodevelopment.

Questionable Reliability of Homocysteine as the Metabolic Marker for Folate and Vitamin B12 Deficiency in Patients with Chronic Obstructive Pulmonary Disease

Summary Background: An increased homocysteine (Hcy) concentration may represent a metabolic marker of folate and vitamin B12 deficiency, both significant public health problems. For different reasons, patients with chronic obstructive pulmonary disease (COPD) are prone to these deficiencies. The study evaluates the reliability of Hcy concentration in predicting folate or vitamin B12 deficiency in these patients. Methods: A group of 50 COPD patients (28 males/22 females, age (x¯±SD=49.0±14.5)

THE CLINICAL IMPORTANCE OF BIOCHEMICAL BONE MARKERS IN PATIENTS WITH ALCOHOLIC AND VIRAL LIVER CIRRHOSIS / KLINIČKI ZNAČAJ BIOHEMIJSKIH KOŠTANIH MARKERA KOD PACIJENATA SA ALKOHOLNOM I VIRUSNOM CIROZOM JETRE

({\rm{\bar x}} \pm {\rm{SD}} = 49.0 \pm 14.5)

Recommedition for Apllication and Determination Tumor Markers of Neuroendocrine System

years was enrolled. A chemiluminescent microparticle immunoassay was applied for homocysteine, folate and vitamin B12 concentration. Kolmogorov-Smirnov, Mann-Whitney U and χ2 tests, Spearman’s correlation and ROC analysis were included in the statistical analysis, with the level of significance set at 0.05. Results: Average (SD) concentrations of folate and vitamin B12 were 4.13 (2.16) μg/L and 463.6 (271.0) ng/L, whereas only vitamin B12 correlated with the Hcy level (P=−0.310 (R=0.029)). Gender related differences were not significant and only a borderline significant correlation between age and folate was confirmed (R=0.279 (P=0.047)). The incidence of folate and vitamin B12 deficiency differed significantly (P=0.000 and P<0.000 for folate and vitamin B12 respectively), depending on the cut-off used for classification (4.4, 6.6 and 8.0 μg/L – folate; 203 and 473 ng/L – vitamin B12). ROC analyses failed to show any significance of hyperhomocysteinemia as a predictor of folate or vitamin B12 deficiency. Conclusion: Reliability of the Hcy concentration as a biomarker of folate or vitamin B12 depletion in COPD patients is not satisfactory, so their deficiency cannot be predicted by the occurrence of HHcy.