The Differences in the Cellular and Plasma Antioxidative Capacity Between Transient and Defined Focal Brain Ischemia: Does it Suggest Supporting Time-Dependent Neuroprotection Therapy?

There are many opened questions about the precocious role of oxidative stress in the physiopathology of the early stage of transitory ischemic attack (TIA) and defined focal brain ischemia, as well as about its correlation with clinical severity, short-lasting and clinical outcome prediction in these conditions. The study evaluates the values of glutathione (GSH), glutathione peroxidase, and superoxide dismutase (SOD) in hemolysates and total thiol content (-SH), advanced oxidation protein products (AOPP), SOD, and malondialdehyde (MDA) in plasma, in TIA and stroke patients in the early stage of their neurological onset. The results are interpreted in view of the potential relationship between tested parameters and clinical severity and clinical outcome prediction. Better hemolysates' and total antioxidant profile with higher values of AOPP were observed in TIA compared to stroke patients (p < 0.05). The stroke patients with initially better clinical presentation showed better antioxidant profile with lower values of AOPP (p < 0.05). In TIA patients, this was observed for GSH, -SH content, and AOPP (p < 0.05), which correlated with a short risk for stroke occurrence in this group (p < 0.01). Beyond MDA values, all tested parameters showed correlation with clinical outcome in stroke patients (p < 0.05). The measurement of oxidative stress in TIA and stroke patients would be important for identifying patients' subgroups which might receive supporting therapy providing better neurological recovery and clinical outcome. That approach might give us an additional view of a short-lasting risk of stroke occurrence after TIA, and its clinical outcome and prognosis.

Short communication: Effect of commercial or depurinized milk diet on plasma advanced oxidation protein products, cardiovascular markers, and bone marrow CD34+ stem cell potential in rat experimental hyperuricemia.

Cardiovascular repair and myocardial contractility may be improved by migration of bone marrow stem cells (BMSC) and their delivery to the site of injury, a process known as BMSC homing. The aim of our study was to examine the dietary effect of a newly patented depurinized milk (DP) that is almost free of uric acid and purine and pyrimidine compounds compared with a standard commercial 1.5% fat UHT milk diet or allopurinol therapy in rat experimental hyperuricemia. Bone marrow stem cell potential (BMCD34(+), CD34-postive bone marrow cells), plasma oxidative stress parameters [advanced oxidation protein products, AOPP) and thiobarbituric acid reactive substances (TBARS)], myocardial damage markers [creatine phosphokinase (CPK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH)], plasma cholesterol, and high-density lipoprotein cholesterol were investigated. The DP milk diet significantly increased the number of BMCD34(+) stem cells compared with commercial UHT milk. Allopurinol given alone also increased the number of BMCD34(+). Hyperuricemia caused a significant increase in all plasma enzyme markers for myocardial damage (CPK, LDH, and AST). A cardioprotective effect was achieved with allopurinol but almost equally with DP milk and more than with commercial milk. Regarding plasma AOPP, TBARS, and cholesterol levels, the most effective treatment was DP milk. In conclusion, the protective role of a milk diet on cardiovascular function may be enhanced through the new depurinized milk diet, which may improve cardiovascular system function via increased bone marrow stem cell regenerative potential, decreased plasma oxidative stress parameters, and decreased levels of myocardial damage markers and cholesterol. New dairy technology strategies focused on eliminating harmful milk compounds should be completely nontoxic. Novel milk products should be tested for their ability to improve tissue repair and function.

Cerebrospinal fluid and plasma oxidative stress biomarkers in different clinical phenotypes of neuroinflammatory acute attacks. Conceptual accession: from fundamental to clinic.

Oxidative stress is revealed as the main contributor in the pathophysiology of neuroinflammation. Analyzing plasma and cerebrospinal fluid (CSF) of patients with different clinical phenotypes of neuroinflammation, defined as clinically isolated syndrome (CIS), and those defined as relapsing remitting multiples sclerosis (RRMS), we tested peripheral and CNS oxidative stress intensity in these neuroinflammatory acute attacks. All obtained values changes were assessed regarding clinical and radiological features of CNS inflammation. The obtained results revealed an increase in malondialdehyde levels in plasma and CSF in CIS and RRMS patients compared to control values (p < 0.05). The obtained values were most prevailed in both study group, CIS and RRMS, in patients with severe clinical presentation (p < 0.05). Measured activities of catalase and total superoxide dismutase were higher in CIS and RRMS patients in plasma compared to control values (p < 0.05), parallel with an increased catalase activity and decrease in superoxide dismutase activity in CSF regarding values obtained in control group (p < 0.05). The positive correlations regarding clinical score were obtained for all tested biomarkers (p < 0.01). Although the positive correlations were observed in MDA levels in plasma and CSF, for both study patients, and their radiological findings (p < 0.01), and a negative correlation in plasma SOD activity and CIS patients' radiological findings (p < 0.01), no other similar correlations were obtained. These findings might be useful in providing the earliest antioxidative treatment in neuroinflammation aimed to preserve total and CNS antioxidative capacity parallel with delaying irreversible, later neurological disabilities.

Diagnostic significance of nitrates and nitrites and L-arginine, in development of hepatorenal syndrome in patients with end stage alcoholic liver cirrhosis.

Hepatorenal syndrome (HRS) represents a complication of the end-stage liver cirrhosis. The aim of the present study was to analyze concentrations of nitrates and nitrites (NO2 + NO3) and L-arginine in patients with liver cirrhosis and HRS as a possible predictive marker for the development of HRS. The research was performed in a group of 28 patients with cirrhosis and HRS, a group of 22 patients suffering from cirrhosis without HRS and a control group comprised of 42 healthy voluntary blood donors. In patients with end-stage alcoholic liver cirrhosis, with HRS, the concentrations of NO2 + NO3 increased and correlated with the degree of cirrhosis progression, compared to patients without HRS and significantly higher compared to the control group. The level of NO2 + NO3 was in a positive correlation with the degree of liver damage de Ritis coefficient (HRS = 0.72; cirrhosis: = 0.55; control = -0.10). Significant positive correlation was found between NO2 + NO3 concentration and inflammatory marker C-reactive protein (HRSC = 0.75; cirrhosis = 0.70, control = -0.25). The correlation between NO2 + NO3 concentration and creatinine concentration in patients with HRS was significantly higher compared to patients without HRS (HRS = 0.82; cirrhosis = 0.32; control = -0.25). By using binary regression analysis, on the basis of clinical criteria of HRS diagnosis, the strongest independent positive predictor for HRS development was NO2 + NO3, associated with 45.02 times higher incidence of HRS, compared to arginine (12.7 times higher incidence), creatinine (13.1 times higher incidence), and AST/ALT ratio (10.55 higher incidence of HRS). Since the determination of NO2 + NO3 represents a reliable and easily applicable method, it may be used as an early predictive marker for HRS development.

Protective effects of glutathione and lipoic acid against cadmium-induced oxidative stress in rat's kidney.

Cadmium is a widespread, toxic industrial pollutant. The proximal tubule of the mammalian kidney is a major target of Cd-induced toxicity. We analyzed the effects of cadmium exposure on the model system of experimental animals, the thiobarbituric acid (TBA)-reactive substance (TBARS) level, and the activity of xanthine oxidase (XO) and catalase in kidney of rats, with and without glutathione and lipoic acid (LA). The experimental animals were classified into six groups, regarding cadmium, glutathione, and LA intake. The concentration of TBARSs in the homogenate was determined by spectrophotometric method according to Nabavi et al. The specific activity of XO was determined spectrophotometrically by the method of Aygul et al. Catalase activity in tissues was determined by spectrophotometric method according to Nabavi et al. The increased level of TBARS and the increased activity of XO in kidney tissue in cadmium poisoning are statistically significant compared to control (p < 0.001). Glutathione and LA applied along with cadmium lowered TBARS concentration and reduced XO activity (p < 0.001). Catalase activity in the kidney tissue was increased in the group, which was administered cadmium (p < 0.001). In conclusion, glutathione and LA, as physiological antioxidants applied with cadmium, have reduced the level of lipid peroxide and the activity of XO, and can be used as protectors in conditions of cadmium poisoning.

Circulating nucleic acids as possible damage-associated molecular patterns in different stages of renal failure.

Chronic renal failure (CRF) is a condition associated with the risk of cardiovascular complications. Systemic inflammatory response, initiated by the pathogen-associated molecular-pattern (PAMP) molecules, exerts many similarities with the damage-associated molecular-pattern (DAMP) molecule-induced systemic response. Up to now, a number of DAMP molecules were identified. We hypothesized that the available circulating nucleic acids, acting as DAMPs, may modulate immunoinflammatory reaction in CRF. Patients with the different stages of chronic kidney disease, kidney transplantation, and patients on dialysis were included in the study. Obtained results about higher concentration of circulating ribonucleic acid (RNA), according to the stages of kidney diseases, may contribute to the hypothesis that damaged kidney tissue releases nucleic acids. Circulating RNAs expressed maximal absorbance peak at 270 nm in spectrophotometric scan analysis, which corresponded to polyC, compared to different standard samples. During in vitro conditions, by using the culture of human residential macrophages, circulating RNA isolated from patients with IV-V-stage renal diseases, patients on hemodialysis, and patients who underwent renal transplantation were able to significantly change signal transduction proteins related to inflammation and antiviral response. They significantly increased the intracellular concentration of active nuclear transcription factor nuclear factor kappa B (NF-kappaB), interferon regulatory factors (IRF)-3, and IRF-7 and significantly decreased melanoma differentiation-associated protein-5 (MDA-5) and p38. In this way, it seems that circulating RNA, acting as DAMP, may contribute to the mechanisms of additional inflammatory reaction, possible immune destruction, and decreased antiviral response, related to complications in kidney diseases.

Protective effect of interferon-alpha on the DNA- and RNA-degrading pathway in anti-Fas-antibody induced apoptosis.

AIM: Fas membrane-associated polypeptide antigen is a receptor molecule responsible for apoptosis-mediated signals. In animal models of acute viral hepatitis, apoptosis of hepatocytes is mediated by Fas-death receptors; therefore, the aim of this study was to evaluate the effect of interferon (IFN)-alpha on apoptotic markers and nuclease activity against different coding and non-coding single and double stranded RNAs during Fas-induced liver apoptosis. METHODS: An in vivo experiment was performed with simultaneous administration of anti-Fas (CD95) antibodies and IFN-alpha, and an in vitro experiment was performed in hepatocyte cultures treated with anti-Fas antibodies and IFN-alpha. RESULTS: Detection of apoptosis using Annexin V-FITC/propidium iodide, Bcl-2 and Bax expression in hepatocyte cultures confirmed the appearance of early apoptotic events and progression toward late apoptosis after anti-Fas antibody treatment. IFN-alpha had a tendency to retard the apoptosis process in Fas-induced apoptosis by increasing the number of viable cells and decreasing the number of cells in late apoptosis, by increasing the percentage of Bcl-2 positive cells, by decreasing the percentage of Bax positive cells, and by decreasing the nuclease activity compared to the anti-Fas antibody treated group. Total DNA and RNA concentration was much reduced in the Fas group and DNA fragmentation assay provided evidence for increased DNA degradation. Enhanced nuclease activity against DNA, rRNA, poly(A), poly(C), poly(U), poly(I:C), and poly(A:U) was manifested in the anti-Fas antibody treated group, except for the inhibitory-bound alkaline RNase. CONCLUSIONS: The results demonstrate that the RNA-degrading pathway in Fas-induced apoptosis can accelerate the liberation of the latent enzyme from the inhibitor complex. IFN-alpha prevented enormous, Fas-ligand induced degradation of all the substrates used in this experimental study, most probably due to similarities in the signal transduction pathways. Investigations of death receptor-induced apoptosis may lead to novel treatment combinations for patients with acute or chronic liver diseases.

The cerebrospinal fluid values of advanced oxidation protein products and total thiol content in patients with amyotrophic lateral sclerosis.

OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is recognized as a progressive neurodegenerative disorder of unknown origin. Oxidative stress (OS) is considered as one of the most challenging hypothesis in the disease pathogenesis. The aim of this study was to contribute to the understanding of what extent there is involvement of OS in ALS. PATIENTS AND METHODS: We assessed Advanced Oxidation Protein Products (AOPP) and total thiol (-SH) groups in cerebrospinal fluid (CSF) of 24 ALS patients (13 of them presented with spinal form while 11 patients had bulbar form) and 20 controls (CG). RESULTS: The obtained AOPP levels in ALS patients were higher than those in CG (p <0.001), while -SH groups showed lower values compared to CG (p<0.001). The AOPP values were higher in ALS patients with bulbar compared with ALS patients with common spinal manifestation (p<0.001). There were no differences in -SH group's levels among these different clinical forms (p>0.05). The negative correlation between AOPP and the levels of -SH groups was confirmed (p <0.01). Significant mild correlations between tested parameters and functional rating scale as well as disease progression index were recorded for both of tested parameters in spinal form of ALS (p<0.01). CONCLUSION: The data presented here clearly support the fact that OS is involved in patophysiology of ALS, where oxidation of -SH groups represents an important aspect of protein oxidation. The CSF AOPP level and -SH groups may serve as potential useful biomarker for functional disorder and progression of the disease in the spinal form of ALS.

Depurinized milk downregulates rat thymus MyD88/Akt/p38 function, NF-κB-mediated inflammation, caspase-1 activity but not the endonuclease pathway: in vitro/in vivo study.

The aim of this study was the evaluation of 15 days dietary regimen of depurinized (DP) milk (obtained using our patented technological procedures) or 1.5% fat UHT milk instead of standard chow diet, on rat thymus and bone marrow MyD88/Akt/p38, NF-κB, caspase-1 and endonuclease pathways, in relation to peripheral blood cell composition. To determine whether the reduced mass of the thymus is a consequence of the direct effect of DP/UHT milk on apoptosis of thymocytes, in vitro Annexin-V-FITC/PI assay was performed. Significant decreases in the thymus wet weight, thymocyte MyD88, Akt-1/phospho-Akt-1 kinase, p38/phospho-p38, NF-κB, caspase-1 activity and CD4+/CD8+ antigen expression were obtained, especially in the DP milk group. The activity of thymocyte alkaline and acid DNase increased in the DP but not in the UHT milk group. The level of IL-6 significantly decreased in DP milk treated group, while the level of total TGF-ß and IL-6 increased in UHT milk group. Significant differences in hematological parameters were obtained in commercial milk fed group. Observed results about prevention of experimental diabetes in DP pretreated groups may suggest that purine compounds, uric acid and other volatile toxic compounds of commercial milk may suppress oral tolerance, probably via IL-6 and TGF-ß cytokine effects.

Antioxidant and proapoptotic effects of anthocyanins from bilberry extract in rats exposed to hepatotoxic effects of carbon tetrachloride.

AIMS: The aim of this research was to determine the hepatoprotective effects of anthocyanins from bilberry extract in rats exposed to carbon tetrachloride (CCl4) by monitoring the parameters of oxidative stress and apoptosis, and by performing the histopathological and morphometric analyses. MAIN METHODS: Animals were divided into four groups: Group I (0.9% NaCl-10days), Group II (bilberry extract, 75mg/kg-10days), Group III (0,9% NaCl-9days, and on the tenth day CCl4-2ml/kg), Group IV (bilberry extract, 75mg/kg-10days and on the tenth day CCl4-2ml/kg). KEY FINDINGS: Bilberry extract led to a significant decrease in the activity of biochemical parameters in serum (AST, GGT, LDH, and ALT), the activity of pro-oxidative enzyme xanthine oxidase, as well as the level of lipid peroxidation in the liver in Group IV compared to Group III (p<0.01). Bilberry extract resulted in a significant increase in the activity of the antioxidant markers-catalase (p<0.05), superoxide dismutase, glutathione S-transferase and glutathione peroxidase (p<0.01), and the concentration of reduced glutathione (p<0.05) in Group IV in relation to Group III. The application of bilberry extract resulted in an increase in the number of apoptotic hepatocytes and the activity of caspase-3 in the liver tissue (p<0.01). The reduction of coagulation necrotic areas was proved (p<0.001) as well as the number of macrovesicular hepatocytes (p<0.01), along with an increased mitotic activity (p<0.01) in Group IV compared to Group III. SIGNIFICANCE: Anthocyanins from bilberry extract have strong antioxidant properties and therefore can be considered as powerful hepatoprotectives in natural products.

Impact of folic acid supplementation on single- and double-stranded RNA degradation in human colostrum and mature milk.

Sufficient intake of folic acid is necessary for normal embryogenesis, fetal, and neonatal development. Folic acid facilitates nucleic acid internalization, and protects cellular DNA from nuclease degradation. Human milk contains enzymes, antimicrobial proteins, and antibodies, along with macrophages, that protect against infections and allergies. However, little to no information is available on the effects of folic acid supplementation on degradation of nucleic acids in human milk. In the present study, we aimed to determine the RNase activity (free and inhibitor-bound) in colostrum and mature milk, following folic acid supplementation. The study design included a total of 59 women, 27 of whom received 400 µg of folic acid daily periconceptionally and after. Folic acid supplementation increased the free RNase and polyadenylase activity following lactation. However, the increased RNase activity was not due to de novo enzyme synthesis, as the inhibitor-bound (latent) RNase activity was significantly lower and disappeared after one month. Folic acid reduced RNase activity by using double-stranded RNA as substrate. Data suggests that folic acid supplementation may improve viral RNAs degradation and mRNA degradation, but not dsRNA degradation, preserving in this way the antiviral defense.

The patients with clinically isolated syndrome and relapsing remitting multiple sclerosis show different levels of advanced protein oxidation products and total thiol content in plasma and CSF.

Advanced oxidation protein products (AOPP) and total thiol (SH) groups levels in plasma and CSF were studied in a cohort of 50 clinically isolated syndrome (CIS) and 57 relapsing remittent multiple sclerosis (RRMS) patients related to 20 control group (CG) patients' values. The obtained results were compared regarding patients demographic, biochemical, clinical (EDSS) and MRI features (total T2 weighted lesions number and Gd enhancement lesion volume). Plasma and CSF AOPP levels in CIS and RRMS patients were higher than those in CG, while SH groups showed lower values compared to CG (p<0.05). Both parameters were higher in CIS than in RRMS patients (p<0.05). Related to EDSS median range, all patients were divided into those with slight or mild and those with severe clinical presentation. AOPP and SH group changes were more pronounced in both, CIS and RRMS patients with higher, compared to those with lower EDSS (p<0.05). AOPP, SH group levels and EDSS positive correlations were observed in both study groups (p<0.01). Both parameters showed the same approach regarding the median range of total T2 weighted lesions and Gd enhancement lesion volume mean values (p<0.05), but no correlation was found between AOPP and SH levels and these patients radiological characteristics (p>0.01). The data support the fact that oxidative stress is always involved in CIS and RRMS pathophysiology, but not always as a disease determinant dependent on its intensity, which might be important for new therapeutic strategies based on antioxidant approach in those patients.

[Patients' diet in "Town" and "City" hospitals in Belgrade].

The roots of hospital foundation in Serbs date from the 12th century, when the hospitals in the monasteries Hilandar (1199) and Studenica (1207) were established. The "Town" Hospital of Belgrade was founded in 1841, which had the status of town and regional hospital until 1881. After that, it was transformed into a general state-owned hospital based on the Law of National Health Protection. The inhabitants of Belgrade obtained a municipal hospital again in 1935, when the "City" Hospital was founded in Zvezdara municipality, named at that period Bulbulder. By researching and observing hospital diet development of municipal hospitals in Belgrade, it was concluded that from the very beginning of the "Town" Hospital functioning there was awareness about its significance, place and role in the overall treatment of patients. Hospital diet, regardless of existing knowledge as the part of medical doctrines of particular time-periods, was often conditioned by limited hospital budgets and under the influence of different social movements and wartime periods

[Assessment of diet therapy effect on blood pressure of obese persons with and without antihypertensive therapy]. / Ispitivanje efekta dijetoterapije na krvni pritisak gojaznih osoba sa i bez antihipertenzivne terapije.

BACKGROUND: Arterial hypertension is a multicausal chronic disease often accompanied by obesity. The aim of this investigation was to examine the effect of diet therapy in the treatment of obese hypertensives with and without antihypertensive therapy. METHODS: The investigation was conducted at the Department of Nutrition on a sample of 110 obese hypertensive patients on diet therapy. Subjects were divided into two groups: the group on diet therapy with antihypertensive drugs E1 (n=78), and the group on diet therapy without pharmacotherapy E2 (n=32). Nourishment state i.e., obesity level was assessed by body mass index (BMI). All the patients belonged to the obese group--BMI > 30 kg/m2. Besides blood pressure values, the following parameters were monitored: serum cholesterol, trygliceride levels and BMI. RESULTS: The obtained results in the group with diet therapy combined with antihypertensive therapy showed highly significant decrease of anthropometrical parameters: body weight (99.14 kg vs. 90.16 kg) (p<0.001) and BMI (37.32 vs. 34.09) (p<0.001); percent body fat (41.97 vs. 38.78) (p<0.001); systolic (154.81 vs. 141.91) (p<0.001), and diastolic pressures (95.42 vs. 87.36) (p<0.001); cholesterol (6.39 vs. 5.99) (p<0.002), and triglycerides concentration (2.69 vs. 2.21) (p<0.019). In the group on single diet therapy, highly significant decrease of body mass (99.33 vs. 90.18) (p<0.001), BMI (34.79 vs. 31.58) (p<0.001), percent body fat (39.27 vs. 36.70) (p<0.001), systolic (148.44 vs. 132.74) (p<0.001), and diastolic pressures (93.97 vs. 82.90) (p<0.001), was achieved, while the differences between initial and final cholesterol and triglyceride concentrations, although observed, were not statistically significant. CONCLUSION: The obtained results implicated that diet therapy significantly helped the normoregulation of both systolic and diastolic blood pressure. Considering this, during physicians' routine practice in the treatment of hypertension, attention should be paid on the reduction of the corresponding level of obesity.