Hyperbaric oxygenation affects the mechanisms of acetylcholine-induced relaxation in diabetic rats.

The effects of hyperbaric oxygenation (HBO2) on acetylcholine-induced vasorelaxation (AChIR) were evaluated in male Sprague-Dawley (SD) rats randomized into four groups: healthy controls (Ctrl), diabetic rats (DM), and control and diabetic rats that underwent hyperbaric oxygenation (Ctrl+HBO2 and DM+HBO2). AChIR was measured in aortic rings, with L-NAME, indomethacin, or MS-PPOH and a combination of inhibitors. mRNA expression of eNOS, iNOS, COX-1 and COX-2 was assessed by qPCR, and protein expression of CYP4A(1-3) by Western blot. Plasma antioxidative capacity and systemic oxidative stress were determined with the ferric reducing ability of plasma (FRAP) and thiobarbituric acid-reactive substances (TBARS) assays, respectively. AChIR was preserved in all groups of rats, but mediated with different mechanisms. In all experimental groups of rats, AChIR was mediated mainly by NO, with the contribution of CYP450 vasodilator metabolites. This effect was the most prominent in the DM+HBO2 group of rats. The TBARS was significantly higher in both DM and DM+HBO2 groups compared to respective controls. eNOS expression was upregulated in the DM+HBO2 group compared to other groups, COX-1 expression was upregulated in the DM+HBO2 group compared to the control. CYP450-4A1 / A2/A3protein expression was significantly higher expressed in both hyperbaric groups compared to their respective controls. In conclusion, HBO2 affected all three vasodilator pathways and shifted AChIR to CYP450 enzymes pathway.

Antioxidant capacity and vasodilatory properties of Mediterranean food: the case of Cannonau wine, myrtle berries liqueur and strawberry-tree honey.

The aim of this work was to use different assays to evaluate the antioxidant and vasodilatory properties of three typical food products from the Mediterranean area and to correlate these activities with their phenolic content. For this purpose, red wines Cannonau, liqueurs obtained by cold maceration of myrtle (Myrtus communis L.) berries and bitter honeys obtained from strawberry-tree flowers (Arbutus unedo L.) were analysed. The total phenolic (TP) content was measured spectrophotometrically with a modified Folin-Ciocalteau method and phenolic compounds were identified and dosed by HPLC-DAD and LC-MS/MS. Antioxidant activities were evaluated with DPPH, FRAP and ABTS assays and the in vitro vasodilatory effects were assessed using norepinephrine precontracted rat aortic rings. Cannonau wines and myrtle liqueurs showed high levels of TP (1978±279 and 1741±150mg GAE/L, respectively), linearly correlated to the results of FRAP, ABTS, and DPPH assays. Their maximal vasodilatory activity was 61.7±4.1% and 53.0±3.0%, respectively. Although strawberry-tree honey contained relatively high levels of phenolic compounds (922±38mg GAE/kg), it did not induce vasodilation, even at the highest dose tested (0.206g/L). These results indicate that foods with high levels of phenolic compounds should be studied using several different biological assays before being recommended to the general public as functional foods.

Can genetics aggravate the health of isolated and remote populations? The case of gout, hyperuricaemia and osteoarthritis in Dalmatia.

INTRODUCTION: The aim of this study was to investigate whether genetics may be considered an additional risk factor for health in isolated and remote populations, compared with their populations of origin. In this study, two remote island population samples from Croatia (from the islands of Vis and the Korcula) were compared with mainland controls from the coastal city of Split. The analyses focused on gout, hyperuricaemia and osteoarthritis, as examples of complex, multifactorial diseases. METHODS: A total of 3006 examinees from all three sites in Dalmatia, Croatia were included in the descriptive part of the study, within a large-scale project of 10,001 Dalmatians. Additionally, a subset of 2428 subjects was genotyped and information on three genomic loci was used in this study. All three loci belong to SLC2A9 gene, considered to have a major role in the regulation of serum uric acid concentration (rs6449213, rs1014290 and rs737267). RESULTS: There was a much a higher prevalence of gout in the isolated populations compared with the mainland sample (3.3% in Vis, 2.2% in Korcula and 1.7% in Split, after age standardization). Furthermore, standardized prevalence of hyperuricaemia (defined as serum uric acid ≥403 mmol/L) was 9.9% in Vis, 5.6% in Korcula and 6.1% in Split. Analysis of the allele frequencies for the three loci of SLC2A9 suggested that in all three instances the prevalence of deleterious genotypes was highest in Vis, followed by Korcula, which had higher or comparable prevalence to the city of Split. Multivariate analysis, adjusted for the main confounder effects indicated that those on the island of Vis, which has the higher degree of isolation, had significantly higher odds ratio for both hyperuricaemia (odds ratio 1.90 95% confidence intervals [1.36-2.64]) and osteoarthritis, but not gout (3.37 [2.14-5.32]). The difference between Split and Korcula included only greater odds for osteoarthritis (1.92 [1.20-3.06]). CONCLUSIONS: Isolated and remote populations that maintain a sufficient level of genetic isolation may suffer not only from consequences of geographic and social isolation, but their population genetic structure may also further contribute to poorer health status and outcomes.

A complex pattern of agreement between oscillometric and tonometric measurement of arterial stiffness in a population-based sample.

OBJECTIVE: Arterial stiffness can be estimated by several noninvasive methods. In a large population-based sample we performed an agreement analysis of the set of arterial stiffness indices (ASIs) measured by tonometric (SphygmoCor) and oscillometric (Arteriograph) techniques. METHODS: Central augmentation index (cAIx) and peripheral augmentation index (pAIx), as well as central SBP (cSBP) were measured in 1012 participants from a population-based study. Data were analyzed using Bland-Altman agreement analysis, multivariate adaptive regression splines and Fisher's linear discriminant analysis. RESULTS: In contrast to high initial correlation between two devices (r = 0.87 for pAIx, 0.88 for cAIx and 0.95 for cSBP), plotting against each other the values of measured ASIs revealed their uneven distribution and grouping into three distinctive clusters of participants. The strongest cluster discriminators were age and DBP (cluster 1: age <40, DBP 70.42 ± 7.41; cluster 2: age >40, DBP 77.36 ± 10.16; cluster 3: age >60, DBP 82.56 ± 9.48). Bland-Altman analysis of clusters showed complex differences in agreement pattern for cAIx and pAIx. For cAIx SphygmoCor gives lower readings, especially in cluster 1, whereas for pAIx Arteriograph gives lower readings in cluster 1 and higher readings in clusters 2 and 3. The agreement for pAIx was better in younger participants and the same for cAIx in older participants. CONCLUSION: ASIs obtained by SphygmoCor and Arteriograph cannot be interchangeably used as they seem to be differently influenced by predictors of arterial stiffness, predominantly by age. Different pattern of pAIx and cAIx agreement across clusters demonstrates importance of distinguishing cAIx and pAIx. Homogeneity of the study population for age should be considered when interpreting results of the studies investigating ASI.

Sex-specific association of anthropometric measures of body composition with arterial stiffness in a healthy population.

BACKGROUND: Anthropometric measures of body composition and arterial stiffness are commonly used as indicators of cardiovascular risk. Little is known, however, about the association of the anthropometric measures with arterial stiffness, especially in a healthy, generally non-obese population. MATERIAL/METHODS: In a sample of 352 healthy subjects (200 premenopausal women), 3 arterial stiffness indices were analyzed (pulse wave velocity, augmentation index and central systolic blood pressure) in relation to 5 anthropometric measures of body composition (body mass index - BMI, body fat percentage by skinfold measurements -%BF, waist circumference - WC, waist-hip ratio - WHpR, and waist-height ratio - WHtR). Data were analyzed using correlation and regression analyses, with adjustment for the following confounders: age, blood pressures, height, heart rate, blood lipids and smoking. RESULTS: Most correlations between anthropometric measures and arterial stiffness indices were significant and positive in both sex groups (r=0.14-0.40, P<0.05). After adjustment for confounding effects, BMI, WC and WHtR remained significant (but inverse) predictors of arterial stiffness (ß from -0.06 to -0.16; P<0.05) in the females, while in the males BMI was the only measure inversely predicting arterial stiffness (ß from -0.09 to -0.13; P<0.05). CONCLUSIONS: Measures of body composition are weak and inverse predictors of arterial stiffness and their influence is sex-dependent. BMI, WC and WHtR were key predictors of arterial stiffness in the females, while BMI was the principal predictor in the males. The associations of anthropometric measures with arterial stiffness are strongly and differently confounded by various factors that have to be taken into account when explaining results of similar studies.

Antioxidant and vasodilatory effects of blackberry and grape wines.

In contrast to the well-described various biological effects of grape wines, the potential effects of commonly consumed blackberry wine have not been studied. We examined in vitro antioxidant and vasodilatory effects of four blackberry wines and compared them with the effects of two red and two white grape wines. Although some blackberry wines had lower total phenolic content relative to the red grape wines, their antioxidant capacity was stronger, which may be related to a higher content of non-flavonoid compounds (most notably gallic acid) in blackberry wines. Although maximal vasodilation induced by blackberry wines was generally similar to that of red wines, blackberry wines were less potent vasodilators. Vasodilatory activity of all wines, in addition to their flavonoid and total phenolic content, was most significantly associated with their content of anthocyanins. No association of vasodilation with any individual polyphenolic compound was found. Our results indicate the biological potential of blackberry wines, which deserves deeper scientific attention.

Comparison of acute effects of red wine, beer and vodka against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans.

OBJECTIVE: We determined and compared acute effects of different alcoholic beverages on oxygen-induced increase in oxidative stress plasma marker and arterial stiffness in healthy humans. METHODS: Ten males randomly consumed one of four tested beverages: red wine (RW), vodka, beer (0.32 g ethanol/kg body wt) and water as control. Every beverage was consumed once, a week apart, in a cross-over design. The volunteers breathed 100% normobaric O(2) between 60th and 90th min of 3h study protocol. Plasma lipid peroxides (LOOH) and uric acid (UA) concentration, blood alcohol concentration (BAC) and arterial stiffness (indicated by augmentation index, AIx) were measured before and 30, 60, 90, 120 and 180 min after beverage consumption. RESULTS: Intake of all alcoholic beverages caused a similar increase of BAC. The oxygen-induced elevation in AIx was similarly reduced in all three groups relative to the control (3.4 ± 1.3%, 5.4 ± 2.2% and 0.2 ± 1.6% vs. 13.7 ± 2.6% for red wine, vodka, beer and control, respectively, 60 min after intake). Exposure to oxygen resulted in increased plasma LOOH in all groups. However, in RW group this increase was lowest (1.1 ± 0.5) in comparison to the vodka (2.1 ± 0.5), beer (1.6±0.3) and control (2.5 ± 0.4µM/L H(2)O(2)). 60 min after intake of RW and beer plasma UA significantly increased (34 ± 4 and 15 ± 3) in contrast to vodka and control (-6 ± 2 and -8 ± 2µmol/L). CONCLUSION: All three alcoholic beverages provided similar protection against oxygen-induced increase in arterial stiffness, probably due to central vasodilatatory effect of alcohol itself, but only RW provided protection against oxygen-induced oxidative stress.

Ethical aspects of human biobanks: a systematic review.

AIM: To systematically assess the existing literature on ethical aspects of human biobanks. METHOD: We searched the Web of Science and PubMed databases to find studies addressing ethical problems in biobanks with no limits set (study design, study population, time period, or language of publication). All identified articles published until November 2010 were included. We analyzed the type of published articles, journals publishing them, involvement of countries/institutions, year of publication, and citations received, and qualitatively assessed every article in order to identify ethical issues addressed by the majority of published research on human biobanking. RESULTS: Hundred and fifty four studies satisfied our review criteria. The studies mainly came from highly developed countries and were all published in the last two decades, with over half of them published in 2009 or 2010. They most commonly discussed the informed consent, privacy and identifiability, return of results to participants, importance of public trust, involvement of children, commercialization, the role of ethics boards, international data exchange, ownership of samples, and benefit sharing. CONCLUSIONS: The focus on ethical aspects is strongly present through the whole biobanking research field. Although there is a consensus on the old and most typical ethical issues, with further development of the field and increasingly complex structure of human biobanks, these issues will likely continue to arise and accumulate, hence requiring constant re-appraisal and continuing discussion.

Thermally treated wine retains vasodilatory activity in rat and guinea pig aorta.

In contrast to the intact wine, cardiovascular effects of the thermally treated wine have not been studied, despite widespread habits of cooking with wine and consumption of mulled wine. Vasodilatory effects of the red wine heated at 75 and 125°C were examined in the isolated rat and guinea pig aorta and compared with the intact and wine dealcoholized without thermal stress. Samples were analyzed for their phenolic content, antioxidant capacity, resveratrol and ethanol contents. Heating-induced degradation of individual phenolic fraction was observed only in the samples treated at 125°C, although total phenolic concentration and related antioxidant activity increased in the thermally treated samples due to the reduction in their volume. All wine samples regardless of treatment caused similar maximal relaxation in both species, but the response was stronger in aortas from guinea pigs. At the lowest concentrations up to 1‰, dealcoholized wine produced vasodilation greater than that produced by intact wine and wines treated at 75 and 125°C, which showed similar vasodilating activity at all concentrations. Our results indicate that wine thermally treated under heating conditions applicable to the preparation of a mulled wine and cooking with wine largely retains vasodilatory activity in vitro despite significant heat-induced changes in its composition.

Antimicrobial effects of wine: separating the role of polyphenols, pH, ethanol, and other wine components.

While the antimicrobial effectiveness of wine is well documented, relative contributions of the wine components to its antimicrobial activity is controversial. To separate the role of wine phenolics, ethanol, and pH from other wine constituents, the antimicrobial effects of intact wine were compared to that of phenols-stripped wine, dealcoholized wine, ethanol, and low pH applied separately and in combination, against 2 common foodborne pathogens, Salmonella enterica serovar Enteritidis and Escherichia coli. All samples were biochemically characterized with respect to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, and pH. Antioxidative activity of the samples corresponded to their total phenolics content. Except for respective controls, pH and ethanol content were similar in all samples. The order of antibacterial activity of the samples was: intact wine > phenols-stripped wine > dealcoholized wine > combination of ethanol and low pH > low pH > ethanol. Separate application of ethanol or low pH showed negligible antibacterial activity while their combination showed synergistic effect. Antibacterial activity of the samples could not be related to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, or pH. Our study indicates that antimicrobial activity of complex solutions such as intact wine cannot be exclusively attributed to its phenolic or nonphenolic constituents, nor can the antimicrobial activity of wine be predicted on the basis of its particular components.

Predictive value of 8 genetic loci for serum uric acid concentration.

AIM: To investigate the value of genomic information in prediction of individual serum uric acid concentrations. METHODS: Three population samples were investigated: from isolated Adriatic island communities of Vis (n=980) and Korcula (n=944), and from general population of the city of Split (n=507). Serum uric acid concentration was correlated with the genetic risk score based on 8 previously described genes: PDZK1, GCKR, SLC2A9, ABCG2, LRRC16A, SLC17A1, SLC16A9, and SLC22A12, represented by a total of 16 single-nucleotide polymorphisms (SNP). The data were analyzed using classification and regression tree (CART) and general linear modeling. RESULTS: The most important variables for uric acid prediction with CART were genetic risk score in men and age in women. The percent of variance for any single SNP in predicting serum uric acid concentration varied from 0.0%-2.0%. The use of genetic risk score explained 0.1%-2.5% of uric acid variance in men and 3.9%-4.9% in women. The highest percent of variance was obtained when age, sex, and genetic risk score were used as predictors, with a total of 30.9% of variance in pooled analysis. CONCLUSION: Despite overall low percent of explained variance, uric acid seems to be among the most predictive human quantitative traits based on the currently available SNP information. The use of genetic risk scores is a valuable approach in genetic epidemiology and increases the predictability of human quantitative traits based on genomic information compared with single SNP approach.

Acute, food-induced moderate elevation of plasma uric acid protects against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans.

We examined the effects of acute, food-induced moderate increase of plasma uric acid (UA) on arterial stiffness and markers of oxidative damage in plasma in healthy males exposed to 100% normobaric oxygen. Acute elevation of plasma UA was induced by consumption of red wine, combination of ethanol and glycerol, or fructose. By using these beverages we were able to separate the effects of UA, wine polyphenols and ethanol. Water was used as a control beverage. Ten males randomly consumed test beverages in a cross-over design over the period of 4 weeks, one beverage per week. They breathed 100% O(2) between 60(th) and 90(th)min of the 4-h study protocol. Pulse wave augmentation index (AIx) at brachial and radial arteries, plasma antioxidant capacity (AOC), thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides (LOOH) assessed by xylenol orange method, UA and blood ethanol concentrations were determined before and 60, 90, 120, 150 and 240 min after beverage consumption. Consumption of the beverages did not affect the AIx, TBARS or LOOH values during 60 min before exposure to hyperoxia, while AOC and plasma UA increased except in the water group. Significant increase of AIx, plasma TBARS and LOOH, which occurred during 30 min of hyperoxia in the water group, was largely prevented in the groups that consumed red wine, glycerol+ethanol or fructose. In contrast to chronic hyperuricemia, generally considered as a risk factor for cardiovascular diseases and metabolic syndrome, acute increase of UA acts protectively against hyperoxia-induced oxidative stress and related increase of arterial stiffness in large peripheral arteries.

Acute tonsillopharyngitis in a family practice in Mostar, Bosnia and Herzegovina.

The aim of this study was to examine the characteristics of acute tonsillopharyngitis diagnosis and treatment in the family care physicians in Mostar, Bosnia and Herzegovina. All patients diagnosed with acute tonsillopharyngitis in the Center of Family Medicine in Mostar in 2005 and 2006 were included in this study. Data were collected from medical records, including age and sex, month in which they visited their physician, symptoms and signs that they had (McIsaac's clinical score was calculated accordingly) and treatment. Results showed that there were no gender differences regarding diagnosis. Mean age was 32.6 +/- 16.9 years. The most patients were recorded during the January-March period. Total of 65.6% patients received antibiotic treatment. Phenoxymethylpenicillin, considered as the recommended antibiotic was used in 46.3% cases only. In conclusion, this first critical assessment of the existing family practice records on treating patients with tonsillopharyngitis suggested that physicians have to be more critical when treating patients with this diagnosis and that the knowledge and treatment of patients with pharyngitis need to be continuously improved in general medical practice.

Genome-wide association study of biochemical traits in Korcula Island, Croatia.

AIM: To identify genetic variants underlying biochemical traits--total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, uric acid, albumin, and fibrinogen, in a genome-wide association study in an isolated population where rare variants of larger effect may be more easily identified. METHODS: The study included 944 adult inhabitants of the island of Korcula, as a part of larger DNA-based genetic epidemiological study in 2007. Biochemical measurements were performed in a single laboratory with stringent internal and external quality control procedures. Examinees were genotyped using Human Hap370CNV chip by Illumina, with a genome-wide scan containing 346027 single nucleotide polymorphisms (SNP). RESULTS: A total of 31 SNPs were associated with 7 investigated traits at the level of P<1.00 x 10(-5). Nine of SNPs implicated the role of SLC2A9 in uric acid regulation (P=4.10 x 10(-6)-2.58 x 10(-12)), as previously found in other populations. All 22 remaining associations fell into the P=1.00 x 10(-5)-1.00 x 10(-6) significance range. One of them replicated the association between cholesteryl ester transfer protein (CETP) and HDL, and 7 associations were more than 100 kilobases away from the closest known gene. Nearby SNPs, rs4767631 and rs10444502, in gene kinase suppressor of ras 2 (KSR2) on chromosome 12 were associated with LDL cholesterol levels, and rs10444502 in the same gene with total cholesterol levels. Similarly, rs2839619 in gene PBX/knotted 1 homeobox 1 (PKNOX1) on chromosome 21 was associated with total and LDL cholesterol levels. The remaining 9 findings implied possible associations between phosphatidylethanolamine N-methyltransferase (PEMT) gene and total cholesterol; USP46, RAP1GDS1, and ZCCHC16 genes and triglycerides; BCAT1 and SLC14A2 genes and albumin; and NR3C2, GRIK2, and PCSK2 genes and fibrinogen. CONCLUSION: Although this study was underpowered for most of the reported associations to reach formal threshold of genome-wide significance under the assumption of independent multiple testing, replications of previous findings and consistency of association between the identified variants and more than one studied trait make such findings interesting for further functional follow-up studies. Changed allele frequencies in isolate population may contribute to identifying variants that would not be easily identified in much larger samples in outbred populations.

Differences in vasodilatory response to red wine in rat and guinea pig aorta.

We examined and compared mechanisms of the red wine (RW)-induced vasorelaxation in guinea pig (GP) and rat aorta. Acetylcholine-induced relaxation of norepinephrine-precontracted aortic rings was stronger in rat aorta than in GP aorta, whereas RW-induced vasorelaxation was stronger in GP aorta. L-nitro-arginine methyl ester (L-NAME) abolished RW-induced vasorelaxation in rat aorta, whereas in GP aorta, it was only reduced by 50%. To examine mechanisms of the L-NAME-resistant relaxation, GP aortic rings were additionally exposed to indomethacin, clotrimazole, and their combination. Indomethacin insignificantly reduced RW-induced relaxation, but in combination with L-NAME, the relaxation was synergistically decreased (80%). After clotrimazole exposure, the relaxation was reduced by 25%, and addition of indomethacin caused no further reduction. Only the combination of L-NAME, indomethacin, and clotrimazole prevented RW-induced vasorelaxation. RW-induced vasorelaxation in KCl-precontracted GP rings was significantly smaller (Emax 78.31% +/- 6.09%) than the RW-induced relaxation in norepinephrine-precontracted rings (Emax 126.01% +/- 2.11%). L-NAME in KCl-precontracted GP rings prevented RW-induced vasorelaxation. In conclusion, different pathways are involved in the RW-induced vasorelaxation in GP aorta, in contrast to rat aorta, in which NO plays main role. Therefore, the uncritical extrapolation of the results from one species to another could be misleading.