Effect of bilberry extract (Vaccinium myrtillus L.) on drug-metabolizing enzymes in rats.

Vaccinium myrtillus L. (bilberry) fruit is a blue-colored berry with a high content of anthocyanins. These bioactive secondary metabolites are considered to play a major role in the health-promoting properties of bilberries. Our in vivo study was designed to assess the possible influence of bilberry extract on drug-metabolizing enzymes (DMEs). Rats were exposed to bilberry extract in drinking water at two concentrations (0.15 and 1.5 g/L). Selected DMEs were determined (mRNA expression and enzymatic activity) after 29 and 58 days in rat liver. In addition, a panel of antioxidant, physiological, biochemical and hematological parameters was studied; these parameters did not demonstrate any impact of bilberry extract on the health status of rats. A significant increase in activity was observed in cytochrome P450 (CYP) 2C11 (131% of control) and CYP2E1 (122% of control) after a 29-day administration, while the consumption of a higher concentration for a longer time led to a mild activity decrease. Slight changes were observed in some other DMEs, but they remained insignificant from a physiological perspective. According to our results, we conclude that the consumption of bilberries as a food supplement should not pose a risk of interacting with co-administered drugs based on their metabolism.

The introduction of simple cardiorespiratory fitness testing in overweight/obese type 2 diabetics: a pilot study.

BACKGROUND: Low level of cardiorespiratory fitness has been recognized as an important independent and modifiable risk factor of increased morbidity and mortality. However, in standard outpatient settings, patients are not routinely screened for fitness and advantages of such testing for the management of type 2 diabetes have not been defined.AimTo describe the toleration of a fast, simple and practicable fitness test (2-min step-in-place test) by overweight/obese type 2 diabetics and their performance indicated by 2-min step-in-place test score (STS). To study short-term anthropometric, functional and metabolic changes following the implementation of the test in the selected population. METHODS: A total of 33 overweight/obese type 2 diabetics underwent, besides routine examination at the outpatient clinic, the fitness test (group A). Patients were asked to increase their regular physical activity with focus on walking without change in diet and chronic medication. Three to four months later, the subjects were tested again. An identical number of age- and sex-matched obese diabetics followed in our outpatient clinic (without fitness testing), was randomly selected from the Hospital Information System (control group B).FindingsAll patients subjected to fitness testing completed the protocol successfully. STS score was found to have a considerable range with differences between males and females at the borderline of statistical significance. The data are compliant with lower aerobic endurance of obese diabetics compared with healthy population. Within study period, the tested group presented with improvements in STS (referring especially to the males) as well as in several laboratory parameters of glucose and lipid homeostasis, glomerular function and subclinical inflammation with no reflection in anthropometry. Group B demonstrated no significant change. In conclusion, 2-min step-in-place test is fast, undemanding and well-tolerated by patients and personnel. Following its validation based on cardiopulmonary exercise testing, the test may prove recommendable for screening or self-monitoring purposes.

Critical evaluation of colon submucosal microdialysis in awake, mobile rats.

Sensors able to record large bowel physiology and biochemistry in situ in awake rodents are lacking. Microdialysis is a mini-invasive technique that may be utilized to continuously deliver or recover low-molecular substances from various tissues. In this experiment we evaluated the feasibility of in vivo microdialysis to monitor extracellular fluid chemistry in the descending colon submucosa of conscious, freely moving rodents. Following surgical implantation of a microdialysis probe, male Wistar rats were housed in metabolic cages where they were analgized and clinically followed for four days with free access to standard diet and water. To assess local microcirculation and probe function, glucose, lactate, glucose-to-lactate ratio and urea clearance were determined in the dialysates from the three postoperative days with focus on the final 24-h period. In an attempt to mitigate the expected tissue inflammatory response, one group of animals had the catheters perfused with 5-aminosalicylic acid-enriched medium with final concentration 1 µmol/L. For verification of probe position and the assessment of the surrounding foreign body reaction, standard histological and immunohistochemical methods were employed. Microdialysis of rat gut is associated with considerable technical challenges that may lead to the loss of probe function and high drop-out rate. In this setting, limited data did not allow to draw any firm conclusion regarding local anti-inflammatory effectiveness of 5-aminosalicylic acid perfusion. Although intestinal microdialysis may be suitable for larger anesthetized animals, low reproducibility of the presented method compromises its routine experimental use in awake and freely moving small-sized rodents.

Protective effect of isoquercitrin against acute dextran sulfate sodium-induced rat colitis depends on the severity of tissue damage.

BACKGROUND: Isoquercitrin (quercetin-3-O-ß-d-glucopyranoside) is a flavonoid that exhibited antioxidant and anti-inflammatory activities in a number of in vitro and in vivo studies. Experimental evidence from rodent models of inflammatory bowel disease is, however, lacking. This study was designed to examine whether isoquercitrin effectively and dose-dependently attenuates acute dextran sulfate sodium (DSS)-induced rat colitis. METHODS: Wistar rats were divided into negative control group (exposed to vehicle only), positive control group (DSS-induced colitis plus vehicle), low isoquercitrin group (DSS pretreated with isoquercitrin 1mg/kg/day) and high isoquercitrin group (DSS with isoquercitrin 10mg/kg/day). Isoquercitrin was administered daily for 14days, and during the last 7days rats drank DSS solution. The effect of isoquercitrin on DSS-induced colitis was assessed clinically (e.g. disease activity index), biochemically (tissue myeloperoxidase activity, local cyclooxygenase-2 expression), using histology (standard hematoxylin-eosin-based histomorphometry, immunohistochemical detection of inducible nitric oxide synthase) and hematology (blood count). RESULTS: Isoquercitrin dose-dependently ameliorated whole colon shortening and mitigated DSS-induced expression of cyclooxygenase-2 and inducible nitric oxide synthase in the descending segment of the organ. However, when different parts of colon were assessed histomorphometrically, the results did not globally support the protective role of this flavonoid. Tissue healing trends observable in the descending colon were not apparent in the rectum, where histological damage was most severe. CONCLUSIONS: We surmise that isoquercitrin may be effective in the prevention of acute colitis. Besides being dose-dependent, the potency of orally administered isoquercitrin may depend on the severity of tissue damage and/or on the site of its action.

Multi-drug intoxication fatality involving atorvastatin: A case report.

Mixed antihypertensive drug intoxication poses a significant risk for patient mortality. In tandem to antihypertensives, hypolipidemic medicines (especially statins) are often prescribed. Among their well-known adverse effects belongs rhabdomyolysis. We report a case of fatal multi-drug overdose in a 65-year-old female alcoholic. The patient was unconscious at admission. Empty blister packs indicated the abuse of 250 tablets of urapidil, 42 tablets of verapamil/trandolapril, 50 tablets of moxonidin, 80 tablets of atorvastatin and 80 tablets of diacerein. Standard measures (gastric lavage, activated charcoal, mechanical ventilation, massive doses of vasopressors, volume expansion, diuretics and alkalinization) failed to provide sufficient drug elimination and hemodynamic support and the sufferer deceased on the fourth day. Dramatic elevations of serum myoglobin (34,020 µg/L) and creatine kinase (219 µkat/L) were accompanied by rise in cardiac troponin I and creatinine. Gas chromatography revealed ethanol 1.17 g/kg (blood) and 2.81 g/kg (urine). Thin layer chromatography and gas chromatography of gastric content and urine verified verapamil, moxonidin and urapidil fragment (diacerein method was unavailable). Atorvastatin and trandolapril concentrations (LC-MS(n)) equaled 277.7 µg/L and 57.5 µg/L, resp. (serum) and 8.15 µg/L and 602.3 µg/L, resp. (urine). Histology confirmed precipitates of myoglobin with acute necrosis of proximal renal tubules in association with striated muscle rhabdomyolysis and myocardial dystrophy. Cardiogenic-distributive shock in conjunction with acute renal failure due to the combined self-poisoning with vasoactive agents and atorvastatin were determined to be this decedent's immediate cause of death. The manner of death was assigned to be suicidal.

Electrochemical sensing of total antioxidant capacity and polyphenol content in wine samples using amperometry online-coupled with microdialysis.

This work describes the method for total antioxidant capacity (TAC) and/or total content of phenolics (TCP) analysis in wines using microdialysis online-coupled with amperometric detection using a carbon microfiber working electrode. The system was tested on 10 selected wine samples, and the results were compared with total reactive antioxidant potential (TRAP), oxygen radical absorbance capacity (ORAC), and chemiluminescent determination of total antioxidant capacity (CL-TAC) methods using Trolox and catechin as standards. Microdialysis online-coupled with amperometric detection gives similar results to the widely used cyclic voltammetry methodology and closely correlates with ORAC and TRAP. The problem of electrode fouling is overcome by the introduction of an electrochemical cleaning step (1-2 min at the potential of 0 V vs Ag/AgCl). Such a procedure is sufficient to fully regenerate the electrode response for both red and white wine samples as well as catechin/Trolox standards. The appropriate size of microdialysis probes enables easy automation of the electrochemical TAC/TCP measurement using 96-well microtitration plates.

Gastric submucosal microdialysis in the detection of rat stomach ischemia--a comparison of the 3H2O efflux technique with metabolic monitoring.

Microdialysis has been utilized for nutritive blood flow measurements, but both the advantages and disadvantages of various approaches have not been evaluated in parallel in the stomach yet. Our aim was to compare the (3)H(2)O efflux technique with biochemical monitoring during temporary celiac artery occlusion in anesthetized rats. Microdialysis probes were implanted in the gastric submucosa and perfused with (3)H(2)O; samples were analyzed for ß-activity, glucose, lactate, pyruvate and glycerol. Gastric mucosa and plasma were subjected to morphometry and analysis of myeloperoxidase, total thiols and lactatdehydrogenase. The most dramatic responses to ischemia were observed in lactate/pyruvate and lactate/glucose (%) ratios (6.1-9.3×, p < 0.0001); the changes in (3)H(2)O efflux and glycerol were less pronounced (1.1-1.7×, p < 0.0001 and < 0.01, respectively). (3)H(2)O efflux correlated best with the lactate/glucose ratio and glucose alone (r = 0.693 and -0.681, respectively, p < 0.0001). A correlation was also found between plasma lactatdehydrogenase and relative glycerol release (r = 0.600, p < 0.05). Myeloperoxidase, lactatdehydrogenase and histology score were increased by ischemia/reperfusion (0.06-0.12 nkat g(-1), p < 0.05, 0.26-0.44 nkat g(-1), p < 0.05 and 1.79-2.33, p < 0.05, respectively), macroscopy and plasma thiols remained unchanged. Microdialysis is useful in monitoring gastric ischemia, metabolic monitoring being superior to the (3)H(2)O efflux technique. The results question the efficacy of the utilized model to produce standardized major gastric damage.

Protein metabolism in leg muscle following an endotoxin injection in healthy volunteers.

The human endotoxin model has been used to study the early phase of sepsis. The aim of the present study was to assess leg muscle protein kinetics after an endotoxin challenge given to healthy human volunteers. Six healthy male subjects were studied in the post-absorptive state before and during 4 h following an intravenous endotoxin bolus (4 ng/kg of body weight). Primed continuous infusion of [(2)H(5)]phenylalanine and [(2)H(3)]3-methylhistidine in combination with sampling from the radial artery, femoral vein and muscle tissue were used to assess leg muscle protein kinetics. Both two- and three-compartment models were used to calculate protein kinetics. In addition 26S proteasome activity and protein ubiquitination were assessed. An increase in the net release of phenylalanine from the leg following the endotoxin challenge was observed; however, this phenylalanine originates from the free intracellular pool and not from protein. Net protein balance was unchanged, whereas both protein synthesis and breakdown were decreased. Degradation rates of contractile proteins were not affected by endotoxin, as indicated by an unchanged rate of appearance of 3-methylhistidine from leg muscle. In addition, proteasome activity and protein ubiquitination were unaffected by endotoxaemia. In conclusion, intravenous endotoxin administration to healthy volunteers resulted in an increased release of free phenylalanine from skeletal muscle, whereas protein balance was unaffected. Both protein synthesis and breakdown were decreased to a similar extent.

The influence of simvastatin, atorvastatin and high-cholesterol diet on acetylcholinesterase activity, amyloid beta and cholesterol synthesis in rat brain.

OBJECTIVE: There is evidence to suppose that cholesterol-lowering medicine might confer protection against dementia, probably via modulation of cholesterol synthesis in the brain. The aim of the present study was to investigate the potential influence of statins and cholesterol diet on selected parameters relevant to Alzheimer's disease pathophysiology. METHODS: For 15 days, rats were orally administered simvastatin (10 or 20mg/kg b.wt.), atorvastatin (10 or 20mg/kg b.wt.), or aqua (control group); and one group was fed high-cholesterol (2%) diet. At the end of experiments brain (and plasma) cholesterol, lathosterol, hydroxymethylglutaryl-coenzyme A reductase protein, acetylcholinesterase activity, amyloid beta (40 and 42) and cholesterol synthesis rate (using the incorporation of deuterium from deuterated water) were determined and statistically compared to those of aqua. RESULTS: Both statins were able to lower cholesterol in the plasma, but none elicited an effect on total brain cholesterol. Significant reductions of brain lathosterol and cholesterol synthesis rate were observed after simvastatin and atorvastatin treatment. Acetylcholinesterase activity, amyloid beta and hydroxymethylglutaryl-coenzyme A reductase levels remained unaffected by the two drugs. CONCLUSIONS: This study brings additional evidence of a role for statins in cholesterol synthesis in the brain. Our data question the relationship between amyloid beta, acetylcholinesterase activity and cholesterol synthesis in the rat brain as well as the assumption about no exchange between peripheral and brain cholesterol pools.

Caffeine does not modulate nutritive blood flow to rat gastric submucosa--a microdialysis study.

BACKGROUND AND AIMS: Coffee irritates the gastric mucosa disrupting its barrier and increasing the risk of peptic ulcers. However, caffeine's contribution to these effects has not yet been elucidated. In this study we looked at the local effect of caffeine on the microcirculation and nitric oxide production in rats together with systemic marker of oxidative stress malondialdehyde as possible mechanisms whereby caffeine might participate in mucosal barrier impairment. MATERIALS AND METHODS: Four groups of rats were anesthetized and administered as a bolus four different intraperitoneal doses of caffeine (0, 1, 10 and 50 mg kg(-1) b.wt.). The gastric submucosal microcirculation and nitric oxide production were then recorded for 2.5 hours by in situ microdialysis using the flow marker ethanol. At the completion of the experiments, plasma caffeine and malondialdehyde levels as well as morphological mucosal injury were determined. RESULTS: There were no major differences in the macro- or microscopic pictures of the mucosa among the groups. Local microcirculatory (ethanol out/in ratio) and nitric oxide monitoring failed to demonstrate statistically significant changes as did measurement of plasma malondialdehyde in response to caffeine injections. CONCLUSIONS: Caffeine per se seems unlikely to contribute to the gastric mucosal barrier injury associated with coffee consumption by alterations in nutritive blood flow, nitric oxide production or aggravation of systemic oxidative stress. This information is relevant for better understanding of the mechanisms involved in caffeine-mediated influences on gastric physiology in relation to the irritant effects of coffee.

Peripheral tissue metabolism during off-pump versus on-pump coronary artery bypass graft surgery: the microdialysis study.

OBJECTIVE: The aim of this study was to monitor and compare metabolic changes in the skeletal muscle during coronary artery bypass grafting surgery with and without cardiopulmonary bypass (CPB) by means of interstitial microdialysis. Glucose, lactate, pyruvate and glycerol were assessed as markers of basic metabolism and tissue perfusion. METHODS: Twenty patients undergoing surgical myocardial revascularization were enrolled in this pilot study. Ten patients were operated on without CPB (group A, off-pump) and 10 patients using normothermic CPB (group B, on-pump). Interstitial microdialysis was performed by a CMA 60 (CMA/Microdialysis AB, Sweden) probe, inserted into the patient's left deltoid muscle. Microdialysis measurements were performed at 30 min intervals. Glucose, lactate, pyruvate and glycerol were measured in samples using a CMA 600 Analyser (CMA/Microdialysis AB, Sweden). Results in both groups were statistically processed and the groups were compared. RESULTS: Both groups were similar with regards to preoperative characteristics. Dynamic changes of interstitial concentrations of the measured analytes were found in off-pump (group A) and on-pump (group B) patients during the operation. There were no significant differences in dialysate concentrations of glucose and lactate between the groups. Significant differences were detected in pyruvate concentrations, lactate-pyruvate ratio and glycerol concentrations between off-pump versus on-pump patients. Pyruvate concentrations were higher in the off-pump group (p<0.05), the lactate-pyruvate ratios indicating the aerobic/anaerobic metabolism status were lower in the off-pump group (p<0.01) and the values of the concentrations of glycerol were lower in the off-pump group (p<0.01). CONCLUSION: Dynamic changes in the interstitial concentrations of the glucose, glycerol, pyruvate and lactate were found in both groups of patients (off-pump and on-pump). The presented preliminary results suggest that extracorporeal circulation during cardiac operations could compromise skeletal muscle energy metabolism.

Zonation of multidrug resistance-associated protein 2 in rat liver after induction with dexamethasone.

BACKGROUND AND AIM: The present study was aimed to evaluate the hepatic zonation of multidrug resistance-associated protein 2 (mrp2), an important drug transporter, and its potential changes during the induction of its expression by known inducer, dexamethasone (DEX). METHODS: The hepatic expression of mrp2 was studied by immunohistochemistry with consequent quantification by measurement of integral optical densities of mrp2 staining in the periportal and perivenous areas of the liver acinus in control and DEX-pretreated rats (1 mg/kg daily per os for 4 days). Overall changes in mrp2 expression and function produced by DEX were monitored using Western blotting and an in vivo clearance study of endogenous-conjugated bilirubin, a mrp2 substrate. RESULTS: In the control animals, a quantitative image analysis revealed the primary periportal localization of mrp2 within the liver acinus with the expression of mrp2 being 16.7-fold of that in the perivenous area. After DEX pretreatment, the expression of mrp2 increased, especially in the perivenous hepatocytes. The overall expression of mrp2 increased 3.2-fold in comparison with the control group. This observation was confirmed by Western blotting, which showed a 1.3-fold increase in the mrp2 protein after DEX pretreatment. The functional consequences of the induced mrp2 protein in the livers of the DEX-pretreated rats were demonstrated by the increased biliary excretion of conjugated bilirubin. CONCLUSION: In conclusion, these results indicate the zonation of mrp2 protein expression primarily to periportal hepatocytes. The induction by DEX produced spatially disproportional changes with an increase in the mrp2 protein being most prominent in the perivenous hepatocytes.

Lithium microdialysis and its use for monitoring of stomach and colon submucosal blood perfusion--a pilot study using ischemic preconditioning in rats.

During shock, exposure of gut to ischemia determines patient's survival. Ischemic preconditioning (ISP) elevates nitric oxide and blood perfusion, whereby it protects organs against subsequent severe ischemia/reperfusion. Using appropriate flow marker, microdialysis may serve to monitor interstitial microcirculation. Hence, our aim was to test the reliability of lithium as a flow marker (lithium microdialysis, LM) on an ISP model. Rats were divided into three groups. Two (ischemic and preconditioned) groups underwent 30 min celiac artery occlusion (CAO) with 2.5 h reperfusion. 25 min before CAO, the latter experienced 5 min ischemia. Sham-operated animals served as controls. LM in stomach and colon submucosa, serum nitric oxide, hepatic and pancreatic enzymes were measured. In stomach, LM indicated a decrease in blood perfusion evoked by CAO (p < 0.01) in both experimental groups. During reperfusion, the ischemic animals showed a restoration of microcirculation, unlike the preconditioned ones, whose blood perfusion failed to regenerate (p < 0.001). For any group, LM showed no microcirculation modification in colon. Serum analytes remained unchanged. We conclude that LM appears to be a potentially suitable indicator of gastrointestinal interstitial microcirculation. However, we failed to demonstrate any beneficial effect of ISP on pancreas, systemic nitric oxide and local/remote microcirculation within studied organs.

[Use of sucrose permeability test (SaLM) for detection of lesions of upper gastrointestinal tract mucosa in upper dyspepsia patients--a pilot study]. / Vyuzití testu propustnosti pro sacharózu (SaLM) v detekci postizení sliznic horní cásti trávicí trubice u pacientu s horním dyspeptickým syndromem--pilotní studie.

INTRODUCTION: Endoscopy, a golden standard with its high diagnostic value, is an invasive and unpleasant method as far as patients are concerned. So far there has been no available non-invasive test in the Czech Republic capable of distinguishing between heavy (i.e. peptic ulcer) and light (i.e. portal gastropathy) lesions of upper gastrointestinal mucosa. AIMS: In this pilot study we decided to test our modification of sucrose permeability test (SaLM test) on upper dyspepsia patients in our conditions. We first needed to compare the results of intestinal permeability obtained from the studied test (containing sucrose, a so called SaLM test) with a formerly established intestinal permeability test (containing glucose, a so called LaMa test) to know, if the new test could replace the old one. Then we wanted to find normal values of sucrose permeability, find a relationship between sucrose permeability and endoscopically verified damage to upper gastrointestinal mucosa and calculate sensitivity and specificity of SaLM test using results of gastroduodenoscopy. After that we tried to suggest possible future benefits of the test for clinical praxis. MATERIALS AND METHODS: A group of 10 young healthy volunteers underwent both SaLM and LaMa tests, which were made methodically indentical to compare the tests as to the results of intestinal permeability. The probands ingested SaLM solution with the following composition: sucrose (25.0 g), lactulose (10.0 g), mannitol (2.0 g), xylose (2.0 g) and water (up to 100 ml). Urine was collected for five hours and the samples were analysed using gas chromatography. From the results normal value of sucrose permeability was calculated, too. After that, 28 patients with upper dyspepsia were included in the study. They were divided into two groups (a group of light lesions with 9 patients and a group of heavy lesions counting 19 patients) according to gastroscopical findings. We compared the results among the three groups. RESULTS: In our volunteers, the intestinal permeability values using LaMa and SaLM tests showed normal distributions. No statistically significant difference (p < 0.05) was found between the tests in regard to the intestinal permeability. The normal value of sucrose permeability was found to be up to 0.10% of the amount taken orally. The permeability for sucrose was significantly higher (p < 0.01) in patients with heavy lesions (0.527 +/- 0.414) versus those with light ones (0.178 +/- 0.090). Moreover, the latter had their sucrose permeability values significantly higher than healthy volunteers (0.088 +/- 0.067), (p < 0.05). Sensitivity and specificity of the test for heavy upper gastrointestinal mucosal damage was 0.95 and 0.33, respectively. CONCLUSION: SaLM test could replace LaMa test without having a significant effect on the intestinal permeability results. It is feasible to study SaLM test on bigger sets of patients and specify it in more detail, since the results of our pilot study (in accordance with many other studies) make it promising for various clinical applications (i.e. in upper dyspepsia patients it might help in deciding about urgency and reasonability of gastroduodenoscopy).

The impairment of gastroduodenal mucosal barrier by coffee.

BACKGROUND: Even though coffee is not considered to be responsible for development of peptic ulcer, it may, however, prolong its healing by increasing acidity of gastric content. In our former work we observed a profound increase in sucrose permeability (above normal values) in healthy volunteers regularly drinking coffee for years. In literature, many factors affecting sucrose permeability have been described so far. None of them, however, studied the effect of coffee. SUBJECTS, MATERIALS AND METHODS: 10 young asymptomatic habitual coffee drinkers were included in the study. The probands underwent SaLM test twice--first time without coffee restriction and second time after 48-hour coffee abstinence. The ingested SaLM solution comprised sucrose (25.0 g), lactulose (10.0 g), mannitol (2.0 g), xylose (2.0 g) and water (up to 100 ml). Urine was collected for five hours and the samples were analysed using gas chromatography. Results were compared with those of 8 young healthy volunteers not drinking coffee. Permeability for sucrose was significantly higher in the group of habitual coffee drinkers in comparison with non-coffee drinkers (p < 0.01). After 48-hour coffee abstinence sucrose excretion decreased significantly (p < 0.05) to a level not differing from that of non-coffee drinkers (p = 0.54). CONCLUSIONS: Our results indicate that coffee may damage gastroduodenal mucosa in habitual coffee drinkers. In a time period of 48 hours the gastroduodenal mucosa is capable of a significant regeneration.