Gas Chromatography-Mass Spectrometry Detection of Thymoquinone in Oil and Serum for Clinical Pharmacokinetic Studies.

Thymoquinone (TQ) is the primary component of Nigella sativa L. (NS) oil, which is renowned for its potent hepatoprotective effects attributed to its antioxidant, anti-fibrotic, anti-inflammatory, anti-carcinogenic, and both anti- and pro-apoptotic properties. The aim of this work was to establish a method of measuring TQ in serum in order to investigate the pharmacokinetics of TQ prior to a targeted therapeutic application. In the first step, a gas chromatography-mass spectrometry method for the detection and quantification of TQ in an oily matrix was established and validated according to European Medicines Agency (EMA) criteria. For the assessment of the clinical application, TQ concentrations in 19 oil preparations were determined. Second, two serum samples were spiked with TQ to determine the TQ concentration after deproteinization using toluene. Third, one healthy volunteer ingested 1 g and another one 3 g of a highly concentrated NS oil 30 and 60 min prior to blood sampling for the determination of serum TQ level. After the successful establishment and validation of the measurement method, the highest concentration of TQ (36.56 g/L) was found for a bottled NS oil product (No. 1). Since a capsule is more suitable for oral administration, the product with the third highest TQ concentration (No. 3: 24.39 g/L) was used for all further tests. In the serum samples spiked with TQ, the TQ concentration was reliably detectable in a range between 5 and 10 µg/mL. After oral intake of NS oil (No. 3), however, TQ and/or its derivatives were not detectable in human serum. This discrepancy in detecting TQ after spiking serum or following oral ingestion may be attributed to the instability of TQ in biomatrices as well as its strong protein binding properties. A pharmacokinetics study was therefore not viable. Studies on isotopically labeled TQ in an animal model are necessary to study the pharmacokinetics of TQ using alternative modalities.

Staphylococcus aureus develops increased resistance to antibiotics by forming dynamic small colony variants during chronic osteomyelitis.

OBJECTIVES: Staphylococcus aureus osteomyelitis often develops to chronicity despite antimicrobial treatments that have been found to be susceptible in in vitro tests. The complex infection strategies of S. aureus, including host cell invasion and intracellular persistence via the formation of dynamic small colony variant (SCV) phenotypes, could be responsible for therapy-refractory infection courses. METHODS: To analyse the efficacy of antibiotics in the acute and chronic stage of bone infections, we established long-term in vitro and in vivo osteomyelitis models. Antibiotics that were tested include ß-lactams, fluoroquinolones, vancomycin, linezolid, daptomycin, fosfomycin, gentamicin, rifampicin and clindamycin. RESULTS: Cell culture infection experiments revealed that all tested antibiotics reduced bacterial numbers within infected osteoblasts when treatment was started immediately, whereas some antibiotics lost their activity against intracellular persisting bacteria. Only rifampicin almost cleared infected osteoblasts in the acute and chronic stages. Furthermore, we detected that low concentrations of gentamicin, moxifloxacin and clindamycin enhanced the formation of SCVs, and these could promote chronic infections. Next, we treated a murine osteomyelitis model in the acute and chronic stages. Only rifampicin significantly reduced the bacterial load of bones in the acute phase, whereas cefuroxime and gentamicin were less effective and gentamicin strongly induced SCV formation. During chronicity none of the antimicrobial compounds tested showed a beneficial effect on bone deformation or reduced the numbers of persisting bacteria. CONCLUSIONS: In all infection models rifampicin was most effective at reducing bacterial loads. In the chronic stage, particularly in the in vivo model, many tested compounds lost activity against persisting bacteria and some antibiotics even induced SCV formation.

GLUT4 in the endocrine pancreas--indicating an impact in pancreatic islet cell physiology?

The glucose transporter GLUT4 is well known to facilitate the transport of blood glucose into insulin-sensitive muscle and adipose tissue. In this study, molecular, immunohistochemical, and Western blot investigations revealed evidence that GLUT4 is also located in the mouse, rat, and human endocrine pancreas. In addition, high glucose decreased and insulin elevated the GLUT4 expression in pancreatic α-cells. In contrast, high glucose increased GLUT4 expression, whereas insulin led to a reduced expression level of the glucose transporter in pancreatic ß-cells. In vivo experiments showed that in pancreatic tissue of type 2 diabetic rats as well as type 2 diabetic patients, the GLUT4 expression is significantly increased compared to the nondiabetic control group. Furthermore, type 1 diabetic rats exhibited reduced GLUT4 transcript levels in pancreatic tissue, whereas insulin treatment of type 1 diabetic animals enhanced the GLUT4 expression back to control levels. These data provide evidence for the existence of GLUT4 in the endocrine pancreas and indicate a physiological relevance of this glucose transporter as well as characteristic changes in diabetic disease.

The insulin-melatonin antagonism: studies in the LEW.1AR1-iddm rat (an animal model of human type 1 diabetes mellitus).

AIMS/HYPOTHESIS: It is well documented that melatonin influences insulin secretion mediated by G-protein-coupled melatonin receptor isoforms MT1 and MT2, which are present in rat and human pancreatic islets, as well as in rat insulinoma cells. Recent investigations have proven that hyperinsulinaemic Goto-Kakizaki (GK) rats, which are a rat model of type 2 diabetic rats, and humans have decreased melatonin plasma levels, whereas a streptozotocin-induced rat model of diabetes developed reduced insulin levels combined with increased melatonin levels. METHODS: Plasma levels of glucose, insulin and melatonin as well as RNA expression of pineal Aanat, Hiomt (also known as Asmt), insulin receptor, adrenoceptor ß1 and the clock genes Per1 and Bmal1 (also known as Arntl) were determined in male and female LEW.1AR1-iddm rats as well as in insulin-substituted LEW.1AR1-iddm rats. RESULTS: Severe hypoinsulinaemia in diabetic LEW.1AR1-iddm rats was associated with decreased body weight and increased melatonin plasma levels combined with mainly elevated expression of Aanat, Hiomt, pineal insulin receptor and adrenoceptor ß1. The changes were normalised by insulin substitution. Diurnal profiles of plasma melatonin and of antagonistic clock genes Per1 and Bmal1 were maintained in diabetic and insulin-substituted rats. CONCLUSIONS/INTERPRETATION: The assumed causal relation between elevated melatonin and reduced insulin levels in LEW.1AR1-iddm rats is supported by the observation that insulin substitution normalised these changes. Further support for this interpretation comes from the observation that in GK rats an increase of plasma insulin was combined with a decrease of plasma noradrenaline (norepinephrine), the most important activator of melatonin synthesis. These relationships between the noradrenergic and insulin pathway support the existence of melatonin-insulin antagonism.

[Magnetically based enhancement of nanoparticle uptake in tumor cells: combination of magnetically induced cell labeling and magnetic heating]. / Magnetisch basierte Steigerung der Nanopartikelaufnahme in Tumorzellen: Kombination von magnetisch induzierter Zellmarkierung und magnetischer Wärmebehandlung.

PURPOSE: Magnetic nanoparticles (MNP) are known to be versatile tools in diagnostic and interventional radiology. The goal of the present study was to assess whether MNP can be selectively accumulated on human adenocarcinoma cells in vitro using an external magnetic field (magnetically induced cell labeling) and whether these labeled tumor cells can then be destroyed after being exposed to an alternating magnetic field (magnetically induced heating). In this context, a long-term goal is to combine these two developing methods to achieve an additive effect in tumor therapy. MATERIALS AND METHODS: BT-474 cells were incubated until confluence. Magnetic nanoparticles (0.32 mg Fe/ml culture medium) were then added and the flask was exposed to an external magnetic field gradient (magnetically induced cell labeling, 56 or 83 mT magnets) for 24 hours in order to label the tumor cells with nanoparticles. Cells without both MNP and magnetic labeling as well as cells with MNP incubation but without magnetic labeling served as controls. After MNP incubation, the magnetically labeled cells (5 x 10 (7) cells/ml) were exposed to an alternating magnetic field for 5.45 minutes (frequency 400 kHz, amplitude 24.6 kA/m). The combination effect of both magnetic labeling and magnetic heating was assessed by determining the temperature increase. The amount of MNP accumulated within the cells was determined by measuring the iron content via atomic absorption spectrometry. For statistical analysis mean values and standard deviations of temperature increases and iron contents were calculated and the differences were analyzed using the Student's t-test. RESULTS: A significant temperature increase (p < 0.01) during magnetic heating of 41.76 +/- 4.60 K was detected after magnetic labeling of the cells (5 x 10 (7) cells/ml, 83 mT) incubated with MNP. In comparison, the cells incubated with MNP but without magnetic labeling revealed a temperature increase of 32.03 +/- 3.33 K, naked cells of only 2.69 +/- 0.34 K. CONCLUSION: The results demonstrated the magnetically based enhancement of cellular uptake of nanoparticles by tumor cells, resulting in the intensification of the generated temperature increase during magnetic heating. Consequently, magnetic nanoparticles are shown to be valuable tools for the combination of magnetically based therapy modalities.

Antioxidant status in acute stroke patients and patients at stroke risk.

BACKGROUND AND PURPOSE: Antioxidant enzymes like copper/zinc superoxide dismutase (SOD), catalase and gluthatione peroxidase (GSHPx) are part of intracellular protection mechanisms to overcome oxidative stress and are known to be activated in vascular diseases and acute stroke. We investigated the differences of antioxidant capacity in acute stroke and stroke risk patients to elucidate whether the differences are a result of chronic low availability in arteriosclerosis and stroke risk or due to changes during acute infarction. METHODS: Antioxidant enzymes were examined in 11 patients within the first hours and days after acute ischemic stroke and compared to risk- and age-matched patients with a history of stroke in the past 12 months (n = 17). Antioxidant profile was determined by measurement of glutathione (GSH), malondialdehyde (MDA), SOD, GSHPx and minerals known to be involved in antioxidant enzyme activation like selenium, iron, copper and zinc. RESULTS: In comparison to stroke risk patients, patients with acute ischemic stroke had significant changes of the GSH system during the first hours and days after the event: GSH was significantly elevated in the first hours (p < 0.01) and GSHPx was elevated 1 day after the acute stroke (p < 0.05). Selenium, a cofactor of GSHPx, was decreased (p < 0.01). GSHPx levels were negatively correlated with National Institutes of Health Stroke Scale (NIHSS) scores on admission (r = -0.84, p < 0.001) and NIHSS scores after 7 days (r = -0.63, p < 0.05). MDA levels showed a trend for elevation in the first 6 h after the acute stroke (p = 0.07). No significant differences of SOD, iron, copper nor zinc levels could be identified. CONCLUSIONS: Differences of antioxidant capacity were found for the GSH system with elevation of GSH and GSHPx after acute stroke, but not for other markers. The findings support the hypothesis that changes of antioxidant capacity are part of acute adaptive mechanisms during acute stroke.

Recurrent vomiting and ethylmalonic aciduria associated with rare mutations of the short-chain acyl-CoA dehydrogenase gene.

We report identification of short-chain acyl-CoA dehydrogenase (SCAD) deficiency in a 12-year-old boy who suffered from recurrent attacks of vomiting once or twice a year from infancy. Growth and development were normal and there were no muscular symptoms. Metabolic screening was performed during a hospitalization at 8 years of age and revealed an increased excretion of ethylmalonic acid (EMA; 45-80 mmol/mol creatinine, normal 0.2-6.6), suggesting a degradation defect of short-chain fatty acids. An increased n-butyrylcarnitine was found in freshly collected serum (0.9 micromol/L; normal <0.4) but not in dry blood spots. Neither of the frequent SCAD gene variants 625G>A and 511C>T was present, but direct sequencing of the promoter and coding regions of the SCAD gene revealed that the patient had mutations on both alleles: 417G>C (Trpl15Cys) and 1095G>T (Gln341His). Neither mutation has been described before in compound heterozygosity or homozygosity. Enzymatic investigations subsequently confirmed a defect of SCAD in both fibroblasts and muscle extracts. Furthermore, expression studies of both mutations demonstrated impaired enzyme function or structure. To our knowledge, this case is the first description of a patient with proven SCAD deficiency presenting with recurrent emesis but without other symptoms, and emphasizes the wide clinical phenotype of this disorder.

[Reference ranges of antioxidant parameters in whole blood (erythrocytes) in a Thüringen region]. / Referenzbereiche antioxidativ wirkender Parameter im Vollblut (Erythrozyten) einer Thüringer Region.

BACKGROUND: The oxidant stress is characterized by measurement of the activities of glutathione peroxidase, superoxiddismutase and also by concentrations of glutathione and selenium in erythrocytes. A standardization of the methods of determination is very important. MATERIAL AND METHODS: In erythrocytes of blood donors (n = 101) the parameters glutathione peroxidase, glutathione, superoxiddismutase and selenium were determined. RESULTS: The following results of the antioxidant parameters in erythrocytes of blood donors were found: Selenium 67.1 +/- 20.1 nmol/mmol Hb, glutathione peroxidase 842 +/- 290 U/mmol Hb, glutathione 108 +/- 48 mumol/mmol Hb, superoxiddismutase 15.8 +/- 6.4 U/mumol Hb. CONCLUSION: Selenium, glutathione peroxidase, glutathione and superoxiddismutase in erythrocytes of blood donors are normally distributed. There are no significant differences between men and women. The use of "own reference values" is necessary because no standardization of the methods of determination exists.

[Sex hormones and antioxidant status]. / Sexualhormone und Antioxidanzienstatus.

Physiologically, an influence of sex steroids on the antioxidative capacity can be seen at least as a short-term effect. First of all the steroid effects are due to the estrogen component rather than the progestin component. The antioxidative potential of natural estrogens is several times higher than that of vitamin E. Long-term studies should be performed to verify this in perimenopausal hormone replacement.