Genetic variants of homocysteine metabolizing enzymes and the risk of coronary artery disease

It is unresolved whether elevated homocysteine in coronary artery disease (CAD) is the cause of arteriosclerosis or its consequence. In contrast, genetic variants of enzymes that metabolize homocysteine cannot be altered by arteriosclerosis. Consequently, their association with CAD would permit to imply causality. We modeled by regression analysis the effect of 11 variants in the methionine cycle upon CAD manifestation in 591 controls and 278 CAD patients. Among the examined variants only the carriership for the c.844ins68 in the cystathionine beta-synthase (CBS) gene was associated with a significantly lowered risk of CAD (OR=0.56; 95% CI=0.35-0.90 in the univariable, and OR=0.41, 95% CI=0.19-0.89 for obese people in the multivariable analysis, respectively). Healthy carriers of the c.844ins68 variant exhibited, compared to the wild type controls, significantly higher postload ratios of blood S-adenosylmethionine to S-adenosylhomocysteine (61.4 vs. 54.9, p=0.001) and of plasma total cysteine to homocysteine (8.6 vs. 7.3, p=0.004). The changes in these metabolites are compatible with an improved methylation status and with enhanced activity of homocysteine transsulfuration. In conclusion, the coincidence of clinical and biochemical effects of a common c.844ins68 CBS variant supports the hypothesis that compounds relating to homocysteine metabolism may play role in the development and/or progression of CAD.     

Characterization of the in vitro metabolic profile of amlodipine in rat using liquid chromatography-mass spectrometry.

In the present study, the metabolic profile of amlodipine, a well-known calcium channel blocker, was investigated employing liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers - a triple-quadrupole (QqQ) and a quadrupole time-of-flight (Q-TOF) mass spectrometer - were utilized to acquire structural information on amlodipine metabolites. The metabolites were produced by incubation of amlodipine with primary cultures of rat hepatocytes. Incubations from rat hepatocytes were analyzed with LC-MS/MS, and 21 phase I and phase II metabolites were detected. Their product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of these metabolites. Mainly phase I metabolic changes were observed including dehydrogenation of the dihydropyridine core, as well as reactions of side chains, such as hydrolysis of ester bonds, hydroxylation, N-acetylation, oxidative deamination, and their combinations. The only phase II metabolite detected was the glucuronide of a dehydrogenated, deaminated metabolite of amlodipine. We propose several in vitro metabolic pathways of amlodipine in rat, based on our analysis of the metabolites detected and characterized.     

Molecular epidemiology of tuberculosis in the Czech Republic, 2004: analysis of M. tuberculosis complex isolates originating from the city of prague, south Moravia and the Moravian-Silesian region

OBJECTIVES: To compare M. tuberculosis complex genotypes from representative regions of the Czech Republic in order to estimate changes in strain prevalence and in the extent of imported drug-resistant tuberculosis. METHODS: Primary M. tuberculosis complex isolates (n=155) and follow-up isolates (n=15) from 155 patients from the first half of 2004 (98 from Prague, 37 from South Moravia and 35 from the Moravian-Silesian region) were genotyped by IS6110-RFLP, spoligotyping, and partly by VNTR-genotyping. RESULTS: Based on IS6110-RFLP, 110 of 155 (71%) primary isolates were unique. Forty-five isolates (29%) were found in 15 clusters comprising two to six patients and all but one cluster were also discriminated by MIRU-VNTR-genotyping. Four clusters comprised patients from different regions, and six were ongoing for several years. An indication of MDR-strain transmission was found in one instance. All four Beijing-type isolates with any resistance were associated with immigration from Eastern Europe. CONCLUSIONS: The molecular epidemiological data of this period-prevalence, population based study and its comparison to earlier investigations point to a low extent of clustering between M. tuberculosis complex isolates in representative regions of the Czech Republic. Few clusters extending geographically and/or over several years were identified, providing a means for an in-depth analysis of risk factors of transmission. Beijing genotype isolates were shown to increase in prevalence to reach 6.5%. Drug resistant isolates of this genotype were associated with immigration of from Eastern Europe, although direct transmission of a resistant isolate was probable only in one of eleven cases.     

Synchronous gastric and sebaceous cancers,a rare manifestation of MLH1-related Muir-Torre syndrome

Muir-Torre syndrome (MTS), a rare variant of the hereditary non polyposis colorectal cancer syndrome, is an autosomal dominant genodermatosis characterised by coincidence of sebaceous gland neoplasms (sebaceous adenoma, epithelioma, or carcinoma) and at least one internal malignancy. The underlying cause of MTS is a germline mutation in DNA mismatch repair genes MSH2, MLH1 and MSH6. We report the case of a 52-year-old caucasian woman with the development of metachronous colon cancer at the age of 38 years, uterine cancer at the age of 43 years, and unique occurrence of synchronous gastric and sebaceous carcinomas related to germline point mutation c. 2194A>T in the last exon of MLH1 gene, resulting in truncated protein in C-terminal region p. Lys732X due to premature stop codon. This mutation, not previously reported in MTS, disrupts the function of MutL complexes presumably by preventing the interaction with PMS1/PMS2 and impairing the endonuclease active site. This case points out the importance of sebaceous neoplasia, especially sebaceous adenocarcinoma, as cutaneous markers of MTS for timely implementation of cancer screening programs.     

Rare allelic variants determine folate status in an unsupplemented European population

The role of folates as coenzymes in 1-carbon metabolism and the clinical consequences of disturbed folate metabolism are widely known. Folate status is a complex trait determined by both exogenous and endogenous factors. This study analyzed the association between 12 genetic variants and folate status in a Czech population with no folate fortification program. These 12 genetic variants were selected from 56 variant alleles found by resequencing the coding sequences and adjacent intronic regions of 6 candidate genes involved in folate metabolism or transport (FOLR1, FOLR2, FOLR3, MTHFR, PCFT, and RFC) from 29 individuals with low plasma and erythrocyte folate concentrations. Regression analyses of a cohort of 511 Czech controls not taking folate supplements revealed that only 2 variants in the MTHFR gene were associated with altered folate concentrations in plasma and/or erythrocytes. In our previous study, we observed that the common variant MTHFR c.665C > T (known as c.677C > T; p.A222V) was associated with decreased plasma folate concentrations. In the present study, we show in addition that the rare variant MTHFR c.1958C > T (p.T653M) is associated with significantly increased erythrocyte folate concentrations (P = 0.02). Multivariate regression analysis revealed that this uncommon variant, which is present in 2% of Czech control chromosomes, explains 0.9% of the total variability of erythrocyte folate concentrations; the magnitude of this effect size was comparable with that of the common MTHFR c.665C > T variant. This result indicates that the rare genetic variants may determine folate status to a similar extent as the common allelic variant.     

Cytotoxicity of copper(II) complexes of N-salicylidene-L-glutamate: modulation by ascorbic acid

Cytotoxic/cytostatic activity of N-salicylidene-L-glutamato diaqua copper(II) complex (CuC) against mice leukemia cells L1210 has been estimated and their bioactivity was enhanced by addition of ascorbic acid. The Cu-complex with isoquinoline ligand (IQ-CuC) had stronger cytostatic effect (IC50 =15.6 microM) than parental complex (CuC) and its cytotoxicity several times increased in the presence of 0.1 mM ascorbic acid (IC50 =1.0 microM). The cytotoxicity has been caused by oxidative stress, enhanced creation of TBARS has been confirmed, and formation of 2',7'-dichlorofluorescein from 2',7'- dichlorodihydrofluorescein has been observed, also. Some hallmarks of apoptotic/necrotic death of L1210 cells have been observed by fluorescent microscopy after dyeing of cell with propidium iodide and Hoechst 33342. In addition, it was confirmed that both complexes in the presence of ascorbic acid cleavaged of pDNA. Although these copper complexes were initially prepared as substances with antioxidant properties we have showed that combined treatment of L1210 cells with IQCuC and ascorbic acid induced strong oxidative stress and death of cells. Our results confirmed that physiological concentration of ascorbic acid increases the cytostatic/cytotoxic efficiency of N-salicylidene-L-glutamato diaqua copper(II) complexes.     

The stereoselective biotransformation of the anti-obesity drug sibutramine in rat liver microsomes and in primary cultures of rat hepatocytes

Sibutramine is an anti-obesity drug sold as a racemic mixture under the trademark Meridia or Reductil. With the aim of evaluating the stereoselectivity in phase I of sibutramine biotransformation, the formation of the main metabolites from R -sibutramine, S -sibutramine and rac-sibutramine was studied in rat microsomes and primary cultures of hepatocytes. A novel analytical method for the determination of sibutramine and its phase I metabolites in culture medium and microsomal incubates using isocratic reversed-phase liquid chromatography with UV detection was developed. Only two metabolites, mono-desmethylsibutramine (M1) and di-desmethylsibutramine (M2), were found in the rat microsomes incubated with sibutramine and NADPH. The kinetics of M1 and M2 formation slightly differed depending on the enantiomeric form of the sibutramine used. The stereoselectivity in sibutramine biotransformation was much more evident in primary cultures of rat hepatocytes. While R-sibutramine incubation led to the formation of M1 and M2 metabolites only, the incubation of S-sibutramine or rac-sibutramine (to a lesser extent) resulted in four major metabolites (M1, M2, M3 and M4) and 2 or 3 minor metabolites. On the basis of our results, R-sibutramine might represent the more advantageous sibutramine enantiomer from the pharmacokinetic standpoint.     

Effects of phenylalkylamines and benzothiazepines on Ca(v)1.3-mediated Ca2+ currents in neonatal mouse inner hair cells

Calcium currents (I(Ca)) in inner hair cells (IHCs) are carried by the Ca(v)1.3 subtype of L-type calcium channels. They play an important role in synaptic transmission of sound-evoked mechanical stimuli. L-type calcium channels are targets of the organic blocker classes dihydropyridines, phenylalkylamines and benzothiazepines. Previously a low sensitivity of the Ca(v)1.3 subtype towards dihydropyridines has been demonstrated. Therefore, this study evaluates the effect of two phenylalkylamines (verapamil and gallopamil) and the benzothiazepine diltiazem on I(Ca) through Ca(v)1.3 channels in mouse IHCs. Whole-cell I(Ca) was measured using the patch-clamp technique in mouse IHCs aged postnatal day 3-7 with 5 mM calcium as a charge carrier. The phenylalkylamines verapamil and gallopamil and the benzothiazepine diltiazem inhibited I(Ca) in IHCs in a concentration-dependent manner. This block was largely reversible. Dose-response curves revealed IC(50) values of 199+/-19 microM for verapamil, 466+/-151 microM for gallopamil and 326+/-67 microM for diltiazem. The inhibition of peak I(Ca) by phenylalkylamines and benzothiazepines was voltage-independent. Verapamil (300 microM) enhanced current inactivation from -20 to +20 mV while diltiazem (300 microM) did so only at very depolarised potentials (+20 mV). In conclusion, the concentrations of phenylalkylamines and benzothiazepine necessary to inhibit 50% of I(Ca) in IHCs were one order larger compared to concentrations which inhibited I(Ca) through Ca(v)1.2 channels in native cells or expression systems. However, inhibitory concentrations were in the same range as those required for block of I(Ca) in turtle hair cells.