Polymorphisms in the CBS gene and homocysteine, folate and vitamin B12 levels: association with polymorphisms in the MTHFR and MTRR genes in Brazilian children.

Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and cystathionine beta-synthase (CBS) genes, involved in the intracellular metabolism of homocysteine (Hcy), can result in hyperhomocysteinemia. The objective of this study was to evaluate prevalence estimates of CBS T833C, G919A and the insertion of 68-bp (844ins68) polymorphisms and their correlation with Hcy, folate and B(12) in 220 children previously genotyped for MTHFR C677T, A1298C, and MTRR A66G. The prevalence of heterozygote children for 844ins68 was 19.5%. The T833C CBS mutation was identified in association with 844ins68 in all the carriers of the insertion. Genotyping for CBS G919A mutation showed that all the children presented the GG genotype. Analysis of Hcy, B(12) and folate, according to the combination of the different genotypes of the C677T and A1298C MTHFR, A66G MTRR, and 844ins68 CBS showed that the 677TT/1298AA/68WW genotype is associated with an increase in Hcy, when compared to the 677CC/1298AC/68WW (P = 0.033) and the 677CT/1298AA/68WW genotypes (P = 0.034). Since B(12) and folate were not different between these groups, a genetic interaction between diverse polymorphisms probably influences Hcy. Our results emphasize the role of genetic interactions in Hcy levels.

Polymorphism C776G in the transcobalamin II gene and homocysteine, folate and vitamin B12 concentrations. Association with MTHFR C677T and A1298C and MTRR A66G polymorphisms in healthy children.

One of the etiologies of hyperhomocysteinemia is decreased vitamin B(12). Genetic variation in the transcobalamin II gene, the transporter of vitamin B(12) to the cells, may produce altered homocysteine levels. We determined transcobalamin II C776G polymorphism, homocysteine, folate and vitamin B(12) levels and analyzed the interactive effect with the methylenetetrahydrofolate reductase C677T and A1298C and methionine synthase reductase A66G polymorphisms in 207 healthy Brazilian children. The prevalence of GG genotype of transcobalamin II C776G polymorphism in this Brazilian population, a highly miscigeneous population was 12.5% and the statistical analysis showed that this population is in Hardy-Weinberg equilibrium, it could be considered representative of the general population. We observed a significant increase in homocysteine in the 776GG vs. 776CC genotype, corroborating the influence of age as a determinant of homocysteine in relation to this polymorphism. When we analyzed vitamin B(12) and its relationship with the C776G polymorphism, we found no significant differences. Only 776CG/66AA or 776GG/66AG genotypes presented a significant increase in homocysteine when compared with other groups. In the multivariate analysis, transcobalamin II C776G (CC/CG vs. GG), methylenetetrahydrofolate reductase C677T (CC/CT vs. TT), folate, gender and age presented statistical significance in relation to the homocysteine. These can be considered independent risk factors for hyperhomocysteinemia in this children group. Our results, if confirmed in other populations, highlight the necessity for investigation of the transcobalamin II C776G polymorphism in the research for hyperhomocysteinemia risk factors.

Paper-based microfluidic devices on the crime scene: A simple tool for rapid estimation of post-mortem interval using vitreous humour.

This paper describes for the first time the use of paper-based analytical devices at crime scenes to estimate the post-mortem interval (PMI), based on the colorimetric determination of Fe2+ in vitreous humour (VH) samples. Experimental parameters such as the paper substrate, the microzone diameter, the sample volume and the 1,10-phenanthroline (o-phen) concentration were optimised in order to ensure the best analytical performance. Grade 1 CHR paper, microzone with diameter of 5 mm, a sample volume of 4 µL and an o-phen concentration of 0.05 mol/L were chosen as the optimum experimental conditions. A good linear response was observed for a concentration range of Fe2+ between 2 and 10 mg/L and the calculated values for the limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 0.9 mg/L, respectively. The specificity of the Fe2+ colorimetric response was tested in the presence of the main interfering agents and no significant differences were found. After selecting the ideal experimental conditions, four HV samples were investigated on paper-based devices. The concentration levels of Fe2+ achieved for samples #1, #2, #3 and #4 were 0.5 ± 0.1, 0.7 ± 0.1, 1.2 ± 0.1 and 15.1 ± 0.1 mg/L, respectively. These values are in good agreement with those calculated by ICP-MS. It important to note that the concentration levels measured using both techniques are proportional to the PMI. The limitation of the proposed analytical device is that it is restricted to a PMI greater than 1 day. The capability of providing an immediate answer about the PMI on the crime scene without any sophisticated instrumentation is a great achievement in modern instrumentation for forensic chemistry. The strategy proposed in this study could be helpful in many criminal investigations.

Insulin-loaded poly(epsilon-caprolactone) nanoparticles: efficient, sustained and safe insulin delivery system.

The aim of this work was to develop an efficient, biodegradable, biocompatible and safe controlled release system using insulin-loaded poly(epsilon-caprolactone) (PCL) nanoparticles. The insulin-loaded PCL nanoparticles were prepared by double emulsion method (water-in-oil-in-water) using Pluronic F68 as emulsifier. Using the double emulsion method a high insulin encapsulation efficiency (90.6 +/-1.6%) with a zeta potential of -29 +/-2.7 mV and average particle size of 796 +/-10.5 nm was obtained. Insulin-loaded PCL nanoparticles showed no toxicity to MIN6 cells. Insulin nanoparticles administered subcutaneously and intraperitoneally in rats reduced glycaemia of basal levels after 15 minutes, and presented a sustainable hypoglycemic effect on insulin-dependent type 1 diabetic rats, showing to be more efficient than unencapsulated insulin. Furthermore, these nanoparticles were not hepatotoxic, as evaluated by the effect over liver cell-death and oxidative stress scavenger system in rats. These results suggest that insulin-loaded PCL nanoparticles prepared by water-in-oil-in-water emulsion method are biocompatible, efficient and safe insulin-delivering system with controlled insulin release, which indicates that it may be a powerful tool for insulin-dependent patients care.

Blood calcium levels in immature rats: influence of extracellular calcium concentration on myocardial calcium handling.

Calcium ions play an important role in several cell functions, from fertilization to cell death. The cytosolic Ca(2+) concentration is much lower than the extracellular concentration ([Ca(2+)](o)). The latter may markedly affect Ca(2+) fluxes across the cell membrane and thus the cellular Ca(2+) load. Thus, when working with preparations in vitro, it is important to keep [Ca(2+)](o) close to the in vivo value. In this study, we determined the calcemia in immature rats, for which values are currently unavailable, and investigated how supraphysiological [Ca(2+)](o) affects myocardial Ca(2+) handling. Blood ionized [Ca(2+)] was similar in neonatal (2-5 days old) and adults Wistar rats (1.28 ± 0.03 and 1.31 ± 0.03 mmol/l; n=6 and 5, respectively, P>0.37), and lower than the [Ca(2+)](o) range often used in experiments with neonatal myocardial preparations. Cytosolic Ca(2+) transients, measured with indo-1 in neonatal ventricular myocytes, were enhanced by an increase in [Ca(2+)](o) from 1.2 to 2 mM, which also increased the Ca(2+) content in the sarcoplasmic reticulum (SR), and changed the pattern of competition between the main transporters that remove Ca(2+) from the cytosol (SR Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger). These observations stress the importance of using physiological [Ca(2+)](o) values for reliability of results. It is expected that the present calcemia data, reported for the first time in immature rats, may contribute to the refinement of in vitro experiments with neonatal rat preparations.

Direct monitoring of drug degradation by easy ambient sonic-spray ionization mass spectrometry: the case of enalapril.

Using enalapril maleate as a test case, the ability of ambient mass spectrometry, namely, via easy ambient sonic-spray ionization mass spectrometry (EASI-MS), to perform direct monitoring of drug degradation has been tested. Two manufacturing processes were investigated (direct compression and wet granulation), and the formation of degradation products was measured via both EASI-MS and high-performance liquid chromatography with ultraviolet detection for a total period of 18 months. Both techniques provide comparable results, which indicate that direct analysis by ambient mass spectrometric techniques presents a viable alternative for drug degradation monitoring with superior simplicity, throughput, and reliability (no sample manipulation), and comparable quantitative results. In terms of qualitative monitoring, the full mass spectra with intact species provided by EASI-MS allow for comprehensive monitoring of known and unknown (or unexpected) degradation products.

New substituted 4-arylaminoquinazolines as potent inhibitors of breast tumor cell lines: in vitro and docking experiments.

The arylquinazolines represent significant advances in the clinical management of breast cancer. Nevertheless some confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety and clinical use. Two 4-arylaminoquinazoline derivatives, recently synthesized, were tested as kinase inhibitors and their citotoxicities showed potent growth inhibitory activity in breast tumor cell lines (MCF-7). The predicted complex structure of quinazoline inhibitors with EGFR protein from molecular docking provided a stereoview of the binding site correlated with structure activity, affording important information about structure-based drug design.

Combined cysteine and homocysteine quantitation in plasma by trap and release membrane introduction mass spectrometry.

Recently, a new and efficient method for total homocysteine (tHcy) quantitation in plasma using trap and release membrane introduction mass spectrometry (T&R-MIMS) with a versatile removable direct introduction membrane probe (DIMP) was described [R. Haddad, M. A. Mendes, N. F. Hoehr and M. N. Eberlin, Analyst, 2001, 126, 1212]. Herein we report on the use of the DIMP-T&R-MIMS technique for total cysteine (tCys) quantitation; hence combined tCys and tHcy quantitation in plasma or serum can be achieved. The method employs Cys and Hcy derivatization with ethyl chloroformate (after disulfide bond reduction with dithiothreitol and protein precipitation with trichloroacetic acid), preconcentration in a capillary silicone membrane, and their thermal desorption to the gas phase inside the ion source region of a mass spectrometer, at a point exactly between the two ionization filaments. Thermal desorption uses the uniform heat radiation provided by the two ionization filaments. The analytes are then ionized by electron ionization and both Cys and Hcy are quantitated by mass spectrometry using selected ion monitoring. For tCys quantitation, good linearity and reproducibility was observed for concentrations ranging from 5 to 350 microM, recovery was near 95%, and the limit of detection (LOD) was of 2 microM. This LOD is well below the mean Cys concentration in plasma, and serum samples from a large group of healthy people showed a mean tCys concentration of 132 +/- 45 microM.

Fast screening of low molecular weight compounds by thin-layer chromatography and "on-spot" MALDI-TOF mass spectrometry.

Fast screening of low-MW compounds is performed by thin-layer chromatography (TLC) followed by direct on-spot matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification with nearly "matrix-free" mass spectra using an UV-absorbing ionic liquid matrix. Owing to minimal background ions from the proton donor triethylamine/alpha-cyano-4-hydroxycinnamic acid ionic liquid matrix, three arborescidine alkaloids, the anesthesics levobupivacaine and mepivacaine, and the antibiotic tetracycline were readily characterized most frequently by the MS detection of their protonated molecules. The technique is fast and sensitive, requires little sample preparation and manipulation, and is therefore suitable for fast screening with TLC separation and MS identification of low-MW compounds, with potential applications in areas such as phytochemistry, synthetic chemistry, and product manufacturing quality monitoring.

Chronic methionine load-induced hyperhomocysteinemia enhances rat carotid responsiveness for angiotensin II.

The purpose of the present study was to investigate the effects of chronic methionine treatment on vascular smooth muscle contractility for angiotensin II (Ang II). Methionine at 0.1, 1 and 2 g/kg body weight was administered daily in the drinking water for 2, 4, 8 and 16 weeks. Rat carotid rings from control and treated animals were placed in an organ chamber containing Krebs solution. Concentration-response curves for Ang II and potassium chloride (KCl) were determined. Methionine-rich diet enhanced the plasmatic homocysteine concentration, and the magnitude of the contractile response to Ang II was increased in carotid rings from treated animals after 8 and 16 weeks. However, the treatment did not alter KCl-induced contraction. In another set of experiments, the rings were incubated with indomethacin and curves for Ang II were obtained. Exposure of the rings to indomethacin inhibited the enhancement in the contractile response to Ang II. The present findings show that chronic methionine treatment enhances homocysteine plasmatic concentration leading to an enhanced Ang-II-induced contraction, which appears to be related to the release of vasoconstrictor prostanoid(s).