Hydroalcoholic leaves extract of Vaccinium ashei Reade promotes cell cycle arrest and apoptosis on T-cell acute lymphoblastic leukemia.

Vaccinium ashei Reade, popularly known as Rabbiteye blueberry, has several therapeutic properties attributed to the phenolic compounds present in its leaves and fruits. Here, we sought to evaluate the effects of the hydroalcoholic extract from V. ashei leaves (Bluegem cultivar, VAB) in T-cell Acute lymphoblastic leukemia (T-ALL). The VAB extract was toxic to T-ALL cells at the ∼60 µg/ml concentration. T-ALL cell death occurred through apoptosis. VAB extract was found to induce micronuclei formation, p53 pathway activation, and cell cycle arrest. Those mutagenic effects were evidenced through microscopy analysis and molecular p53 pathway activation. A series of phenolic compounds were identified in VAB extract by mass spectrometry, such as vanillic acid, catechin, caffeic acid, chlorogenic acid, rutin, coumaric acid, taxifolin, quercetin and naringenin, some of which are presumed to induce DNA damage. In conclusion, the V. ashei leaves extract may have important secondary metabolites with antileukemic properties.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present study aimed to identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

Validation of direct method to quantify dexamethasone in human aqueous humor by LC-MS/MS.

AIM: Dexamethasone (Dex) has been used for the treatment of ocular diseases, presenting concentrations up to 150 ng/ml in the biological matrix. Drug sampling has been performed from aqueous humor (AH), which the volume varies from 50 to 200 µl, becoming a challenge for analytical analyses. RESULTS: We developed and validated a direct and sensitive method by LC-MS/MS for Dex measurement in human AH, using water as surrogate matrix to reduce the sample volume applied in some validation assays. With accuracies of 99.6% and precision within 15% in the range of 0.1-150 ng/ml, Dex had an 87-day stability. CONCLUSION: Our method is robust and sensitive enough to be applied in bioequivalence studies with human AH using only 20 µl of biological sample.

High sensitivity method validated to quantify estradiol in human plasma by LC-MS/MS.

17ß-Estradiol (E2) is an endogenous steroid in the human body. Its measurement is important for health and human biology understanding. However, E2 concentration in human plasma is in the range of pg/mL, which makes it difficult to detect. In this way, LC-MS/MS has been shown the most sensitive tool, although E2 is a weakly ionizable molecule. In this work, we validated a more sensitive and accurate method for E2 quantification in human plasma. Our extraction step ensured a cleaner chromatography, resulting in a precise measurement and highly reproducible method in the range of 2-150pg/mL. Moreover, we proved a long stability for E2 in several conditions. All results indicate that our developed method is robust and sensitive enough to apply in bioequivalence studies for E2 measurement in human plasma, even at very low concentrations.

Small molecule specific run acceptance, specific assay operation, and chromatographic run quality assessment: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization teams.

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.

Stability: recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

This paper provides a comprehensive overview of stability-related aspects of quantitative bioanalysis and recommends science-based best practices, covering small and large molecules as well as chromatographic and ligand-binding assays. It addresses general aspects, such as the use of reference values, transferability and treatment of failing stability results, and also focuses on specific types of stability assessment: bench-top, freeze/thaw and long-term frozen stability, stock stability, extract stability, stability in whole blood, tissue and urine, and stability of endogenous analytes, in special matrix types and in incurred samples.

Baseline resolution of isomers by traveling wave ion mobility mass spectrometry: investigating the effects of polarizable drift gases and ionic charge distribution.

We have studied the behavior of isomers and analogues by traveling wave ion mobility mass spectrometry (TWIM-MS) using drift-gases with varying masses and polarizabilities. Despite the reduced length of the cell (18 cm), a pair of constitutional isomers, N-butylaniline and para-butylaniline, with theoretical collision cross-section values in helium (ΩHe ) differing by as little as 1.2 Å(2) (1.5%) but possessing contrasting charge distribution, showed baseline peak-to-peak resolution (Rp-p ) for their protonated molecules, using carbon dioxide (CO2), nitrous oxide (N2O) and ethene (C2H4 ) as the TWIM drift-gas. Near baseline Rp-p was also obtained in CO2 for a group of protonated haloanilines (para-chloroaniline, para-bromoaniline and para-iodoaniline) which display contrasting masses and theoretical ΩHe , which differ by as much as 15.7 Å(2) (19.5%) but similar charge distributions. The deprotonated isomeric pair of trans-oleic acid and cis-oleic acid possessing nearly identical theoretical ΩHe and ΩN2 as well as similar charge distributions, remained unresolved. Interestingly, an inversion of drift-times were observed for the 1,3-dialkylimidazolium ions when comparing He, N2 and N2O. Using density functional theory as a means of examining the ions electronic structure, and He and N2-based trajectory method algorithm, we discuss the effect of the long-range charge induced dipole attractive and short-range Van der Waals forces involved in the TWIM separation in drift-gases of differing polarizabilities. We therefore propose that examining the electronic structure of the ions under investigation may potentially indicate whether the use of more polarizable drift-gases could improve separation and the overall success of TWIM-MS analysis.

DHFR 19-bp deletion and SHMT C1420T polymorphisms and metabolite concentrations of the folate pathway in individuals with Down syndrome.

BACKGROUND: Down syndrome (DS) results from the presence and expression of three copies of the genes located on chromosome 21. Studies have shown that, in addition to overexpression of the Cystathionine ß-synthase (CBS) gene, polymorphisms in genes involved in folate/homocysteine (Hcy) metabolism may also influence the concentrations of metabolites of this pathway. AIM: Investigate the association between Dihydrofolate reductase (DHFR) 19-base pair (bp) deletion and Serine hydroxymethyltransferase (SHMT) C1420T polymorphisms and serum folate and plasma Hcy and methylmalonic acid (MMA) concentrations in 85 individuals with DS. METHODS: Molecular analysis of the DHFR 19-bp deletion and SHMT C1420T polymorphisms was performed by polymerase chain reaction (PCR) by difference in the size of fragments and real-time PCR allelic discrimination, respectively. Serum folate was quantified by chemiluminescence and plasma Hcy and MMA by liquid chromatography-tandem mass spectrometry. RESULTS: Individuals with DHFR DD/SHMT TT genotypes presented increased folate concentrations (p=0.004) and the DHFR II/SHMT TT genotypes were associated with increased MMA concentrations (p=0.008). In addition, the MMA concentrations were negatively associated with age (p=0.04). CONCLUSION: There is an association between DHFR DD/SHMT TT and DHFR II/SHMT TT combined genotypes and folate and MMA concentrations in individuals with DS.

Polymorphism C1420T of Serine hydroxymethyltransferase gene on maternal risk for Down syndrome.

Recent researches have investigated the factors that determine the maternal risk for Down syndrome (DS) in young woman. In this context, some studies have demonstrated the association between polymorphisms in genes involved on folate metabolism and the maternal risk for DS. These polymorphisms may result in abnormal folate metabolism and methyl deficiency, which is associated with aberrant chromosome segregation leading to trisomy 21. In this study, we analyzed the influence of the polymorphism C1420T in Serine hydroxymethyltransferase (SHMT) gene on maternal risk for DS and on metabolites concentrations of the folate pathway (serum folate and plasma homocysteine and methylmalonic acid). The study group was composed by 105 mothers with DS children (case group) and 185 mothers who had no children with DS (control group). The genotype distribution did not show significant statistical difference between case and control mothers (P = 0.24) however a protective effect between genotypes CC (P = 0.0002) and CT (P < 0.0001) and maternal risk for DS was observed. Furthermore, the SHMT C1420T polymorphism (rs1979277) does not affect the concentration of metabolites of folate pathway in our DS mothers. In conclusion, our data showed a protective role for the genotypes SHMT CC and CT on maternal risk for DS. The concentrations of metabolites of folate pathway did not differ significantly between the genotypes SHMT.

Grape juice concentrate prevents oxidative DNA damage in peripheral blood cells of rats subjected to a high-cholesterol diet.

The goal of the present study was to investigate whether subchronic treatment with grape juice concentrate is able to protect liver and peripheral blood cells against cholesterol-induced injury in rats. The effects of the grape juice concentrate treatment on histopathological changes, immunohistochemistry for cyclo-oxygenase-2 (COX-2), and basal and oxidative DNA damage induced by H2O2 using a single-cell gel (comet) assay were evaluated. Male Wistar rats (n 18) were divided into three groups: group 1--negative control; group 2--cholesterol at 1 % (w/w) in their diet, treated for 5 weeks; group 3--cholesterol at 1 % in their chow, treated for 5 weeks, and grape juice concentrate at 222 mg/d in their drinking-water in the final week only. The results indicated that the treatment with grape juice concentrate did not show remarkable differences regarding liver tissue in group 3 compared with group 2. However, grape juice concentrate was able to decrease oxidative DNA damage induced by H2O2 in peripheral blood cells, as depicted by the tail moment results. COX-2 expression in the liver did not show statistically significant differences (P>0·05) between groups. Taken together, the present results suggest that the administration of subchronic grape juice concentrate prevents oxidative DNA damage in peripheral blood cells.

Antioxidant potential of rat plasma by administration of freeze-dried jaboticaba peel (Myrciaria jaboticaba Vell Berg).

The effect of the intake of freeze-dried jaboticaba peel powder on the antioxidant potential of rats' blood plasma was investigated in two experiments. In the first, 35 male rats, divided into 5 groups, received 7 mg of anthocyanins/100 g of body weight, by gavage. The blood was withdrawn 0, 30, 60, and 120 min after gavage. The antioxidant capacity was evaluated in plasma by ORAC and TEAC. There was no significant difference in the plasmatic antioxidant potential among the groups. In the second experiment, 40 male rats were divided into 4 groups that consumed, respectively, 0, 1, 2, and 4% of freeze-dried jaboticaba peel powder added to their diet. An increase in the plasmatic antioxidant potential was observed for groups that received 1 and 2% of jaboticaba peel powder (1.7 times by TEAC method and 1.3 times by ORAC); however, the group that received 4% of the powder did not show antioxidant effects according to the methods used.

Visualizing inhibition of fatty acid synthase through mass spectrometric analysis of mitochondria from melanoma cells.

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria.

19-base pair deletion polymorphism of the dihydrofolate reductase (DHFR) gene: maternal risk of Down syndrome and folate metabolism.

CONTEXT AND OBJECTIVE: Polymorphisms in genes involved in folate metabolism may modulate the maternal risk of Down syndrome (DS). This study evaluated the influence of a 19-base pair (bp) deletion polymorphism in intron-1 of the dihydrofolate reductase (DHFR) gene on the maternal risk of DS, and investigated the association between this polymorphism and variations in the concentrations of serum folate and plasma homocysteine (Hcy) and plasma methylmalonic acid (MMA). DESIGN AND SETTING: Analytical cross-sectional study carried out at Faculdade de Medicina de São José do Rio Preto (Famerp). METHODS: 105 mothers of individuals with free trisomy of chromosome 21, and 184 control mothers were evaluated. Molecular analysis on the polymorphism was performed using the polymerase chain reaction (PCR) through differences in the sizes of fragments. Folate was quantified by means of chemiluminescence, and Hcy and MMA by means of liquid chromatography and sequential mass spectrometry. RESULTS: There was no difference between the groups in relation to allele and genotype frequencies (P = 0.44; P = 0.69, respectively). The folate, Hcy and MMA concentrations did not differ significantly between the groups, in relation to genotypes (P > 0.05). CONCLUSIONS: The 19-bp deletion polymorphism of DHFR gene was not a maternal risk factor for DS and was not related to variations in the concentrations of serum folate and plasma Hcy and MMA in the study population.

19-base pair deletion polymorphism of the dihydrofolate reductase (DHFR) gene: maternal risk of Down syndrome and folate metabolism / Polimorfismo de deleção de 19 pares de bases do gene dihidrofolato redutase (DHFR): risco materno para síndrome de Down e metabolismo do folato

CONTEXT AND OBJECTIVE: Polymorphisms in genes involved in folate metabolism may modulate the maternal risk of Down syndrome (DS). This study evaluated the influence of a 19-base pair (bp) deletion polymorphism in intron-1 of the dihydrofolate reductase (DHFR) gene on the maternal risk of DS, and investigated the association between this polymorphism and variations in the concentrations of serum folate and plasma homocysteine (Hcy) and plasma methylmalonic acid (MMA). DESIGN AND SETTING: Analytical cross-sectional study carried out at Faculdade de Medicina de São José do Rio Preto (Famerp). METHODS: 105 mothers of individuals with free trisomy of chromosome 21, and 184 control mothers were evaluated. Molecular analysis on the polymorphism was performed using the polymerase chain reaction (PCR) through differences in the sizes of fragments. Folate was quantified by means of chemiluminescence, and Hcy and MMA by means of liquid chromatography and sequential mass spectrometry. RESULTS: There was no difference between the groups in relation to allele and genotype frequencies (P = 0.44; P = 0.69, respectively). The folate, Hcy and MMA concentrations did not differ significantly between the groups, in relation to genotypes (P > 0.05). CONCLUSIONS: The 19-bp deletion polymorphism of DHFR gene was not a maternal risk factor for DS and was not related to variations in the concentrations of serum folate and plasma Hcy and MMA in the study population.

CONTEXTO E OBJETIVO: Polimorfismos em genes do metabolismo do folato podem modular o risco materno para síndrome de Down (SD). Este estudo avaliou a influência do polimorfismo de deleção de 19 pares de base (pb) no íntron 1 do gene dihidrofolato redutase (DHFR) no risco materno para SD e investigou a associação entre esse polimorfismo e variações nas concentrações de folato sérico, homocisteína (Hcy) e ácido metilmalônico (MMA) plasmáticos. TIPO DE ESTUDO E LOCAL: Estudo transversal analítico realizado na Faculdade de Medicina de São José do Rio Preto (Famerp). MÉTODOS: 105 mães de indivíduos com trissomia livre do cromossomo 21 e 184 mães controles foram avaliadas. A análise molecular do polimorfismo foi realizada pela reação em cadeia da polimerase (PCR) por diferença de tamanho dos fragmentos. O folato foi quantificado por quimioluminescência, e Hcy e MMA foram determinados por cromatografia líquida/espectrometria de massas sequencial. RESULTADOS: Não houve diferença entre os grupos em relação às frequências alélica e genotípica (P = 0,44; P = 0,69, respectivamente). As concentrações de folato, Hcy e MMA não mostraram diferença significativa entre os genótipos, entre grupos (P > 0,05). CONCLUSÕES: O polimorfismo de deleção de 19 pb do gene DHFR não é um fator de risco materno para SD e não está relacionado com variações nas concentrações de folato sérico, Hcy e MMA plasmáticos na população estudada.

Mass spectrometry fingerprinting of media used for in vitro production of bovine embryos.

Using the bovine species as a biological model, direct infusion chip-based nano-electrospray ionization mass spectrometry (nano-ESI-MS) fingerprinting in the positive ion mode is used to obtain fast chemical profiles of media used for in vitro production of bovine embryos. Nano-ESI-MS fingerprinting is useful for characterization and routine quality control requiring no sample pre-separation, being able to differentiate four different media (IVM, IVF, SOF and HSOF) via principal component analysis (PCA). For media stored at +4 degrees C for up to 45 days, no significant (p>0.05) variation was observed in cleavage and blastocyst rate development, as well as in the nano-ESI-MS chemical profiles. For media exposed to a heat shock (60 degrees C for 3 h), no significant decrease (p>0.05) in embryo development rates was observed, but nano-ESI-MS profiles were quite distant from fresh control media in the PCA. For frozen media (-70 degrees C for 2 months), again no significant variation (p>0.05) in embryo development was noticed, but nano-ESI-MS profiles from all media were significantly affected. These results indicate that nano-ESI(+)-MS fingerprinting was able to characterize different media based on their specific chemical profile. The technique seems therefore applicable as a routine quality control assay, detecting, for example, compositional changes after temperature variations that may affect post-transfer embryo viability.