Sensitive LC-MS/MS method for quantitation of levodopa and carbidopa in plasma: application to a pharmacokinetic study.

AIM: The combination of levodopa with carbidopa has been used for treatment of Parkinson's disease being an important therapy in dopamine level control in the brain. Both are very polar compounds becoming a challenge for analysis by LC-MS/MS. MATERIALS & METHODS: In this work, it was developed and validated a sensitive bioanalytical method by UHPLC-MS/MS for simultaneous levodopa and carbidopa quantification in human plasma using a fast protein precipitation method. Moreover, the bioanalytical method was applied to a pharmacokinetic study in healthy volunteers. RESULTS/CONCLUSION: The results demonstrated a sensitive and adequate method for application to pharmacokinetic/bioequivalence studies.

Chronic use of pravastatin reduces insulin exocytosis and increases ß-cell death in hypercholesterolemic mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases ß-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.

Correlation between Mitochondrial Reactive Oxygen and Severity of Atherosclerosis.

Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS) generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression) with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.

Analysis and characterisation of bovine oocyte and embryo biomarkers by matrix-assisted desorption ionisation mass spectrometry imaging.

In the field of 'single cell analysis', many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na](+) (m/z 725) and phosphatidylcholine [PC (32:0)+Na](+) (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development.

S-nitrosoglutathione inhibits inducible nitric oxide synthase upregulation by redox posttranslational modification in experimental diabetic retinopathy.

PURPOSE: Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR. METHODS: Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 µM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19). RESULTS: In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation. CONCLUSIONS: In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.

Rapid and simultaneous in situ assessment of aflatoxins and stilbenes using silica plate imprinting mass spectrometry imaging.

A fast and direct combination of techniques for simultaneous mycotoxin and phytoalexin identification in peanut skin and kernel is described. Silica Plate Imprinting Laser Desorption/Ionization Mass Spectrometry Imaging (SPILDI-MSI) is a powerful technique that exhibits great advantages, such as solvent-free and matrix-free characteristics, as well as no sample preparation or separation steps. It also permits accurate identification of mycotoxins and phytoalexins with unique fingerprint profiles in just a few seconds. Results are expressed as chemical images of the 4 identified types of aflatoxins (B1, B2, G1 and G2) and a stilbenoid (resveratrol). Also, SPILDI-MSI allows the comparison between the spatial distribution of aflatoxins and resveratrol found in kernel and skin. This novel application has proven to be useful for instantaneous qualitative assessment of aflatoxins and stilbenoids both in the peanut skin and kernel and offers precise tracking of fungal contamination in nuts and other foodstuffs.

Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI).

Lipid pathways play important biological roles in mammalian embryology, directing early developmental pathways to differentiation. Phospholipids and triglycerides, among others, are the main composing lipids of zona pellucida in several embryo species. Lipid analysis in embryos by mass spectrometry usually requires sample preparation and/or matrix application. This novel approach using silica plate laser desorption/ionization mass spectrometry imaging (SP-LDI-MSI) allows direct single-cell imaging and embryo region discrimination with no matrix coating. Its application is herein described for two- and eight-cell embryos. Lipid biomarkers for blastomere and intact zona pellucida are reported and corroborated by both fragmentation reactions (MS/MS) and images. Results obtained in this work are understood to be of great use for further developments on in vitro bovine fertilization. Since much of the processes can be monitored by characteristic biomarkers, it is now possible to precisely identify cell division errors during early embryo stages, as well as evaluate pre-implantation conditions.

Molecular characterization of noroviruses and HBGA from infected Quilombola children in Espirito Santo State, Brazil.

Noroviruses (NoV) are the main etiological agents of gastroenteritis outbreaks worldwide and susceptibility to NoV infection has been related to the histo-blood group antigen (HBGA). This study aimed to determine the prevalence of NoV strains and to evaluate the HBGA phenotype and genotype of children from semi-isolated Quilombola communities, descendents of black slaves in Brazil. A total of 397 children up to eleven years old, with and without diarrhea, from Quilombola Communities in the Espirito Santo State, Brazil, were investigated for the presence of NoV from August 2007 to September 2009. Feces were collected from all the children, and blood from the NoV positive children. NoV was screened by reverse transcription-PCR with primers for the RNA-dependent RNA polymerase region; genogroup was determined by PCR with primers for the C and D regions and genotyped by sequencing. HBGA phenotype was performed by gel-spinning and FUT2 and FUT3 were analyzed by PCR or sequencing analysis. NoV were detected in 9.2% (12/131) of diarrheic and 1.5% (4/266) of non-diarrheic children (p<0.05, Fisher's exact test). GI and GII genogroups were present in 12.5% and 87.5% of the samples, respectively. The following genotypes were characterized: GII.4 (25%), GII.12 (25%), GII.6 (12.5%) and GI.1 (6.3%), GI.3 (12.5%) and GI.4 (6.3%). Children infected with NoV showed the A (n = 6), O (n = 6), and B (n = 2) HBGA phenotypes, and 13 of them were classified as secretors (Se) and one as a non secretor (se). Mutations of Se (40), (171,216,357,428,739,960) were found for the FUT2 gene and mutations of Le (59, 202, 314) for the FUT3 gene. The only se child was infected by NoV GI, whereas the Se children were indiscriminately infected by GI or GII. This study showed rates of NoV infection in symptomatic and asymptomatic Quilombola children consistent with other studies. However, children under 12 months were seven times more affected than those between 1 and 5 years old. GII.12 was as frequent as GII.4 and GI.1 and GI.4 were described for the first time in Brazil. Owing to the small number of cases studied, no clear pattern of susceptibility and/or HBGA resistance could be inferred.

T-cell autoreactivity to Hsp in human transplantation may involve both proinflammatory and regulatory functions.

Heat shock proteins (Hsp) are moving from the category of basically intracellular chaperone molecules to important proteins in both innate and acquired immune responses, with great potential for clinical application as immunomodulators. Both proinflammatory and regulatory Hsp-reactive T cells have been described in animal models of autoimmune diseases. To investigate the role of autoreactivity to Hsp60 and Hsp70 in human transplantation, we analyzed, sequentially, peripheral blood mononuclear cell proliferation and cytokine production before and at different time points after renal transplantation, as well as the modulation of proliferation to Hsp in the presence of exogenous cytokines. Proliferation to Hsp60 and Hsp70 in the pretransplantation (pre-Tx) period was significantly associated with rejection episodes in the first months post-Tx. In contrast, IL-4 production was significantly associated with absence of rejection. Addition of exogenous IL-4 distinctly modulated the proliferative response to Hsp60; inhibiting proliferation in 83% of patients in the early post-Tx period (0-6 months), in which rejection episodes occurred, and inducing proliferation in 62.5% of patients in the later period (>12-24 months), when no rejection was observed. Characterization of autoreactive anti-Hsp60 regulatory T cells may permit new approaches to control the proinflammatory response to the graft, as well as aggressive autoimmunity.

Rotavirus strain diversity in Rio de Janeiro, Brazil: characterization of VP4 and VP7 genotypes in hospitalized children.

Rotavirus strains from 91 patients treated at a children's hospital from 1996 to 1998 in Rio de Janeiro, Brazil, were characterized by electropherotyping, reverse transcription-PCR amplification for P and G genotypes, and Southern hybridization. Results obtained showed that following predominant [P],G type combination: P[4], G2 (21 per cent), P[8], G1 (17 per cent), P[8], G3 (13 per cent), which are prevalent throughout the world. However, an unexpected number of cases were associated with uncommon genotypes: P[8], G2 (13 per cent), P[8], G5 (11 per cent), P[8], G9 (7 per cent), P[8], G10 (4 per cent), P[6], G4 (3 per cent), P[6], G3 (1 per cent), P[4], G9 (1 per cent), and P[6], G9 (1 per cent). Mixed infections with more than one type were identified in only two cases and 16 per cent of the samples were not G and/or P typeable. A subset of G types was confirmed by Southern hybridization and chemiluminescent detection. Rotavirus seasonal distribution was observed between April and July. The contribution of the results obtained in the present investigation corroborates the required epidemiological surveillance for rotavirus infection in Brazil.