Glutaric Acid Administration Impairs Energy Metabolism in Midbrain and Skeletal Muscle of Young Rats

A genetic mice model of glutaric acidemia type I (GAI) has recently been developed, however affected animals do not develop the striatal damage characteristic of patients with this disorder. Therefore, the initial aim of the present work was to induce high glutaric acid (GA) concentrations in rat brain similar to those found in GAI patients through subcutaneous injection of GA. High brain GA concentrations (up to 0.60 μmol/g ≅ 0.60mM) were achieved by a single subcutaneous injection of saline-buffered GA (5 μmol/g body weight) to Wistar rats of 7–22 days of life. GA brain levels were about 10-fold lower than in plasma and 5-fold lower than in skeletal and cardiac muscles, indicating that the permeability of the blood brain barrier to GA is low. We also aimed to use this model to investigate neurochemical parameters in the animals. Thus, we evaluated the effect of this model on energy metabolism parameters in midbrain, in which the striatum is localized, as well as in peripheral tissues (skeletal and cardiac muscles) of 22-day-old rats. Control rats were treated with saline in the same volumes. We verified that CO₂ production from glucose was not altered in midbrain of rats treated with GA, indicating a normal functioning of the tricarboxylic acid cycle. Creatine kinase activity was also not changed in midbrain, skeletal and cardiac muscles. In contrast, complex I–III activity of the respiratory chain was inhibited in midbrain (25%), while complexes I–III (25%) and II–III (15%) activities were reduced in skeletal muscle, with no alterations found in cardiac muscle. These data indicate that GA administration moderately impairs cellular energy metabolism in midbrain and skeletal muscle of young rats.     

Polycaprolactone-poly(ethylene glycol) multiblock copolymers as potential substitutes for di(ethylhexyl) phthalate in flexible poly(vinyl chloride) formulations

New high-molecular-weight hydrophobic/hydrophilic segmented copolymers of poly(ester ether carbonate) structure, containing poly(epsilon-caprolactone) (PCL) and poly(ethylene glycol) (PEG) segments in their main chain, were synthesized and characterized. These copolymers were obtained by a two-step chain-extension reaction carried out in the presence of alpha,omega-dihydroxy-oligoPCL of molecular weight 1250 and PEG samples of molecular weight 150, 400, 600, 1000, and 2000. The molecular structures of all synthesized materials were characterized by means of (1)H NMR and (13)C NMR spectroscopy, their molecular weights were determined by means of size exclusion chromatography, and their thermal properties were obtained by means of differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The poly(ester ether carbonate)s of this study are partly or totally miscible at least up to 50 wt % with poly(vinyl chloride) (PVC) and could be used to produce flexible PVC formulations. The miscibility between PVC and the poly(ester ether carbonate)s reported in this paper was investigated by means of DSC and DMA analysis. PVC blends were also analyzed by determining their swellability and the amount of extractables in aqueous media. By comparison purposes, the chain-extension product of PCL1250, that is, PCL polycarbonate, was also synthesized and characterized. The results obtained demonstrated that the copolymers with shortest PEG segment length, i.e. PEG150, 400, and 600, give the best results in terms of miscibility with PVC and lead to blends with maximum resistance to extraction by water. Therefore, they represent, in principle, good substitutes for low-molecular-weight, leachable PVC plasticizers, such as di(ethylhexyl) phthalate.     

Evidence that oxidative stress is increased in patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a hereditary disorder of peroxisomal metabolism biochemically characterized by the accumulation of very long chain fatty acids (VLCFA), particularly hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in different tissues and in biological fluids. The disease is clinically characterized by central and peripheral demyelination and adrenal insufficiency, which is closely related to the increased concentrations of these fatty acids. However, the mechanisms underlying the brain damage in X-ALD are poorly known. Considering that free radical generation is involved in various neurodegenerative disorders, like Parkinson disease, multiple sclerosis and Alzheimer's disease, in the present study we evaluated various oxidative stress parameters, namely chemiluminescence, thiobarbituric acid reactive species (TBA-RS), total radical-trapping antioxidant potential (TRAP), and total antioxidant reactivity (TAR) in plasma of X-ALD patients, as well as the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes and fibroblasts from these patients. It was verified a significant increase of plasma chemiluminescence and TBA-RS, reflecting induction of lipid peroxidation, as well as a decrease of plasma TAR, indicating a deficient capacity to rapidly handle an increase of reactive species. We also observed a significant increase of erythrocytes GPx activity and of catalase and SOD activities in fibroblasts from the patients studied. It is therefore proposed that oxidative stress may be involved in pathophysiology of X-ALD.     

Induction of Oxidative Stress by Chronic and Acute Glutaric Acid Administration to Rats

1. Glutaric acidemia type I (GA I) is a neurometabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase, which leads to tissue accumulation of predominantly glutaric acid (GA) and also 3-hydroxyglutaric acid to a lesser amount. Affected patients usually present progressive cortical atrophy and acute striatal degeneration attributed to the toxic accumulating metabolites. 2. In the present study, we determined a number of oxidative stress parameters, namely chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total antioxidant reactivity (TAR), glutathione (GSH) levels, and the activities of catalase and glutathione peroxidase (GPx), in various tissues from rats chronically exposed to GA or to saline (controls). High GA concentrations, similar to those found in glutaric aciduria type I, were induced in the brain by three daily subcutaneous injections of saline-buffered GA (5 μmol/g body weight) to Wistar rats of 5–22 days of life. The parameters were assessed 12 h after the last GA administration in different brain structures, skeletal muscle, heart, liver, erythrocytes, and plasma. The lipid peroxidation parameters chemiluminescence and/or TBA-RS measurements were found significantly increased in midbrain, liver, and erythrocytes of GA-injected rats. The activity of GPx was significantly reduced in midbrain and markedly increased in liver. TAR measurement was significantly reduced in midbrain and liver. Furthermore, GSH levels were reduced in liver and heart. We also investigated the acute in vivo effect of GA administration on the same oxidative stress parameters in cerebral structures and erythrocytes from 22-day-old rats. We found that TBA-RS values were significantly increased in erythrocytes, TAR levels were markedly decreased in midbrain and cerebellum, and GPx activity mildly reduced in the midbrain. 3. These data showing an imbalance between antioxidant defences and oxidative damage, particularly in midbrain, liver, and erythrocytes from GA-injected rats, indicate that oxidative stress might be involved in GA toxicity and that the midbrain, where the striatum is located, is the brain structure more susceptible to GA chronic and acute exposition.     

NOD2 and CCDC122-LACC1 genes are associated with leprosy susceptibility in Brazilians

Leprosy is a complex disease with phenotypes strongly influenced by genetic variation. A Chinese genome-wide association study (GWAS) depicted novel genes and pathways associated with leprosy susceptibility, only partially replicated by independent studies in different ethnicities. Here, we describe the results of a validation and replication study of the Chinese GWAS in Brazilians, using a stepwise strategy that involved two family-based and three independent case–control samples, resulting in 3,614 individuals enrolled. First, we genotyped a family-based sample for 36 tag single-nucleotide polymorphisms (SNPs) of five genes located in four different candidate loci: CCDC122-LACC1, NOD2, TNFSF15 and RIPK2. Association between leprosy and tag SNPs at NOD2 (rs8057431) and CCDC122-LACC1 (rs4942254) was then replicated in three additional, independent samples (combined OR_AA = 0.49, P = 1.39e?06; OR_CC = 0.72, P = 0.003, respectively). These results clearly implicate the NOD2 pathway in the regulation of leprosy susceptibility across diverse populations.     

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background

Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis.

Methodology/Principal Findings

Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results.

Conclusions/Significance

The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.     

A Very Low Geno2pheno False Positive Rate Is Associated with Poor Viro-Immunological Response in Drug-Na?ve Patients Starting a First-Line HAART

Background

We previously found that a very low geno2pheno false positive rate (FPR ≤2%) defines a viral population associated with low CD4 cell count and the highest amount of X4-quasispecies. In this study, we aimed at evaluating whether FPR ≤2% might impact on the viro-immunological response in HIV-1 infected patients starting a first-line HAART.

Methods

The analysis was performed on 305 HIV-1 B subtype infected drug-naïve patients who started their first-line HAART. Baseline FPR (%) values were stratified according to the following ranges: ≤2; 2–5; 5–10; 10–20; 20–60; >60. The impact of genotypically-inferred tropism on the time to achieve immunological reconstitution (a CD4 cell count gain from HAART initiation ≥150 cells/mm³) and on the time to achieve virological success (the first HIV-RNA measurement <50 copies/mL from HAART initiation) was evaluated by survival analyses.

Results

Overall, at therapy start, 27% of patients had FPR ≤10 (6%, FPR ≤2; 7%, FPR 2–5; 14%, FPR 5–10). By 12 months of therapy the rate of immunological reconstitution was overall 75.5%, and it was significantly lower for FPR ≤2 (54.1%) in comparison to other FPR ranks (78.8%, FPR 2–5; 77.5%, FPR 5–10; 71.7%, FPR 10–20; 81.8%, FPR 20–60; 75.1%, FPR >60; p = 0.008). The overall proportion of patients achieving virological success was 95.5% by 12 months of therapy. Multivariable Cox analyses showed that patients having pre-HAART FPR ≤2% had a significant lower relative adjusted hazard [95% C.I.] both to achieve immunological reconstitution (0.37 [0.20–0.71], p = 0.003) and to achieve virological success (0.50 [0.26–0.94], p = 0.031) than those with pre-HAART FPR >60%.

Conclusions

Beyond the genotypically-inferred tropism determination, FPR ≤2% predicts both a poor immunological reconstitution and a lower virological response in drug-naïve patients who started their first-line therapy. This parameter could be useful to identify patients potentially with less chance of achieving adequate immunological reconstitution and virological undetectability.     

High-performance liquid chromatographic isolation and fast atom bombardment mass spectrometric identification of Di-N-desethylamiodarone, a new metabolite of amiodarone in the dog

Amiodarone is an antiarrhythmic drug which has received considerable attention in recent years. It has been suggested that the unusual pharmacodynamic characteristics of this drug may be due in part to the influence of active metabolites. Using fast atom bombardment (FAB) mass spectrometry we have identified a new metabolite of amiodarone, the di-N-desethyl analog (DDEA). This metabolite was present in the blood of dogs treated with the parent drug, and showed a greater affinity for myocardium than did the parent drug. The unique features of FAB mass spectrometry over electron impact mass spectrometry was an essential element in facilitating the identification of this new metabolite. Whether or not this metabolite has pharmacologic activity or is responsible for some of the side effects occurring during amiodarone administration is not known.     

Chronobiology of blood pressure in childhood: implications for tracking

Chronobiology, in its methodological evolution, developed data series analyses paying particular attention to blood pressure (BP), because of the importance of this biorhythmic variable for assessing the risk of developing hypertension. An example of the potentiality of the chronobiologic procedures is given in the present report which deals with the inferential analysis of the BP 24-h patterns in 3-12 year-old children. By using the chronobiologic methodology, time-qualified standards for BP are calculated. Rhythmometric parameters for circadian rhythm of systolic and diastolic components of BP are also computed. Data presented are a tangible outcome for emphasizing the introduction of chronobiology in epidemiology and pediatrics in order to optimize the primary prevention and care of hypertension taking as reference the chronobiologic standards of BP.