Amanda Barden

l-Carnitine Blood Levels and Oxidative Stress in Treated Phenylketonuric Patients

Aimsl-Carnitine exerts an important role by facilitating the mitochondrial transport of fatty acids, but is also a scavenger of free radicals, protecting cells from oxidative damage. Phenylketonuria (PKU), an inborn error of phenylalanine (Phe) metabolism, is currently treated with a special diet consisting of severe restriction of protein-enriched foods, therefore potentially leading to l-carnitine depletion. The aim of this study was to determine l-carnitine levels and oxidative stress parameters in blood of two groups of PKU patients, with good and poor adherence to treatment. Methods Treatment of patients consisted of a low protein diet supplemented with a synthetic amino acids formula not containing Phe, l-carnitine, and selenium. l-Carnitine concentrations and the oxidative stress parameters thiobarbituric acid reactive species (TBARS) and total antioxidant reactivity (TAR) were measured in blood of the two groups of treated PKU patients and controls. Results We verified a significant decrease of serum l-carnitine levels in patients who strictly adhered to the diet, as compared to controls and patients who did not comply with the diet. Furthermore, TBARS measurement was significantly increased and TAR was significantly reduced in both groups of phenylketonuric patients relatively to controls. We also found a significant negative correlation between TBARS and l-carnitine levels and a significant positive correlation between TAR and l-carnitine levels in well-treated PKU patients. Conclusions Our results suggest that l-carnitine should be measured in plasma of treated PKU patients, and when a decrease of this endogenous component is detected in plasma, supplementation should be considered as an adjuvant therapy.

Effect of short- and long-term exposition to high phenylalanine blood levels on oxidative damage in phenylketonuric patients

Phenylketonuria is the most frequent disturbance of amino acid metabolism. Treatment for phenylketonuric patients consists of phenylalanine intake restriction. However, there are patients who do not adhere to treatment and/or are not submitted to neonatal screening. These individuals are more prone to develop brain damage due to long‐lasting toxic effects of high levels of phenylalanine and/or its metabolites. Oxidative stress occurs in late‐diagnosed phenylketonuric patients, probably contributing to the neurological damage in this disorder. In this work, we aimed to compare the influence of time exposition to high phenylalanine levels on oxidative stress parameters in phenylketonuric patients who did not adhere to protein restricted diet. We evaluated a large spectrum of oxidative stress parameters in plasma and erythrocytes from phenylketonuric patients with early and late diagnosis and of age‐matched healthy controls. Erythrocyte glutathione peroxidase activity and glutathione levels, as well as plasma total antioxidant reactivity were significantly reduced in both groups of patients when compared to the control group. Furthermore, protein oxidative damage, measured by carbonyl formation and sulfhydryl oxidation, and lipid peroxidation, determined by malondialdehyde levels, were significantly increased only in patients exposed for a long time to high phenylalanine concentrations, compared to early diagnosed patients and controls. In conclusion, exposition to high phenylalanine concentrations for a short or long time results in a reduction of non‐enzymatic and enzymatic antioxidant defenses, whereas protein and lipid oxidative damage only occurs in patients with late diagnosis.

Oxidative stress in plasma from maple syrup urine disease patients during treatment

Maple Syrup Urine Disease (MSUD) is an autossomal recessive metabolic disorder caused by a deficiency of branched-chain α-keto acid dehydrogenase complex activity leading to accumulation of the branched-chain amino acids leucine, isoleucine and valine and their corresponding branched-chain α-keto acids. Affected patients usually present hypoglycemia, ketoacidosis, convulsions, poor feeding, coma, psychomotor delay and mental retardation. Considering that the pathophysiology of MSUD is still poorly understood, in this study we evaluated some parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBARS), total antioxidant reactivity (TAR) and total antioxidant status (TAS) in plasma from treated MSUD patients presenting high and low plasma leucine levels. We verified a significant increase of TBARS (lipid peroxidation) and a decrease of TAR (capacity to rapidly react with free radicals) in plasma from treated MSUD patients with low and with high plasma levels of leucine compared to the control group. It was also verified that TAS (quantity of tissue antioxidants) was not altered in plasma from treated MSUD patients with low and high blood leucine levels. Finally, we found no correlation between leucine, valine and isoleucine levels with the various parameters of oxidative stress. These results are indicative that increased lipid oxidative damage and decreased antioxidant defenses occur in plasma of MSUD patients and that the accumulating branched-chain amino acids are probably not directly associated to oxidative stress in this disorder.

Oxidative stress parameters in diabetic rats submitted to forced swimming test: the clonazepam effect

Diabetes-associated depression may occur due to changes in the quality of life imposed by treatment, or may be a consequence of the biochemical changes accompanying the disease. We evaluated the oxidative stress from diabetic animals submitted to an experimental model of depression and the effects of clonazepam. Male Wistar rats were induced to diabetes with streptozotocin and submitted to forced swimming test. Clonazepam was administered 24, 5 and 1 h before test. The animals were sacrificed by decapitation, and plasma and erythrocytes were separated, as well as hippocampus, cortex and striatum. Reactive species of thiobarbituric acid (TBARS) and total antioxidant reactivity (TAR) as well as antioxidant enzyme activities catalase (CAT) and superoxide dismutase (SOD) were evaluated. Results showed a significant increase of TBARS and a significant decrease of TAR in plasma from diabetic animals, which was altered by clonazepam. There were no effects of CAT and SOD activities in erythrocytes from tested animals. The results observed in hippocampus showed a significant increase of TBARS from diabetic rats, altered by clonazepam, and no one alteration was verified in TAR. The significant increase of TBARS and the significant decrease of TAR in cortex from diabetic rats were not altered by clonazepam administration. There were no alterations of TBARS and TAR in striatum from tested animals. Besides, clonazepam reverses the immobility in diabetic rats. Considering the action of clonazepam, it is suggested that it could be an alternative therapeutic for depression to diabetic patients, once it could give a protection against free radicals.

Stability-Indicating RP-LC Method for the Determination of Vildagliptin and Mass Spectrometry Detection for a Main Degradation Product

A simple, precise and stability-indicating reversed-phase liquid chromatography method was developed and validated for the determination of vildagliptin (VLG) in pharmaceutical dosage form. The chromatographic separation was obtained within 6 min and was linear in the range of 20-80 µg/mL (r(2) = 0.9999). Limit of detection and limit of quantitation were 0.63 and 2.82 µg/mL, respectively. The method was validated in accordance with International Conference on Harmonization acceptance criteria for specificity, linearity, precision, accuracy, robustness and system suitability. Stress studies were carried out and no interference of the degradation products was observed. The excipients did not interfere in the determination of VLG. Furthermore, the main degradation product obtained from the stress studies (thermal, oxidative and alkaline hydrolysis) was evaluated for mass spectrometry and its molecular structure was predicted. The proposed method was successfully applied for the quantitative analysis of VLG in tablet dosage form, which will help to improve quality control and contribute to stability studies of pharmaceutical tablets containing this drug.

Erythrocyte glutathione peroxidase activity and plasma selenium concentration are reduced in maple syrup urine disease patients during treatment

Maple syrup urine disease (MSUD) is an inherited disorder caused by a deficiency of the branched‐chain α‐keto acid dehydrogenase complex activity. In the present study we evaluated selenium levels in plasma from MSUD patients at diagnosis and under treatment and the activities of glutathione peroxidase, catalase and superoxide dismutase in erythrocytes from treated patients. We verified that MSUD patients present a significant selenium deficiency at diagnosis, which becomes more pronounced during treatment, as well as a decrease of erythrocyte glutathione peroxidase activity during treatment. In contrast, erythrocyte catalase and superoxide dismutase activities were not altered in these patients. Our present results suggest that the reduction of an important antioxidant enzyme activity may be partially involved in the pathomechanisms of this disorder and that plasma selenium levels must be corrected through dietary supplementation in MSUD patients.

Quantitative evaluation of besifloxacin ophthalmic suspension by HPLC, application to bioassay method and cytotoxicity studies.

Besifloxacin (BSF) is a synthetic chiral fluoroquinolone developed for the topical treatment of ophthalmic infections. The present study reports the development and validation of a microbiological assay, applying the cylinder-plate method, for determination of BSF in ophthalmic suspension. To assess this methodology, the development and validation of the method was performed for the quantification of BSF by high performance liquid chromatography (HPLC). The HPLC method showed specificity, linearity in the range of 20-80 µg mL(-1) (r=0.9998), precision, accuracy and robustness. The microbiological method is based on the inhibitory effect of BSF upon the strain of Staphylococcus epidermidis ATCC 12228 used as a test microorganism. The bioassay validation method yielded excellent results and included linearity, precision, accuracy, robustness and selectivity. The assay results were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9974) in the range of 0.5-2.0 µg mL(-1), precise (inter-assay: RSD=0.84), accurate (101.4%), specific and robust. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Students t test, which indicated that there was no statistically significant difference between these two methods. These results confirm that the proposed microbiological method can be used as routine analysis for the quantitative determination of BSF in an ophthalmic suspension. A preliminary stability study during the HPLC validation was performed and demonstrated that BSF is unstable under UV conditions. The photodegradation kinetics of BSF in water showed a first-order reaction for the drug product (ophthalmic suspension) and a second-order reaction for the reference standard (RS) under UVA light. UVA degraded samples of BSF were also studied in order to determine the preliminary in vitro cytotoxicity against mononuclear cells. The results indicated that BSF does not alter the cell membrane and has been considered non-toxic to human mononuclear cells in the experimental conditions tested.

A simultaneous assay method using capillary zone electrophoresis for a fixed dose combination of vildagliptin and metformin hydrochloride in coated tablets

In the present study, a quick and reliable validated capillary zone electrophoresis (CZE) method was established for the simultaneous determination of vildagliptin (VLG) and metformin hydrochloride (MET) in pharmaceutical dosage form. Successful separation of the drugs by the CZE method was achieved in a fused silica capillary by applying a potential of 25 kV (positive polarity) and hydrodynamic injection by 50 mbar for 5 s. The selected running buffer consisted of 25 mM sodium tetraborate decahydrate solution (array pH 9.0) with UV/photodiode (PDA) detection at 207 nm and 250 nm for VLG and MET respectively. Specificity, linearity, precision, accuracy, limit of detection and limit of quantitation and robustness were established for VLG and MET in accordance with International Conference on Harmonization (ICH) guidelines. The specificity and stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using PDA detection. Electrophoretic separation was obtained within 10 min and the method was linear in the range of 30–60 μg mL−1 and 300–600 μg mL−1 for VLG and MET, respectively (R2 > 0.9980). Precision of the method was reflected by RSD lower than 2.95% and accuracy results around 100% for both VLG and MET measurement. Moreover, the Plackett–Burman experimental design was used to evaluate robustness, producing results within the acceptable range.