Analysis of cerebrospinal fluid γ-aminobutyric acid by capillary electrophoresis with laser-induced fluorescence detection.

The measurement of γ-aminobutyric acid (GABA) is suitable for investigating various neurological disorders. In this study, a sensitive and selective method for free GABA quantification in cerebrospinal fluid (CSF) has been standardised. This method is based on CE with LIF detection using 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a derivatisating agent. The reaction conditions (NBD-F concentration, pH, temperature and reaction time) and the electrophoretic parameters (run buffer composition and pH and separation voltage) were optimised to obtain the maximum derivatisation efficiency and electrophoretic resolution. The best resolution was obtained using 200 mM sodium borate, 10 mM SDS, 8.5 mM ß-CD, pH 10 and 20 kV voltage. The method was linear in the concentration range of 2.5-1000 nM with good inter- and intra-assay precision values. The effects of CSF handling on free GABA concentrations were also evaluated. Our results show that the time delay between CSF collection and freezing strongly increases the CSF GABA values. Age-related reference values were established in 55 paediatric controls. The influence of antiepileptic therapy on free CSF GABA was studied in 38 neuropaediatric patients. Significantly, higher GABA values were obtained in patients taking valproic acid or vigabatrin therapy, which are antiepileptic drugs that modulate GABA metabolism.

Predisposition to epilepsy in fragile X syndrome: does the Val66Met polymorphism in the BDNF gene play a role?

Epilepsy is detected in about 23% of patients with fragile X syndrome (FXS). Absence or reduced levels of the fragile X mental retardation protein (FMRP), a global regulator of translation in neurons and an important factor in synaptic plasticity, produce the observed epileptic patterns. The brain-derived neurotrophic factor (BDNF) gene is a specific regulator of synaptic plasticity, and disturbances in its function cause dendrite abnormalities similar to those observed in FXS. A putative reciprocal regulation of FMRP and BDNF has been hypothesized. The Val66Met polymorphism in the BDNF gene may be involved in the alteration of normal secretion of the mature peptide and may modulate the epileptic phenotype observed in some patients with FXS. We investigated the relationship of this Met66 allele to the prevalence of epilepsy in 77 patients with FXS. No association was observed between this polymorphism and epilepsy in our group of patients. Therefore, it should not be considered a biomarker for developing epilepsy in patients with FXS.

Glutamine effects on heat shock protein 70 and interleukines 6 and 10: Randomized trial of glutamine supplementation versus standard parenteral nutrition in critically ill children.

BACKGROUND & AIMS: To determine whether glutamine (Gln) supplementation would have a role modifying both the oxidative stress and the inflammatory response of critically ill children. METHODS: Prospective, randomized, double-blind, interventional clinical trial. Selection criteria were children requiring parenteral nutrition for at least 5 days diagnosed with severe sepsis or post major surgery. Patients were randomly assigned to standard parenteral nutrition (SPN, 49 subjects) or standard parenteral nutrition with glutamine supplementation (SPN + Gln, 49 subjects). RESULTS: Glutamine levels failed to show statistical differences between groups. At day 5, patients in the SPN + Gln group had significantly higher levels of HSP-70 (heat shock protein 70) as compared with the SPN group (68.6 vs 5.4, p = 0.014). In both groups, IL-6 (interleukine 6) levels showed a remarkable descent from baseline and day 2 (SPN: 42.24 vs 9.39, p < 0.001; SPN + Gln: 35.20 vs 13.80, p < 0.001) but only the treatment group showed a statistically significant decrease between day 2 and day 5 (13.80 vs 10.55, p = 0.013). Levels of IL-10 (interleukine 10) did not vary among visits except in the SPN between baseline and day 2 (9.55 vs 5.356, p < 0.001). At the end of the study, no significant differences between groups for PICU and hospital stay were observed. No adverse events were detected in any group. CONCLUSIONS: Glutamine supplementation in critically-ill children contributed to maintain high HSP-70 levels for longer. Glutamine supplementation had no influence on IL-10 and failed to show a significant reduction of IL-6 levels.

Thiamine transporter-2 deficiency: outcome and treatment monitoring.

BACKGROUND: The clinical characteristics distinguishing treatable thiamine transporter-2 deficiency (ThTR2) due to SLC19A3 genetic defects from the other devastating causes of Leigh syndrome are sparse. METHODS: We report the clinical follow-up after thiamine and biotin supplementation in four children with ThTR2 deficiency presenting with Leigh and biotin-thiamine-responsive basal ganglia disease phenotypes. We established whole-blood thiamine reference values in 106 non-neurological affected children and monitored thiamine levels in SLC19A3 patients after the initiation of treatment. We compared our results with those of 69 patients with ThTR2 deficiency after a review of the literature. RESULTS: At diagnosis, the patients were aged 1 month to 17 years, and all of them showed signs of acute encephalopathy, generalized dystonia, and brain lesions affecting the dorsal striatum and medial thalami. One patient died of septicemia, while the remaining patients evidenced clinical and radiological improvements shortly after the initiation of thiamine. Upon follow-up, the patients received a combination of thiamine (10-40 mg/kg/day) and biotin (1-2 mg/kg/day) and remained stable with residual dystonia and speech difficulties. After establishing reference values for the different age groups, whole-blood thiamine quantification was a useful method for treatment monitoring. CONCLUSIONS: ThTR2 deficiency is a reversible cause of acute dystonia and Leigh encephalopathy in the pediatric years. Brain lesions affecting the dorsal striatum and medial thalami may be useful in the differential diagnosis of other causes of Leigh syndrome. Further studies are needed to validate the therapeutic doses of thiamine and how to monitor them in these patients.