Robust measurement of vitamin A status in plasma and blood dried on paper.

Vitamin A deficiency is the leading cause of preventable blindness in children and increases the risk of disease and death from severe infections. In addition, fat soluble vitamin A and associated retinoids directly regulate the expression of genes involved in fatty acid metabolism. Conventional methods for measuring vitamin A involve venipuncture, centrifugation and refrigeration all of which make measuring vitamin A in nutritional surveys expensive. We aimed to develop a simple and robust system for measurement of retinol (biomarker for vitamin A) using dried blood spot (DBS) samples. Low recoveries and inconsistent results reported by others were found to be due to poor extraction efficiency rather than retinol instability. Maintaining acid conditions during extraction resulted in recoveries >95% with <6.5% of coefficient of variation. Using isocratic high performance liquid chromatography, separation was achieved in <3.5 min. Detector response was linear (R(2)=0.9939) within a range of 0.05-2 µg/mL, with a limit of quantification of 0.05 µg/mL. Retinol in DBS was shown to be stable (>95%) at room temperature for up to 10 weeks. DBS values for retinol were highly correlated with venous blood samples from 24 healthy subjects (r=0.9724) and were consistent with results from a commercial laboratory. This simple and reliable method for the determination of vitamin A status should prove particularly valuable for population studies and large clinical trials.

Lentiviral-mediated gene therapy for murine mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a heritable glycosaminoglycan (GAG) storage disorder which is characterised by lysosomal accumulation of heparan sulphate, secondary to a deficiency of sulphamidase (heparan-N-sulphatase, N-sulphoglucosamine sulphohydrolase, EC No. 3.10.1.1.). There is currently no treatment for affected individuals who experience progressive CNS deterioration prior to an early death. As a first step towards developing gene therapy as a treatment for MPS IIIA, an MPS IIIA mouse model was used to examine the efficacy of intravenous lentiviral-mediated gene therapy. Five-week-old mice were injected with virus expressing murine sulphamidase and analysed 6 months after treatment. Transduction by the lentiviral vector was highest in the liver and spleen of treated animals, and sulphamidase activity in these tissues averaged 68% and 186% of normal, respectively. Storage was assessed using histochemical, chemical and mass spectrometric analyses. Storage in most somatic tissues was largely normalised, although chondrocytes were an obvious exception. Histologically, improvement of lysosomal storage within the brain was variable. However, beta-hexosaminidase activity, which is abnormally elevated in MPS IIIA, was significantly reduced in every treated tissue, including the brain. Total uronic acid was also significantly reduced in the brains of treated mice. The level of a disaccharide marker (hexosamine-N-sulphate[alpha-1,4]hexuronic acid; HNS-UA) of heparan sulphate storage was also decreased in the brains of treated mice, albeit non-significantly. These results suggest that lentiviral-mediated somatic gene transfer may affect not only the somatic, but possibly also the CNS pathology, found in MPS IIIA.