ABSTRACT: Mucopolysaccharidosis IVA (MPS IVA), or Morquio Syndrome type A, is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS), resulting in excessive lysosomal storage of keratan sulfate in many tissues and organs. This accumulation causes a severe skeletal dysplasia with short stature, and affects the eye, heart and other organs, with many signs and symptoms. Morquio A syndrome is estimated to occur in 1 in 200,000 to 300,000 live births. Clinical trials with enzyme replacement therapy for this disease are in progress, and it is probable that the treatment, when available, would be more effective if started early. We describe an innovative fluorometric method for the assay of GALNS in dried blood spots (DBS).
We used dried blood spots (DBS) as the enzyme source and compared it with leukocytes samples, having studied 25 MPS IVA patients and 54 healthy controls. We optimized the assay conditions, including incubation time and stability of DBS samples. To eppendorf type tubes containing a 3-mm diameter blood spot we added elution liquid and substrate solution. After 2 different incubations at 37°C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of the enzyme activity. Results in DBS were compared to the ones obtained in leukocytes using the standard technique.
The fluorescent methodology was validated in our laboratory and the assay was found sensitive and specific, allowing reliable detection of MPS IVA patients. The use of DBS simplifies the collection and transport steps, and is especially useful for testing patients from more remote areas of large countries, and when samples need to cross country borders.
This assay could be easily incorporated into the protocol of reference laboratories and play a role in the screening for MPS IVA, contributing to earlier detection of affected patients.
Clinica chimica acta; international journal of clinical chemistry 06/2011; 412(19-20):1805-8. · 2.54 Impact Factor
ABSTRACT: L-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.
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.
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.
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.
Cellular and Molecular Neurobiology 10/2008; 29(2):211-8. · 1.97 Impact Factor
ABSTRACT: Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays.
Fluorometric methods were used to evaluate the activities of arylsulfatase B, alpha-N-acetylglucosaminidase, chitotriosidase, alpha and beta-galactosidases, beta-glucosidase, beta-glucuronidase, total hexosaminidases, hexosaminidase A, alpha-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 degrees C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured.
Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays.
Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.
Clinica Chimica Acta 11/2006; 372(1-2):98-102. · 2.54 Impact Factor