Biyotinidaz Enzim Eksikliğinin Değerlendirilmesinde Spektrofotometrik ve Florometrik Yöntemlerin Karşılaştırılması

Abstract

Biotin, a water-soluble vitamin, is used as a co-factor by enzymes involved in carboxylation reactions. It functions as the carboxyl carrier for biotin-dependent carboxylases which catalyze gluconeogenesis, fatty acid metabolism and amino acid catabolism, thus biotin plays an essential role in maintaining metabolic homeostasis. Biotinidase catalyses the recycle of biotin from endogenous and dietary sources. Biotinidase deficiency is an autosomal recessively inherited disorder of biotin recycling that is associated with neurologic and cutaneous consequences whenremain untreated. In central Anatolia where marriages between relatives are very common (26%),the insidance of biotinidase deficiency is high. Fortunately, the clinical features of the disorder can be ameliorated or prevented by administering pharmacological doses of the vitamin biotin.Neonatal screening forbiotinidase deficiency is conducted in many countries including our country. Spectrophotometric or fluorometric assays are used to measure biotinidase activity. The enzyme activity is determinedspectrophotometrically by measuring the hydrolysis of "n-biotinyl-p-aminobenzoate" substrate while "biotinyl-6-aminoquinoline"is used as a substratein the fluorometricmethod. In this study, we aim to set fluorometric method in our hospital and compare the results of the colorimetric and fluorometric methods, additionally to evaluate the advantages and disadvantages of both methods. Study group were chosen among the BTD deficiency suspected newborn, children and parents (n=52) who applied to Hacettepe University Pediatric Metabolism Unit, for the BTD activity check up. Ethical approval was takenfrom Hacettepe UniversityMedical FacultyClinical Research Ethics Committee (date: 23.01.2013and number: LUT 12/178-12).Reference range for fluorometric method is 3,53-3,79 U/L and for spectrophotometric method is 4,4-12 U/L. According to our results, biotinidase activity is stable for 2 hours at room temperature and at 4°C, 2 months at -20°C and several months at -80°C. Genetic and clinical results showed that 25% of the total patients have complete BTD deficiency and treated with 10 mg/day biotin while 15,38% of the patients have partiel BTD deficiency and had 5 mg/day biotin treatment. When area under the ROC curve was measured for fluorometric and spectrophotometric method, it was found as 0,960±0,25 and 0,927±0,41 respectively. Fluorometric method showed 100% sensitivity and 97% specificity whereas spectrophotometric method showed 90,5% sensitivity and 93,7% specificity. As a result, we can conclude that fluorometric method is superior to spectrophotometric method due to higher sensitivity and specificity.