Comparison of Spectrophotometric and Fluorimetric Methods in Evaluation of Biotinidase Deficiency Translated title: POREĐENJE SPEKTROMETRIJSKOG I FLUORIMETRIJSKOG METODA U PROCENI NEDOSTATKA BIOTINIDAZE

Summary

Background

Biotin, a water-soluble vitamin, is used as a co-factor by enzymes involved in carboxylation reactions. Biotinidase (BTD) catalyzes the recycling of biotin from endogenous and dietary sources. Biotinidase deficiency (BD) is an autosomal recessively inherited disorder of biotin recycling that is associated with neurologic and cutaneous consequences when untreated. The aim of the study was to compare the results of spectrophotometric and fluorimetric methods, as well as to evaluate the advantages and disadvantages of both methods in current research practices.

Methods

Study group was chosen among the BD suspected newborn, children and parents (n = 52) who applied to Hacettepe University Pediatric Metabolism Unit.

Results

BTD activity is stable for 2 hours at room temperature and at 4 °C, and for 4 months at –20 °C and –80 °C. Genetic and clinical results showed that 25% of the total number of patients had complete BD which was treated with 10 mg/day biotin, while 15.38% of the patients had partial BD, and they were prescribed biotin 5 mg/day. The area under the ROC curve was 0.960±0.25 and 0.927± 0.41 for the fluorimetric and spectrophotometric method, respectively. Fluorimetric method showed 100% sensitivity and 97% specificity, whereas spectrophotometric method showed 90.5% sensitivity and 93.7% specificity.

Conclusions

Fluorimetric method is superior to the spectrophotometric method due to higher sensitivity and specificity.

Kratak sadržaj

Uvod

Enzimi uključeni u reakcije karboksilacije koriste biotin, vitamin rastvorljiv u vodi, kao kofaktor. Biotinidaza (BTD) katališe recikliranje biotina iz endogenih izvora i iz ishrane. Nedostatak biotinidaze (NB) predstavlja autozomni recesivni nasledni poremećaj recikliranja biotina koji se u slučaju nelečenja povezuje s neurološkim i kožnim posledicama. Cilj ove studije bio je da se uporede rezultati spektrofotometrijskog i fluorimetrijskog metoda, kao i da se procene prednosti i mane oba ova metoda u današnjoj istraživačkoj praksi.

Metode

Studijsku grupu smo izabrali između novorođenčadi, dece i roditelja (n=52) sa sumnjom na NB koji su se javili Pedijatrijskom odeljenju za metabolizam Univerziteta Hacettepe.

Rezultati

Aktivnost BTD stabilna je 2 sata na sobnoj temperaturi i na 4 °C, a 4 meseca na –20 °C i –80 °C. Genetski i klinički rezultati pokazuju da 25% ukupnog broja obolelih ima potpuni NB, koji je tretiran sa 10 mg biotina dnevno, dok 15,38% obolelih ima delimični NB, tretiran sa 5 mg biotina dnevno. Oblast ispod ROC krive iznosila je 0,960±0,25 za fluorimetrijski, odnosno 0,927±0,41 za spektrofotometrijski metod. Fluorimetrijski metod pokazao je osetljivost od 100% i specifičnost od 97%, dok je spektrofotometrijski metod pokazao osetljivost od 90,5% i specifičnost od 93,7%.

Zaključak

Fluorimetrijski metod je superioran u odnosu na spektrofotometrijski zbog svoje veće osetljivosti i specifičnosti.