Article pubs.acs.org/ac
Quantitative Analytical Method for the Determination of Biotinidase Activity in Dried Blood Spot Samples Eszter Szabó,† Ildikó Szatmári,† László Szőnyi,† and Zoltán Takáts*,‡ †
1st Department of Pediatrics, Semmelweis University, Budapest, 1085 Hungary Faculty of Medicine, Department of Surgery & Cancer, Imperial College, SW7 2AZ London, U.K.
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ABSTRACT: Biotinidase activity assay is included in most newborn screening protocols, and the positive results are confirmed by quantitative enzyme activity measurements. In our study, we describe a new quantitative analytical method for the determination of biotinidase activity using the blood sample deposited onto filter paper as the assay medium, by predepositing N-biotinyl-p-aminobenzoic acid onto the standard sample collection paper. The analysis of the assay mixture requires a simple extraction step from a dried blood spot followed by the quantification of product by LC-MS. The method provides a simple and reliable enzyme assay method that enables the rapid diagnosis of biotinidase deficiency (BD). Out of the measured 36 samples, 13 were healthy with lower enzyme activities, 16 were patients with partial BD, and 7 were patients with profound BD with residual activity below 10%. Expression of enzyme activity in percentage of mean activity of negative controls allows comparison of the different techniques. The obtained results are in good agreement with activity data determined from both dried blood spots and serum samples, giving an informative diagnostic value.
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profound consequences of untreated disease. The early detection of the disease and administration of pharmacological doses of biotin are necessary to prevent physical and neurological abnormalities of affected infants; therefore, many countries include BD in their newborn screening program.11 Due to the yet incomplete screening in a variety of countries, only rough estimates of incidence data are available, ranging from 1:33,000 to 1:500,0009 for profound biotinidase deficiency (0−10% of the mean normal activity) and from 1:14,00012 to 1:129,00013 for partial deficiency (10−30% activity). For BD patient stratification, quantitative enzyme activity measurements are necessary. There are various methods for the determination of biotinidase activity using different substrates, biological samples, and analytical techniques. The biotinidase enzyme hydrolyzes most artificially synthesized biotin conjugates such as biotinyl-p-aminobenzoate (B-PABA) or biotin-6-amidoquinoline. The most commonly used assay for biotinidase deficiency screening colorimetrically determines the quantity of p-aminobenzoate (PABA) produced from the hydrolysis of B-PABA.14−16 A semiquantitative newborn screening method for biotinidase deficiency was developed by Heard et al.17 In this case, normal biotinidase activity yields purple coloration resulting from the coupling reaction of the liberated PABA from B-PABA
iotinidase deficiency (BD) is an autosomal recessive metabolic disorder caused by the impaired activity of the biotinidase enzyme (biotin−amidohydrolase; EC 3.5.1.12) that hydrolyzes endogenous and dietary biocytin or short biotinylated peptides, liberating biotin for reutilization.1,2 Biotin, a water-soluble B-complex vitamin, plays an important role in human metabolism.3,4 It is the coenzyme for four integral carboxylase enzymes: acetyl CoA carboxylase, a pivotal enzyme in the synthesis of fatty acids; pyruvate carboxylase, which plays a role in the initial step of gluconeogenesis; propionyl CoA carboxylase, which catabolizes the amino acids valine, isoleucine, methionine, and threonine, as well as the odd-chain fatty acids and the side chain of cholesterol; and β-methylcrotonyl CoA carboxylase, involved in the catabolism of leucine.5 An insufficient amount of biotin in the body, caused by biotinidase deficiency, leads to activation of alternative metabolic pathways, resulting in the accumulation of abnormal quantities of related organic acids. The high concentration of organic acid metabolites can result in ketolactic acidosis and hyperammonemia. Individuals with untreated profound or partial biotinidase deficiency usually develop neurological symptoms, including seizures, hypotonia, feeding problems, ataxia, cognitive deficits, optic atrophy, and sensorineural hearing loss.6−8 Biotinidase deficiency can be treated efficiently by biotin supplementation. Based on the results of worldwide newborn screening, the estimated incidence of this disorder is 1:60,000.9 The importance of early detection of enzyme deficiencies has increased the significance of detection of biotinidase enzyme activity,10 due to the straightforward treatment option and the © XXXX American Chemical Society
Received: August 5, 2015 Accepted: September 23, 2015
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DOI: 10.1021/acs.analchem.5b02996 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry with N-1-naphthylethylendiamine dihydrochloride. The last step of this colorimetric assay determines the concentration of a free aromatic amino group containing species in the sample freed by hydrolysis. Each sample is then analyzed using an automated colorimetric plate reader device. Samples showing extinction at 546 nm higher than the cutoff value are considered to have normal biotinidase activity; those with insufficient extinction are considered to have abnormal activity. One of the problems of this method is that the presence of any free aromatic amino group results in the formation of an azodye-type chromophore with N-1-naphthylethylendiamine. Therapeutic drugs containing aromatic amino groups (e.g., sulfonamides), which may be administered to the infant or to the mother immediately before parturition, might interfere with the biotinidase assay. A further important disadvantage of this method is that the extinction has to be determined within 30 min after the final reaction was initiated because of the instability of the chromophore.17 An alternative method for the biotinidase activity assay is based on a semiquantitative fluorometric technique using biotin-6-amidoquinoline as the substrate. In this method, the chromophore formation reaches a plateau within 2 min after the addition of the final reagent and is stable in time due to the adsorption of the azo dye on the coexisting denatured proteins. By the use of this method, no increase in mean and maximum adsorbance was detected in the group of newborn children who were being treated with potentially interfering drugs at the time of blood sampling.18,19 Several special and laborious assays were developed; for example, the biotinidase enzyme activity was historically determined in urine and serum using the native substrate biocytin in an improved agar plate method employing Lactobacillus plantarum ATCC 8014. The major disadvantage of these methods is that they lack overall robustness.10,20 The use of dried blood spots (DBS) obtained from heel or finger pricks and spotted onto filter paper for the collection and analysis of human blood dates back to the early 1960s when Dr. Robert Guthrie used these specimens at first to determine blood phenylalanine levels in newborns for the detection of phenylketonuria.21 Introduction of this novel approach for blood collection made population screening of newborns and other clinical tests feasible. The use of DBS for enzyme assays offers a particular advantage over using conventional samples (e.g., tedious leukocyte isolation, tissue biopsy), however, only for enzymes present in the circulating blood. DBS samples are easy to collect, and only small blood volumes are required (
