Diagnosis of tetrahydrobiopterin deficiency using filter paper blood spots: further development of the method and 5 years experience

Abstract

In every newborn with even mild hyperphenylalaninemia (HPA) tetrahydrobiopterin (BH 4 ) deficiencies need to be excluded as soon as possible. Differential diagnosis is most commonly performed by analysis of urinary neopterin and biopterin. In 2005 a new method for the measurement of neopterin, biopterin and other pterins in dried blood spot (DBS) on filter paper was introduced. In order to evaluate the usefulness of this method as a standard tool for differential diagnosis of HPAs we analyzed neopterin, biopterin, pterin and dihydropteridine reductase activity in DBS from 362 patients with HPA over the period of five years. Age-dependent reference values were established for the HPA population. Sixty-four patients with BH 4 deficiency (27 patients with 6-pyruvoyl-tetrahydropterin synthase deficiency, seven with GTP cyclohydrolase I deficiency, and 30 with dihydropteridine reductase) were identified. Reference values for neopterin and biopterin in DBS were calculated for each of the variants. 6-pyruvoyl-tetrahydropterin synthase and GTP cyclohydrolase I deficiency can be diagnosed by neopterin and biopterin analysis alone, while for diagnosis of dihydropteridine reductase deficiency additional determination of enzyme activity from the same DBS is essential. Regarding test sensitivity, the interpretation of neopterin and biopterin concentration per hemoglobin is more valid than the interpretation of neopterin and biopterin per liter. Percentage of biopterin, of the sum of neopterin and biopterin should always be calculated. In addition, determination of hemoglobin concentration is essential as a measure for efficient extraction of neopterin and biopterin. Although the measurement of neopterin and biopterin in urine is more sensitive due to the higher concentrations present, our data prove the usefulness of their measurement from DBS for the routine diagnosis of BH 4 deficiencies.

Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficiencies

“…The use of DBS on filter paper (Guthrie card) is, however, more practical and allows measurement of pterins, DHPR activity, and amino acids from a single specimen [10]. It is important to know that patients with classic PKU excrete more pterins in urine compared with healthy controls and the amount of excreted metabolites is directly proportional to blood Phe levels.…”

“…Modified according to Opladen et al [10]. Dried blood spots (DBS) or random urine (U) can be used for the differential diagnosis and depending on the profile of neopterin (Neo), biopterin (Bio), and primapterin (Pri) and dihydropteridine reductase (DHPR) activity in DBS, diagnosis of following BH4 deficiencies can be established: GTP cyclohydrolase I (GTPCH) deficiency (low or no detectable neopterin and biopterin), 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency (high neopterin and low or no detectable biopterin), dihydropteridine reductase (DHPR) deficiency (normal neopterin and normal or elevated biopterin and no DHPR activity), and pterins-4a-carbinolamine dehydratase (PCD) deficiency (elevated neopterin, low-normal biopterin, and elevated primapterin).…”

An international survey of patients with tetrahydrobiopterin deficiencies presenting with hyperphenylalaninaemia

“…The diagnostic work-up of BH 4 deficiencies includes the analysis of pterins (neopterin, biopterin, and primapterin) in urine or dried blood spots (DBSs) and the measurement of DHPR activity in the DBSs. In patients where abnormal pterins or reduced DHPR enzyme activity is found the analysis of biogenic amines, pterins, and folates in CSF should follow for further differentiation of the underlying disease (Blau and Burgard 2006;Opladen et al 2011a). The BH 4 loading test is an additional useful tool for the differential diagnosis of BH 4 deficiencies.…”

Water-Soluble Vitamins and Nonnutrients

“…Deficiencies may be detected by measuring tetrahydrobiopterin concentrations in the blood or in dried blood spots (Opladen et al, 2011). Such assessment is particularly important for newborn screening because BH4 deficiency can cause PKU and responds well to treatment.…”