Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper

doi:10.1016/j.ymgme.2005.09.011

Molecular Genetics and Metabolism

Volume 86, Supplement, December 2005, Pages 96-103

https://doi.org/10.1016/j.ymgme.2005.09.011 Get rights and content

Abstract

Tetrahydrobiopterin (BH₄) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH₄ deficiencies, and to identify different variants of BH₄ deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH₄ deficiencies in urine and for the monitoring of BH₄ pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.

Introduction

Tetrahydrobiopterin (BH₄) is the essential co-factor/co-substrate of phenylalanine hydroxylases (PAH) and several other monooxygenases [1]. Measurement of pterins in different biological fluids is the most common method for the screening and differential diagnosis of inborn errors of BH₄ metabolism. Five distinct genetic defects are known to cause hyperphenylalaninemia (HPA), including the classical form of Phenylketonuria (PKU) with a defect in the apo-enzyme PAH or a defect in four out of five BH₄ co-factor-synthesizing or regenerating enzymes [2]. Either of two defects in biosynthesis of BH₄, i.e., GTP cyclohydrolase I (GTPCH) or 6-pyruvoyl-tetrahydropterin synthase (PTPS) or defects in regeneration, i.e., pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) may be responsible for BH₄ deficiency. BH₄ deficiency is a severe but treatable disease and early detection in newborns is essential to avoid irreversible brain damage.

According to the current protocol, the following investigations should be performed in all newborns with HPA (blood phenylalanine >120 μmol/L): 1. analysis of pterins (neopterin, biopterin, and primapterin) in urine; 2. measurement of DHPR activity in dried blood spots from Guthrie card; and 3. analysis of phenylalanine and tyrosine in plasma or blood before and after BH₄ loading with 20 mg/kg body weight. Tests 1 and 2 are essential for all newborns and identify primarily variants of BH₄ deficiency in older children due to characteristic pterin patterns: GTPCH deficiency with low neopterin and biopterin, PTPS deficiency with high neopterin and only traces of biopterin, PCD deficiency with high neopterin, moderately biopterin and high primapterin, and DHPR deficiency with normal or moderate elevated neopterin and high biopterin. Test 2 identifies patients with DHPR deficiency, which can sometimes be missed by test 1 if the urine is collected under low-protein diet. Test 3 is useful in all forms of BH₄ deficiency and can also detect patients with BH₄-responsive PAH deficiency [3].

With the introduction of tandem mass-spectrometry (TMS) in newborn screening, blood sample collection on filter paper became a routine procedure. The aim of this study was to test the use of filter paper blood spots (Guthrie cards) instead of urine for screening of BH₄ deficiencies and to study the pharmacokinetics of BH₄ by monitoring blood concentrations of pterins following oral loading test in patients with HPA. The new method should enable simultaneous measurement of pterins (neopterin, biopterin, isoxanthopterin, and pterin), DHPR activity, and amino acids from a single Guthrie card specimen. The main advantage of using Guthrie cards instead of urine is the easy handling and sample collection, and the less expensive shipping of the samples at room temperature.

Section snippets

Materials and methods

Pterins were purchased from Schircks Laboratories (Jona, Switzerland). All other chemicals were of the highest quality available.

Results

BH₄ is extremely unstable in collected blood and about 30–40% is readily decomposed to pterin. Thus, in this study total biopterin was calculated as the sum of biopterin and pterin (biopterin + pterin).

Discussion

Dried blood spots on filter paper (Guthrie cards) were introduced in the early 1960s for the newborn screening of few common and treatable inherited metabolic diseases, including PKU [5]. With the introduction of TMS, a number of new tests were developed for blood spots and blood collection on filter paper became a practical alternative for measurement of metabolites such as amino acids and acylcarnitines in serum, plasma, or even urine. A minimal sample volume is required and samples can be

Acknowledgment

This work was supported by the Swiss National Science Foundation Grant No. 310000-107500/1.

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Development of a new UPLC-ESI-MS/MS method for the determination of biopterin and neopterin in dried blood spot
2017, Clinica Chimica Acta
Citation Excerpt :
This method could be widely used since LC-MS/MS has become more and more available in recent years and it has already been introduced into routine laboratories, especially for newborn screening [6]. Driven by the advantages of using DBS in 2005 a HPLC method for the measurement of Neo, Bio and pterin in dried blood spot (DBS) was described [4]. It was applied to a large population of patients with HPA [5] demonstrating to be very useful in the diagnosis of BH4 deficiencies: pterins pattern obtained by HPLC analysis of DBS of patients with PTPS and GTPCH deficiency showed a sensitivity of 96.3.
(6R)-5,6,7,8-tetrahydrobiopterin (BH4) deficiencies are rare inherited defects of synthesis or regeneration of BH4. Due to the resulting hyperphenylalaninemia (HPA), some of them are detected by newborn screening and require the assessment of the pattern of neopterin (Neo) and biopterin (Bio) excretion in urine to be confirmed. Aim of present study was to develop a method for the measurement of these diagnostic biomarkers in dried blood spot (DBS).
After DBS extraction, samples were filtered and injected into the UPLC column coupled with a tandem mass spectrometer working in positive electrospray ionization.
The chromatographic separation was accomplished in 6 min. The LoQ was 0.57 and 1.45 nmol/l of blood for Neo and Bio respectively and the response was linear over the range 0–100 nmol/l of blood. The within- and between-day imprecision was < 6.4 and 10.8% respectively. Reference ranges for newborns, infants and children/adult were established. The method was tested in 11 patients affected by BH4 defects.
The assessment of Neo and Bio in DBS is reliable and sensitive and may be proposed as a second tier test for the newborns with hyperphenylalaninemia (HPA) as well as a new potential diagnostic tool for symptomatic subjects with BH4 deficiencies.
Diagnosis of aromatic l-amino acid decarboxylase deficiency by measuring 3-O-methyldopa concentrations in dried blood spots
2014, Clinica Chimica Acta
Inherited defects that affect the synthesis or metabolism of neurotransmitters cause severe motor dysfunction. The diagnosis of these diseases, including aromatic l-amino-acid decarboxylase (AADC) deficiency, typically requires cerebrospinal fluid (CSF) neurotransmitter analysis. However, 3-O-methyldopa (3-OMD), which is a catabolic product of l-dopa that accumulates in individuals with AADC deficiency, can be detected in blood.
3-OMD concentrations were measured in dried blood spots (DBSs). One 3.2-mm punch was eluted with 90% methanol containing a deuterated internal standard (3-OMD-d3), and then analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS).
3-OMD in DBSs was shown to be stable for more than 28 days at 37 °C. We measured DBS 3-OMD concentrations in controls and patients with AADC deficiency. 3-OMD concentrations in normal newborns and children decreased with age. Patients with AADC deficiency revealed > 15-fold increase of DBS 3-OMD concentrations. Archive newborn screening DBS samples, obtained from 6 patients with AADC deficiency, revealed more than 19-fold increase of 3-OMD concentrations.
We demonstrated that DBS 3-OMD concentrations were highly elevated in newborns and children with AADC deficiency. Because 3-OMD is stable in DBS, this method can be used for both high risk and newborn screening of AADC deficiency.
Pitfalls in phenylalanine loading test in the diagnosis of dopa-responsive dystonia
2013, Molecular Genetics and Metabolism
Citation Excerpt :
Phe and Tyr in DBS were analyzed using tandem mass spectrometry (TMS) following standard methods. Biopterin in plasma was measured as described previously [14], but can be measured in DBS as well [15]. Detailed patient's case report will be published elsewhere.
Phenylalanine (Phe) loading test is a useful tool in the differential diagnosis of dopa-responsive dystonia due to autosomal dominant or recessive GTP cyclohydrolase I (GTPCH) deficiency or autosomal recessive sepiapterin reductase (SR) deficiency. In these patients hepatic phenylalanine hydroxylase system is compromised due to subnormal tetrahydrobiopterin (BH₄) levels and hydroxylation of phenylalanine (Phe) to tyrosine (Tyr) is reduced with elevated Phe/Tyr ratio 1–2 h after oral Phe administration (100 mg/kg bw) administration. In healthy persons there is only a modest increase in Tyr production and blood Phe normalizes after 4 h. We report on a challenge with Phe (100 mg/kg bw) in a patient with dopa-responsive dystonia while on therapy with BH₄ and l-dopa. During Phe challenge Phe concentration remained below the normal range while a transient mild hypertyrosinemia was observed, leading to an extremely low Phe/Tyr ratio. A repeated test, after BH₄ withdrawal, reversed the findings and resulted normal. These data suggest activation of hepatic phenylalanine hydroxylase by BH₄. Thus, the Phe loading test should not be performed during substitution with BH₄.
Pulmonary hypertension in the newborn GTP cyclohydrolase I-deficient mouse
2011, Free Radical Biology and Medicine
Tetrahydrobiopterin (BH4) is a regulator of endothelial nitric oxide synthase (eNOS) activity. Deficient levels result in eNOS uncoupling, with a shift from nitric oxide to superoxide generation. The hph-1 mutant mouse has deficient GTP cyclohydrolase I (GTPCH1) activity, resulting in low BH4 tissue content. The adult hph-1 mouse has pulmonary hypertension, but whether such condition is present from birth is not known. Thus, we evaluated newborn animals’ pulmonary arterial medial thickness, biopterin content (BH4 + BH2), H₂O₂ and eNOS, right ventricle-to-left ventricle + septum (RV/LV + septum) ratio, near-resistance pulmonary artery agonist-induced force, and endothelium-dependent and -independent relaxation. The lung biopterin content was inversely related to age for both types, but significantly lower in hph-1 mice, compared to wild-type animals. As judged by the RV/LV + septum ratio, newborn hph-1 mice have pulmonary hypertension and, after a 2-week 13% oxygen exposure, the ratios were similar in both types. The pulmonary arterial agonist-induced force was reduced (P < 0.01) in hph-1 animals and no type-dependent difference in endothelium-dependent or -independent vasorelaxation was observed. Compared to wild-type mice, the lung H₂O₂ content was increased, whereas the eNOS expression was decreased (P < 0.01) in hph-1 animals. The pulmonary arterial medial thickness, a surrogate marker of vascular remodeling, was increased (P < 0.01) in hph-1 compared to wild-type mice. In conclusion, our data suggest that pulmonary hypertension is present from birth in the GTPCH1-deficient mice, not as a result of impaired vasodilation, but secondary to vascular remodeling.
New insights into tetrahydrobiopterin pharmacodynamics from Pah<sup>enu1/2</sup>, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency
2010, Biochemical Pharmacology
Citation Excerpt :
Venous blood samples (approximately 40 μl) were collected on filter cards before and 20, 40, 60, 120, and 180 min after injection. Total biopterin (BH4, dihydrobiopterin, and biopterin) was quantified by High Pressure Liquid Chromatography (HPLC) as previously described [29,30]. Biopterin clearance was determined by non-linear regression of a double exponential Bateman function as described by Koch et al. [31].
Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH₄) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah^enu1/2 bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH₄ treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH₄ pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using ¹³C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah^enu1/1, and Pah^enu1/2 mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah^enu1/1 and Pah^enu1/2 indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH₄. In conclusion, our findings show a significant impact of the genotype on the response to BH₄ in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.
Genotype-predicted tetrahydrobiopterin (BH<inf>4</inf>)-responsiveness and molecular genetics in Croatian patients with phenylalanine hydroxylase (PAH) deficiency
2009, Molecular Genetics and Metabolism
Specific mutations in the gene encoding phenylalanine hydroxylase (PAH), located on chromosome 12q22-24.1, are linked to tetrahydrobiopterin (BH₄; sapropterin)-responsive phenylketonuria (PKU). Diagnosis is usually done through the newborn screening for PKU, followed by a BH₄ loading test. So far, more than 60 mutant alleles, presenting with a substantial residual PAH activity (average ∼47%), were identified in more than 500 patients worldwide. We investigated the predictive value of BH₄-responsive PAH mutations in Croatian population. From a group of 127 PKU patients, 62 were selected (based on the genotype) as potentially BH₄-responsive and 39 loaded with BH₄ (20 mg/kg). The overall frequency of BH₄-responsiveness (>30% blood phenylalanine reduction within 24 h) was 36% (14 out of 39 patients with 23 different genotypes), significantly less than expected. The best responders were patients with mild hyperphenylalaninemia (4/4; 100%), followed by mild PKU (8/9; 89%), and classical PKU (2/26; 8%). The most common BH₄-responsive genotypes were p.E390G/p.R408W and p.P281L/p.E390G. These genotypes correspond for approximately >30% residual PAH activity. The p.E390G mutation was 100% associated with BH₄-responsiveness, regardless of the second allele (p.R408W, p.P281L, p.F55Lfs, p.L249P). With regard to the predicted relative PAH activity of recombinantly expressed mutant alleles, there was a significant (p < 0.002) difference between BH₄-responders and non-responders.
In a general Croatian PKU population, disease-causing mutations were identified on 226 alleles (99%). There were 35 different mutations: 21 missense, 8 splice site, 3 nonsense, 2 single nucleotide deletions, and 1 in-frame deletion. Four mutations are reported for the first time: p.E76D, p.L333P, p.G346E, and IVS8-2A > G. Five mutations accounted for over two-thirds of investigated alleles: p.L48S, p.R261Q, p.P281L, p.E390G, and p.R408W. Thus, the Croatian PKU population seems to be more homogenous than some other Mediterranean or Central European populations.
This study reveals the importance of a full genotype for the prediction of BH₄-responsiveness. In contrast to previous assumption and with exception of the p.E390G mutation, single allele mutations are not reliable for the selection of potential PKU candidates for pharmacological therapy with BH₄.

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Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgment

Mol. Genet. Metab.

Clin. Chim. Acta

Tetrahydrobiopterin biosynthesis, regeneration, and functions

Biochem. J.

Disorders of tetrahydrobiopterin and related biogenic amines

Disorders of phenylalanine and tetrahydrobiopterin