Metabolism and regulation of tetrahydrobiopterin and its implications for BH4-responsive hyperphenylalaninemia and BH4-deficiencies

The autosomal recessive inherited, metabolic disorder phenylketonuria (PKU) is caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH) – a key enzyme in the catabolism of phenylalanine. PKU patients present with noxiously increased concentrations of the amino acid phenylalanine in the plasma, leading to a so-called hyperphenylalaninemia. This disorder is treatable by avoiding uptake of phenylalanine. As this amino acid is naturally ubiquitously present in normal nutrition, patients have to follow a very strict and artificial diet in order to evade the grave consequences, such as severe mental retardation, they would have to expect otherwise. In the 1970s it was realised that there exists another cause of hyperphenylalaninemia which led to the detection of tetrahydrobiopterin deficiency. The connecting link between these two disor-ders is tetrahydrobiopterin (BH4), the natural cofactor of phenylalanine hydroxylase and other members of the aromatic amino acid hydroxylases, enzymes pivotal for catecholamine and serotonin biosynthesis. BH4 serves also as an essential cofactor for other enzymes (nitric oxide synthase and glyceryl-ether monooxygenase) and has additional functions on a cellular level. In recent years an other new variant of hyperphenylalaninemia/PKU was described (BH4-responsive HPA/PKU). Patients with this type of PKU are characterised by a marked reduction and normalisation of the increased phenylalanine concentration after oral loading with BH4. This finding opened a new perspective for a pharmacological treatment of so-called BH4-responsive HPA/PKU, as an alternative to the phenylalanine restricted diet which has notoriously a low compliance. With BH4 tested in medication of BH4-responsive HPA/PKU patients and being already used to treat BH4-deficient patients, our interest in the effects of exogenous BH4 on the organism was aroused. We started a project to study the influence of BH4, its metabolism and regulation. In the course of our study, we developed a new method for the measurement of different pterins (neopterin, biopterin, and pterin) in dried blood spots, which could be of use as an alternative in the screening for BH4 deficiencies. We identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using this method. Extensive pharmacokinetic studies of BH4 have been performed in animal models but only few parameters are known from studies in humans. With the dried blood spots method we analysed the pharmacokinetics of orally administered BH4 in 71 patients with hyperphenylalaninemia and calculated a rapid absorption- (1.1 h) and distribution phase (2.5 h) and a slower elimination phase (46 h). Previous findings of others, that BH4-responsiveness was higher among patients with mild PAH mutations, could be confirmed.In a third part we looked at the molecular genetics of BH4 responsive HPA/PKU patients. By virtue of data mining in the BIOPKU database we identified 60 different mutations associated with BH4 responsiveness and analysed the frequency of potentially responsive genotypes and their dispersal in Europe. We estimated an average of 55% responsive amongst HPA/PKU patients in Europe. We also studied the outcome and made a long-term follow-up of 36 patients with BH4 deficiency, 26 with a 6-pyruvoyl-tetrahydropterin deficiency and 10 with dihydropteridine reductase deficiency, the two most common forms of BH4-deficiency. Our data suggested that diagnosis within the first month of life is essential for a good outcome and that low 5-hydroxyindolacetic acid and homovanillic acid values in cerebrospinal fluid could be an indicator for the ongoing developmental impairment, even in absence of neurological symptoms. In a last part, we investigated in the effects of BH4 on the metabolism and regulation of enzymes, either directly involved in the biosynthesis and regeneration of BH4 or otherwise associated with BH4 metabolism. The experiments were performed employing various cell lines, which were treated by supplementation of BH4 and other agents with either stimulating or inhibiting effects. In most investigated cell lines, after supplementation with cytokines, a significant and strong up-regulation (~50-fold) of the gene expression of GCH1 was found, the gene encoding for GTP cyclohydrolase I, the first and rate limiting enzyme in BH4 de novo biosynthesis. Furthermore, the expression of AKR1B1, involved in alternative pathway, was found to be upregulated (~4-fold). A slight but significant reduction of the transcription of QDPR, coding for the enzyme dihydropteridine reductase, was observed after supplementation with sepiapterin, known to be taken up quickly by cell cultures and intracellularly converted to BH4