Hans-Christoph Curtius

Neopterin in clinical practice

Neopterin and the closely related biopterin belong to a group of pteridine compounds containing a 2-amino, 4-0~0, pyrimidino-pyrazino-(pterin) ring, with a 3-carbon side-chain on carbon 6. Pteridines were first described as pigments of insects and lower vertebrates [ll. During the past ten years increased concentrations of neopterin in body fluids have been reported in patients with three groups of disorders the hyperphenylalaninaemias [2], in malignancy 131 and in several inflammatory disease states [3,4]. Since then a rapidly increasing number of publications appeared, indicating a positive correlation between neopterin concentrations and activity of prognosis of a variety of disorders. On the basis of these findings it has been proposed that measurement of neopterin should be used more widely in clinical practice. Others, however, felt, that the clinical utility of neopterin measurements is limited. This, because of the wide range of stimuli that increase neopterin concentrations, of the consistent overlap between neopterin results in patients and in controls and because of the relatively small difference between disease categories [5]. This articles will critically review present knowledge of neopterin biosynthesis and use of neopterin assays in clinical practice.

7-SUBSTITUTED PTERINS : FORMATION DURING PHENYLALANINE HYDROXYLATION IN THE ABSENCE OF DEHYDRATASE

Previously we described a new form of human hyperphenylalaninemia characterized by the formation of 7-substituted pterins. We present evidence strongly suggesting that the 7-substituted pterins are formed by rearrangement of 6-substituted pterins. This rearrangement occurs during the phenylalanine hydroxylase reaction cycle which normally involves the enzymes phenylalanine hydroxylase, pterin-4a-OH-dehydratase, and q-dihydropterin reductase, specifically in the absence of dehydratase activity. We conclude that formation of 7-substituted pterins in humans is a consequence of an absence of dehydratase activity, which might result from a genetic defect. A chemical mechanism for this rearrangement is presented. Our results also suggest that tetrahydroneopterin can be a cofactor for the phenylalanine hydroxylase system in vivo.

BIOSYNTHESIS OF TETRAHYDROBIOPTERIN: POSSIBLE INVOLVEMENT OF TETRAHYDROPTERIN INTERMEDIATES

The biosynthetic pathway of tetrahydrobiopterin (BH(4)) from dihydroneopterin triphosphate (NH(2)P(3)) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH(2)P(3) was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH(4) to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH(4) biosynthesis under normal in vivo conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH(2)P(3) may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH(4) by sepiapterin reductase. This proposed pathway can explain ho the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH(4) biosynthesis in vivo .

Human liver 6-pyruvoyl tetrahydropterin reductase is biochemically and immunologically indistinguishable from aldose reductase

6-Pyruvoyl tetrahydropterin reductase has been implicated in the biosynthesis of tetrahydrobiopterin. Using immunochemical and biochemical techniques the purified human liver enzyme was shown to be identical to aldose reductase. This suggests that 6-pyruvoyl tetrahydropterin reductase may play an additional role in the reduction of aldehydes derived from the biogenic amine neuro-transmitters and corticosteroid hormones as well as in the pathogenesis of diabetic complications, as has been postulated for aldose reductase.

Purification of 6-pyruvoyl-tetrahydropterin synthase from human liver

The enzyme which catalyzes the first step in the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin has been purified approx. 40,000-fold from human liver to apparent homogeneity. The enzyme has a native molecular weight of approximately 83,000 and consists of four identical subunits, each of which has a molecular weight of approximately 19,000. It contains carbohydrates and is remarkably stable to heat treatment. In the presence of purified sepiapterin reductase, Mg2+, and NADPH, this enzyme catalyzes efficiently the formation of tetrahydrobiopterin from dihydroneopterin triphosphate. This indicates that these two proteins are sufficient for the overall conversion.

Hyperphenylalaninaemia presumably due to carbinolamine dehydratase deficiency: Loading tests with pterin derivatives

Primapterinuria, a recently discovered variant of hyperphenylalaninaemia, is characterized by the excretion of 7-substituted pterins in the patients urine (Curtius et al 1988). Although 9 patients have already been diagnosed worldwide (Dhondt et al 1987, 1988; Blaskovics and Giudici 1988; Blau et al 1988), the exact metabolic defect is still hypothetical. Patients with primapterinuria show an increased ratio of neopterin to biopterin excretion, excretion of subnormal levels of biopterins, and normal levels of biogenic amines in CSF (Blau et al 1989; Curtius et al 1990a)

Bestimmung von freien aminosäuren im augenkammerwasser des menschen bei homocystinurie-patienten und kontrollfällen

Abstract The concentrations of amino acids in the aqueous humor of the eye were determined by column chromatography. Six control subjects and 2 patients with homocystinuria were studied. The concentrations of amino acids in the aqueous humor of the control subjects were compared with the concentrations in plasma of normal subjects. For most amino acids the concentrations in the aqueous humor were similar to those found in plasma, but valine and asparagine-glutamine in the aqueous humor were relatively high, proline, glycine, glutamic acid and ornithine relatively low. In the patients with homocystinuria a considerable amount of homocystine was detected and methionine was markedly elevated. The small amounts of aqueous humor available for analysis make a modification of the amino acid analyzer and of the recording system necessary.

Characterization of human pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha, and of the Cys81-mutant involved in hyperphenylalaninæmia

Human pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-l a (in the following abbreviated PC D) is a small protein, which has two apparent functions: It assists the phenylalanine hydroxylase (PAH) reaction in that it catalyzes the dehydration of the intermediate pterin-4a-carbinolamine (1), and it has been proposed to induce dimerization of the named factorla (2). The amino acid sequence of the protein has been solved (3), and its 3D-structure has been reported (4). Its CysSIArg mutant has been proposed to cause hyperphenylalaninremia (5) and to be at the origin of the high content of 7-pterins in the patient. We report here on some properties of PCD which has been expressed in E. coli and purified, as well as of two CysSI mutants (Ser and Arg). Details of the present work have been submitted to EJB for publication (6).

Pterin−4a−carbinolamine dehydratase from Pseudomonas aeruginosa: characterization, catalytic mechanism and comparison to the human enzyme

The three-dimensional structure of pterin-4a-carbinolamine dehydratase (PCD) from Pseudomonas aeruginosa has been solved. Based on this we have investigated the roles of putative active center residues through functional replacement by site-directed mutagenesis. Three histidines, His73, His74 and His91, appear to be involved in dehydration catalysis. The three-dimensional positions of these residues match those of corresponding histidines at the active center of human PCD. Based on the coincidence of catalytic parameters, and on the similar effects induced by the mutations, it is concluded that the substrate binding mode and the reaction mechanisms of bacterial and human PCD are basically identical.

Progress in the Study of Biosynthesis and Role of 7-Substituted Pterins: Function of Pterin-4a-Carbinolamine Dehydratase

A new form of atypical phenylketonuria (PKU) was discovered in 1988. Characteristic for this transient hyperphenylalaninemia is the excretion of 7-substituted pterins in patients’ urine, i.e. L-primapterin (7-isomer of L-biopterin), D- or L-ana- pterin (7-isomer of D- or L- neopterin) and 6-oxo-L- or D-primapterin (7-isomer of 7-oxo-L- or D-biopterin) (1,2,3).