7-8-dihydrobiopterin and 7-8-dihydroneopterin

In humans, genetic defects of the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor in hydroxylation reactions, are associated with severe neurological disorders. The diagnosis of these conditions relies on the determination of BH4, dihydrobiopterin (BH2), and dihydroneopterin (NH2) in cerebrospinal fluid (CSF). As MS/MS is less sensitive than fluorescence detection (FD) for this purpose, the most widely used method since 1980 involves two HPLC runs including two differential off-line chemical oxidation procedures aiming to transform the reduced pterins into their fully oxidized fluorescent counterparts, biopterin (B) and neopterin (N). However, this tedious and time-consuming two-step indirect method underestimates BH4, BH2, and NH2 concentrations. Direct quantification of BH4 is essential for studying its metabolism and for monitoring the efficacy of BH4 supplementation in patients with genetic defects. Here we describe a single step method to simultaneously measure BH4, BH2, B, NH2, and N in CSF by HPLC coupled to FD after postcolumn coulometric oxidation. All target pterins were quantified in CSF with a small volume (100 μL), and a single filtration step for sample preparation and analysis. As compared to the most widely used method in more than 100 CSF samples, this new assay is the easiest route for accurately determining in a single run BH4, BH2, and NH2 in CSF in deficit situations as well as for monitoring the efficacy of the treatment.

Topics: Adult; Biopterins; Child, Preschool; Chromatography, High Pressure Liquid; Female; Filtration; Fluorescence; Humans; Infant; Infant, Newborn; Light; Male; Mass Spectrometry; Metabolic Diseases; Neopterin; Nuclear Magnetic Resonance, Biomolecular; Time Factors; Ultraviolet Rays; Young Adult

7,8-Dihydrobiopterin (H(2)Bip) and 7,8-dihydroneopterin (H(2)Nep) belong to a class of heterocyclic compounds present in a wide range of living systems. H(2)Bip accumulates in the skin of patients suffering from vitiligo, whereas H(2)Nep is secreted by human macrophages when the cellular immune system is activated. We have investigated the photochemical reactivity of both compounds upon UV-A irradiation (320-400 nm), the chemical structures of the products and their thermal stability. The study was performed in neutral aqueous solutions. The reactions were followed by UV/Visible spectrophotometry and HPLC and the products were analyzed by means of electrospray ionization mass spectrometry and (1)H-NMR. Excitation of H(2)Bip and H(2)Nep leads to the formation, in each case, of two main isomeric dimers. The latter compounds undergo a thermal process that may consist in a retro [2 + 2]-cycloaddition and hydrolysis to yield the reactant (H(2)Bip or H(2)Nep) and a product that has incorporated a molecule of H(2)O.

Topics: Biopterins; Chromatography, High Pressure Liquid; Dimerization; Humans; Isomerism; Macrophages; Magnetic Resonance Spectroscopy; Neopterin; Photolysis; Spectrometry, Mass, Electrospray Ionization; Temperature; Ultraviolet Rays

Pterins belong to a class of heterocyclic compounds present in a wide range of living systems and accumulate in the skin of patients affected by vitiligo, a depigmentation disorder. The study of the emission of 7,8-dihydropterins is difficult because these compounds are more or less unstable in the presence of O(2) and their solutions are contaminated with oxidized pterins which have much higher fluorescence quantum yields (Φ(F)). In this work, the emission properties of six compounds of the dihydropterin family (6-formyl-7,8-dihydropterin (H(2)Fop), sepiapterin (Sep), 7,8-dihydrobiopterin (H(2)Bip), 7,8-dihydroneopterin (H(2)Nep), 6-hydroxymethyl-7,8-dihydropterin (H(2)Hmp), and 6-methyl-7,8-dihydropterin (H(2)Mep)) have been studied in aqueous solution. The fluorescence characteristics (spectra, Φ(F), lifetimes (τ(F))) of the neutral form of these compounds have been investigated using the single-photon-counting technique. Φ(F) and τ(F) values obtained lie in the ranges 3-9 × 10(-3) and 0.18-0.34 ns, respectively. The results are compared to those previously reported for oxidized pterins.

Topics: Biopterins; Neopterin; Oxidation-Reduction; Oxygen; Pterins; Quantum Theory; Solutions; Spectrometry, Fluorescence; Water

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.

Topics: Aminohydrolases; Animals; Biopterins; Folic Acid; GTP Cyclohydrolase; Kinetics; Liver; Neopterin; Oxidation-Reduction; Pteridines; Pterins; Rats; Xanthopterin

Luminol-dependent chemiluminescence of normal human monocytes activated by zymosan is demonstrated to be inhibited by tetrahydrobiopterin in a concentration-dependent manner. The reduced pterins tetrahydrobiopterin, dihydrobiopterin, and dihydroneopterin are all shown to be readily oxidized by the hydroxyl radical. The susceptibility of reduced pterins to free radical attack may explain the inhibition of chemiluminescence observed and an additional role of reduced pterins as free radical scavengers in tissues is considered.

Topics: Ascorbic Acid; Biopterins; Dithioerythritol; Free Radicals; Humans; Hydrogen Peroxide; In Vitro Techniques; Luminescent Measurements; Macrophages; Monocytes; Neopterin; Oxidation-Reduction; Pteridines