7-8-dihydroneopterin and sapropterin

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

Pteridines are heterocyclic compounds which are synthesized and released by human monocytes/macrophages following stimulation by interferon-gamma. Their concentration in various body fluids proved to be indicative for the stimulation of the cellular immune system, and determination of pteridines has become an important diagnostic tool. We show that pteridine derivatives, namely neopterin (N), 7,8-dihydroneopterin (NH2), and 5,6,7,8-tetrahydrobiopterin (BH4) increase intracellular calcium (Cai) in human monocytic cells. Significant increases of Cai are observed at 10 nmol/l NH2, at 100 nmol/l BH4 and at 1 mol/l N, i.e. at concentrations encountered in vivo. At a concentration of 1 mumol/l, Cai is increased (from a control value of 145 +/- 7 nmol/l) to 464 +/- 62 nmol/l (NH2), 340 +/- 41 nmol/l (BH4) and 344 +/- 46 nmol/l (N), respectively. The increase of Cai depends on the presence of extracellular calcium and is likely to be due to activation of a calcium channel. We show that the absence of extracellular calcium or the addition of lanthanum ions to the extracellular fluid fully reverses the pteridine-induced increase of Cai. According to these observations, pteridines may mimic the effects of other inflammatory mediators on monocytic cells and seem to be involved in the crosstalk of immunocompetent cells.

Topics: Biopterins; Calcium; Cell Line; Egtazic Acid; Humans; Lanthanum; Monocytes; Neopterin; Pteridines

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

Topics: Aminohydrolases; Biopterins; GTP Cyclohydrolase; Humans; Infant, Newborn; Leukocyte Count; Liver; Male; Neopterin; Phenylalanine; Pteridines; Pterins; T-Lymphocytes