title: Simultaneous determination of all forms of biopterin and neopterin in cerebrospinal fluid.
abstract: 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.
authors.affiliation: LETIAM, GCAPS, EA 4041, IUT d'Orsay, Université Paris Sud 11, Plateau de Moulon. 91400 Orsay, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Guibal
authors.forename: Nathalie
authors.initials: N
authors.lastname: Lévêque
authors.forename: Diane
authors.initials: D
authors.lastname: Doummar
authors.forename: Nicolas
authors.initials: N
authors.lastname: Giraud
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Roze
authors.forename: Diana
authors.initials: D
authors.lastname: Rodriguez
authors.forename: Rémy
authors.initials: R
authors.lastname: Couderc
authors.forename: Thierry
authors.initials: T
authors.lastname: Billette De Villemeur
authors.forename: Fathi
authors.initials: F
authors.lastname: Moussa
mesh_terms: D000328:Adult; D001708:Biopterins / Q000031:analogs & derivatives / Q000134:cerebrospinal fluid*; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005374:Filtration; D005453:Fluorescence; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008027:Light; D008297:Male; D013058:Mass Spectrometry; D008659:Metabolic Diseases / Q000134:cerebrospinal fluid; D019798:Neopterin / Q000031:analogs & derivatives / Q000134:cerebrospinal fluid*; D019906:Nuclear Magnetic Resonance, Biomolecular; D013997:Time Factors; D014466:Ultraviolet Rays; D055815:Young Adult
chemical_list: C000976:7,8-dihydroneopterin; D001708:Biopterins; D019798:Neopterin; C017226:7,8-dihydrobiopterin; C003402:sapropterin