Simultaneous Determination of All Forms of Biopterinand Neopterin in Cerebrospinal Fluid

摘要

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 coulometricoxidation. All target pterins were quantified in CSF with a smallvolume (100 μL), and a single filtration step for sample preparationand analysis. As compared to the most widely used method in more than100 CSF samples, this new assay is the easiest route for accuratelydetermining in a single run BH4, BH2, and NH2 in CSF in deficit situationsas well as for monitoring the efficacy of the treatment.