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.

Pterin determination in cerebrospinal fluid: state of the art

“…Besides, more recently, some authors compared the two proposed chemical oxidation procedures iodine vs. M n O 2 , and they concluded that the oxidation with iodine results in the most accurate determination of N and B in CSF [25]. However, it is now well established that chemical oxidation methods are to underestimate amounts of reduced forms of N and B [9,15].…”

“…Recently, the effects of pH on the separation and the electro-oxidation of the targeted pterins have been extensively studied [9]. The authors observed that high oxidation yields (≥50%) of BH2 and NH2 can be obtained irrespective of the pH range between pH 3 and pH 8.…”

“…The chromatographic behavior of pterins present in CSF is [1][2][3][4], and [9]). GTP, Guanosine triphosphate; GTPCH, guanosine triphosphate cyclohydrolase; NH 2 , dihydroneopterin; N, neopterin; PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; PCD, pterin-4α-carbinolamine dehydratase; DHPR, dihydropteridin reductase; BH 2 , dihydrobiopterin; DHFR, dihydrofolate reductase; B, biopterin.…”

“…Their separation can be easily achieved by reversed phase or HILIC modes ( Figure 3) [8,9,12,[15][16][17].…”

“…The correct diagnosis of these conditions can be achieved through the analysis of HVA, 5-HIAA, 3-OMD, 5-OHT, neopterin, and B in the CSF (Table 1). However, while the analysis of neurotransmitter metabolites can be easily achieved by liquid chromatography coupled to electrochemical detection (ECD) [7] or to mass spectrometry (MS) [8] most of available analytical methods of pterins in CSF cannot allow direct quantification of BH4 [9]. This review will focus on the methods and advances for monitoring pterins in CSF.…”