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.

Figure 1

Metabolism pathway of tetrahydrobiopterin (BH₄). N (neopterin); B (biopterin); BH₂ (dihydrobiopterin); NH₂ (dihydroneopterin). GTP (guanosine triphosphate); GTPCH (guanosine triphosphate cyclohydrolase EC 3.5.4.16); PTPS (6-pyruvoyl-tetrahydropterin synthase EC 4.2.3.12); SR (sepiapterin reductase EC 1.1.1.153); PCD (pterin-4α-carbinolamine dehydratase EC 3.5.4.16); DHPR (dihydropteridin reductase EC 1.5.1.34). (Dashed arrows: nonenzymatic.)

Figure 2

Pterin separation as a function of stationary phase and pH. (a) Atlantis dC18 (N_BH2 = 19 230); (b) Eclipse XDB C18 (N_BH2 = 11 510); (c) XTerra (N_BH2 = 2080); (d) Zic-HILIC (N_NH2 = 16 250). N_BH2 and N_NH2 = theoretical plate number for BH2 or NH2, respectively. Mobile phase for panels (a)–(c): pH 7.4, 0.05 M sodium citrate/methanol (97/3, v/v). Mobile phase for panel (d): pH 7.4, 0.2 M ammonium formiate/acetonitrile (20/80, v/v). Flow rate 0.6 mL/min at 30 °C and UV detection at 260 nm for all columns (AU = absorbance units).

Figure 3

Fluorescence detection after electrochemical oxidation of NH2, BH2 and BH4. (a) Hydrovoltammograms at pH 7.4. (b) Oxidation yields as a function of pH at +400 mV. (a and b) HPLC conditions: Atlantis dC18 (4.6 × 150 mm, 3 μm); mobile phase: pH 7.4, 0.05 M sodium citrate/methanol (97/3, v/v); flow rate: 0.6 mL/min at 30 °C; detection: λ_ex 350 nm; λ_em 450 nm after coulometric oxidation. (c) Retention factors as a function of pH on the same column and with the same mobile phase as for (a) and (b). Flow rate 0.6 mL/min at 30 °C and UV detection at 260 nm. X₂ is an interfering compound coeluting with B (See Figure 4).

Figure 4

Chromatographic profiles of a standard mixture and a pooled CSF sample (n = 5) under the proposed chromatographic conditions: column = Atlantis dC18 (4.6 × 150 mm, 3 μm); mobile phase: pH 7.4, 0.05 M sodium citrate/methanol (97/3, v/v); flow rate: 0.6 mL/min at 30 °C, detection: λ_ex 350 nm, λ_em 450 nm (FU: fluorescence units (arbitrary units)). (a) Standard mixture after postcolumn oxidation at +600 mV. Pooled CSF sample (b1, dashed line) without oxidation; (b2, solid line) after off-line chemical oxidation according to ref (25); (c) after postcolumn coulometric oxidation at +400 mV; and (d) after postcolumn oxidation at +600 mV allowing the disappearance of the interfering peaks X₁ and X₂.

Figure 5

Chromatographic profiles of a standard mixture and a pooled CSF sample (n = 5) under the proposed chromatographic conditions: column = Atlantis dC18 (4.6 × 150 mm, 3 μm); mobile phase: pH 7.4, 0.05 M sodium citrate/methanol (97/3, v/v); flow rate: 0.6 mL/min at 30 °C; postcolumn oxidation at +600 mV detection: λ_ex 350 nm, λ_em 450 nm (FU: fluorescence units (arbitrary units)). (a) Standard mixture; (b) pooled CSF without internal standard; (c) pooled CSF with lumazine as internal standard (IS).

Figure 6

(a) Relationship between age and BH4 (y = −0.95x + 40.24; R² = 0.106), BH2 (y = −0.057x + 10.00; R² = 0.009), and NH2 (y = −0.15x + 16.53; R² = 0.009) concentrations. (Inset: patients over 20 years.) (b) Comparison between B and N levels in 99 CSF samples determined by the proposed method (BH4 + BH2 = total biopterin) or by off-line chemical oxidation according to ref (25) . (c) Comparison between total biopterin (BH4+BH2) and NH2 as determined with the proposed method (see Figure 4); B₁, B₂ and N₁, N₂ refer to biopterin and neopterin levels as determined by offline chemical oxidation after calibration of the method with B and N calibrators or with BH4 and NH2 calibrators, respectively. Asterisk (*) indicates significant difference (p < 0.01) as compared to both methods used to determine (BH4 + BH2, and B1, and NH2, and N1, respectively).

Figure 7

Chromatographic profile obtained with the proposed method for CSF samples from patients with various metabolic disorders. (a) CSF sample from a patient with tyrosine hydroxylase deficiency. (b and c) CSF sample from a patient with amino acid decarboxylase deficiency showing two additional peaks corresponding to 3 OMD (3-ortho-methyl dopa) and 5 OHT (5-hydroxytryptophan). (d) CSF sample from a patient with sepiapterin reductase deficiency. (e) CSF sample from a patient with a secondary defect in BH4 synthesis. (f) CSF sample from a patient suffering from leukodystrophy of unknown origin. HPLC conditions: Atlantis dC18 (4.6 × 150 mm, 3 μm). Mobile phase: pH 7.4, 0.05 M sodium citrate/methanol (97/3, v/v). Flow rate: 0.6 mL/min at 30 °C. Oxidation: + 600 mV. Detection: λ_ex 350 nm; λ_em 450 nm.

Figure 8

Chromatographic profiles of a CSF sample (1, solid line) before and (2, dashed line) after spiking with with B (5 nM), N (5 nM), BH2 (20 nM), NH2 (20 nM), and BH4 (50 nM), showing the feasibility of the proposed method under UHPLC conditions. Chromatographic conditions: Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm); flow rate: 0.5 mL/min at 30 °C. The mobile phase and the detection conditions are the same as for the proposed method (see Figure 4). X₁ and X₂ are unknown peaks.