Autoxidation Kinetics of Tetrahydrobiopterin-Giving Quinonoid Dihydrobiopterin the Consideration It Deserves

Abstract

In humans, tetrahydrobiopterin (H4Bip) is the cofactor of several essential hydroxylation reactions which dysfunction cause very serious diseases at any age. Hence, the determination of pterins in biological media is of outmost importance in the diagnosis and monitoring of H4Bip deficiency. More than half a century after the discovery of the physiological role of H4Bip and the recent advent of gene therapy for dopamine and serotonin disorders linked to H4Bip deficiency, the quantification of quinonoid dihydrobiopterin (qH2Bip), the transient intermediate of H4Bip, has not been considered yet. This is mainly due to its short half-life, which goes from 0.9 to 5 min according to previous studies. Based on our recent disclosure of the specific MS/MS transition of qH2Bip, here, we developed an efficient HPLC-MS/MS method to achieve the separation of qH2Bip from H4Bip and other oxidation products in less than 3.5 min. The application of this method to the investigation of H4Bip autoxidation kinetics clearly shows that qH2Bip's half-life is much longer than previously reported, and mostly longer than that of H4Bip, irrespective of the considered experimental conditions. These findings definitely confirm that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Keywords: biogenic amines; dopamine; hydroxylation; pterins; quinonoid dihydrobiopterin; serotonin; tetrahydrobiopterin; transient intermediate.

Figure 1

Autoxidation pathways of tetrahydrobiopterin (H4Bip) according to Refs. [2,18,19,20,21,22,23]. qH2Bipquinonoid dihydrobiopterin; H2Bip7,8-dihydro-biopetrin; H2Ptr—dihydro-pterin; H2XPtr—dihydro-xanthopterin; Bip—biopterin; Ptr—pterin; DHPR—dihydro-pteridine reductase.

Figure 2

Chromatographic profiles of H4Bip working solutions prepared and incubated in 0.1 M ammonium formate buffer at (a) pH 2.8 at time 0; (b) pH 2.8, after 1 h of incubation; (c) pH 2.8, after 10 h of incubation (zoom-in on the peak of residual qH2Bip); (d) pH 5.4 after 1.6 h of incubation; (e) pH 7.4 after 49.4 h of incubation. Chromatographic conditions are detailed in the experimental section.

Figure 3

H4Bip autoxidation kinetics as a function of pH and type of buffer (0.1 M ammonium buffer): (a–c) pH ≤ 3.0; (d–f) pH 7.4; and (g–i) pH 5.4. (a) pH 2.8, formate; (b) pH 3.0, acetate; (c) pH 2.8, citrate; (d) pH 7.4, formate; (e) pH 7.4, acetate; (f) pH 7.4, citrate; (g) pH 5.4, formate; (h) pH 5.4, acetate; and (i) pH 5.4, citrate. R.A.—Relative abundance. R.A. is expressed as the percentage of each peak-area ratio (PAR) (PAR—peak area of the considered product/IS peak area) relative to the total sum of all PARs.

Figure 4

Autoxidation kinetics as a function of H4Bip concentration: (a) H4Bip autoxidation (0.5, 1.0, and 2.0 µM); (b) qH2Bip degradation.