Determination Of Pterins Research Articles

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

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.

Determination of pterins in urine by HPLC with UV and fluorescent detection using different types of chromatographic stationary phases (HILIC, RP C8, RP C18)

Pterins are a class of potential cancer biomarkers. New methods involving hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) high-performance liquid chromatography have been developed for analysis of eight pterin compounds: 6,7-dimethylpterin, pterin, 6-OH-methylpterin, biopterin, isoxanthopterin, neopterin, xanthopterin, and pterin-6-carboxylic acid. The effect of mobile phase composition, buffer type, pH and concentration on retention using HILIC, C8 and C18 RP stationary phases were examined. Separation of pterins on RP and HILIC stationary phase was performed and optimized. Eight pterins were successfully separated on HILIC Luna diol-bonded phases, Aquasil C18 RP column and LiChrospher C8 RP column. Determination and separation of the pterins from urine samples were performed on HILIC Luna and LiChrospher C8 RP columns which were chosen as the most appropriate ones. Finally, LiChrospher C8 RP column with fluorescence detection was selected for further validation of the method. The optimum chromatographic condition was mobile phase methanol (A)/phosphoric buffer pH 7, 10mM (B), isocratic elution 0–15min 5% A flow=0.5ml/min 15–17min. 5% A, flow=0.5–1ml/min the linearity (R2>0.997) and retention time repeatability (RSD%<1) were at satisfactory level. The precision of peak areas expressed as RSD in % was between 0.55 and 14. Pterins detection limits varied from 0.041ng/ml to 2.9ng/ml. Finally, HPLC method was used for the analysis of pterins in urine samples with two different oxidation procedures. Concentration levels of pterin compounds in bladder cancer patients and healthy subjects were compared.

46] Determination of pterins by liquid chromatography/electrochemistry

Publisher Summary This chapter focuses on determination of pterins by liquid chromatography/electrochemistry. Pterins are of interest because of their possible involvement in a number of diseases. Pterins are obtained from the sources such as pterin, pterin-6-carboxylic acid, 6-methylpterin, and xanthopterin. A property of the pterins essential to their biological role as enzyme cofactors is their electrochemistry. Fully oxidized pterins can be reversibly reduced to 5,8-dihydropterins which rapidly tautomerize to 7,8-dihydropterins. These 7,8-dihydropterins can either be further reduced to 5,6,7,8-tetrahydropterins or be oxidized back to the fully oxidized state. Dual-electrode detector uses two working electrodes placed adjacent to each other and normal to the direction of flow—that is, in a parallel-adjacent configuration. Using this detector, one electrode is operated at an oxidizing potential to detect the dihydro- and tetrahydropterins while the other electrode is operated at a reducing potential to detect the oxidized pterins, thus the three stable oxidation states of the pterins can be detected simultaneously. For the determination of pterins in urine, a 25-cm chromatography column thermostatted at 30° is used. The mobile phase contains 5% methanol (v/v). A flow rate of 1.0 ml/min is employed.

Determination of pterins in biological samples by liquid chromatography/electrochemistry with a dual-electrode detector.

Determination of pterins in biological samples by liquid chromatography/electrochemistry with a dual-electrode detector.

The determination of pterins in biological samples by liquid chromatography/electrochemistry

A method is presented for determining the concentrations of several oxidized pterins in a variety of biological samples. This methodology employs reverse-phase "ion-pair" liquid chromatography with electrochemical detection. Detection limits below a picomole have been achieved with responses linear over four orders of magnitude. This methodology was employed to study the excretion pattern of pterins in human urine. The concentration of several pterins in mouse brain and liver samples were also determined. The direct detection of tetrahydrobiopterin was demonstrated.

Diagnosis of variants of hyperphenylalaninemia by determination of pterins in urine

Assessment of urinary pterins is proposed as a rapid method for recognition of the variants of hyperphenylalaninemia. This is achieved by means of oxidation of pterins by iodine in acidic and alkaline solutions and then by high performance liquid chromatography on a cation-exchange column with fluorimetric detection. In biopterin-synthetase deficiency, only neopterin accumulated; in dihydropteridine-reductase (DHPR) deficiency and in phenylketonuria, high levels of pterins are found, but BH 4 levels, absent in the former and high in the latter, allow a differential diagnosis. Phenylalanine loads in the controls also lead to increased elimination of pterins, but with a pattern different from that found in phenylketonuria. This method can be used before dietary treatment and thus can be proposed for all newly detected hyperphenylalaninemic babies.