Establishment of mesuring Tetrahydrobiopterin in Develpmental Delay Patients' Cerebrospinal Fluid

Tetrahydrobiopterin (BH4) is an endogenous factor in human body, one of its important functions is to act as a coenzyme for certain amino acid hydroxylases. Tetrahydrobioterin participates in the synthesis of neurotransmitters such as dopamine and serotonin. Deficiency in BH4 will lead to the disturbance of homeostasis of certain amino acids

in the body, especially dopamine and serotonin, and is called tetrahydrobiopterin deficiency (BH4 deficiency).

Tetrahydrobiopterin deficiency is one of the Department of Health-declared rare diseases in Taiwan. Clinically, it correlates closely with developmental delays in children Early diagnosis is beneficial for victims. Presently, there is no analytical method applied routinely to measure the amount of pterins in cerebrospinal fluids in Taiwan.

The present study tried to employ and optimize applicable analytical conditions, including buffer systems, pH value, flow rate, and voltage (electrochemical detection), by using high-performance liquid chromatography (HPLC) and liquid chromatography/ tandem mass spectrometry (LC/MS-MS). The application of suitable assay methods to the measurement of patients’ cerebrospinal fluid (CSF) samples was also attempted.

The established analytical conditions in the study are as follows.

The first was for the simultaneous detection of neopterin and biopterin by reversed-phase HPLC with fluorescence detector.

Chromatographic conditions were as follows: column 150 x 4.6 mm packed with 5 μm C18, mobile phase 5 mM citric buffer, pH adjusted to 5.2, flow rate 0.5 mL/min, excitation wavelength 350 nm and emission wavelength 448 nm. The detection limit was 200 pg.

The second was for the determination of BH4 by using reversed-phase HPLC with electrochemical detector. Chromatographic conditions were as follows: column 150 x 4.6 mm packed with 5 μm C18, mobile phase 10mM phosphoric buffer, pH adjusted to 2.5, flow rate 0.4 mL/min, applied voltage was 700 mV. The detection limit was 80 pg.

The third was for the simultaneously detection of neopterin, biopterin, BH4, and BH2 by utilizing LC/MS-MS, using reversed-phase 5 μm C18 column (250 x 4.6 mm, I.D.) for separation. The mobile phase consisted of 5% methanol in water (pH2.5), with a flow-rate of 0.8 mL/min. The detection limit was 1 pg.

The analytical methods described above were applied to the assay of human CSF samples. During Nov. 10, 2004 to Dec. 31, 2005, fifty-four patients with developmental delays were recruited into the study from the outpatient and inpatient services of the NTUH Department

of Pediatrics. CSF samples were collected for analysis.

Results showed that neopterin, biopterin, BH4, and BH2 were not detectable in all CSF samples by employing LC/MS-MS, even though our detection limit was comparable to previous Western reports. One possible explanation is that the CSF pterin levels of our population was significantly lower than the Caucasian population and, thus, the quantification was hampered by undesirable detection limit. For aiding in the diagnosis, treatment, and monitoring of BH4 deficiency and therapy, larger-scale trial with both healthy and diseased subjects are needed for setting up reference values for our population in the future.