Tetrahydrobiopterin測定方法之建立及其於發育遲緩兒童腦脊髓液含量探討

Abstract

Tetrahydrobiopterin（BH4）屬人體之內生性物質，其重要生理功能之ㄧ係做為體內某些胺基酸代謝所需之輔酶，參與神經傳導物質dopamine與serotonin生合成之過程，缺乏時將會導致體內特定胺基酸與神經傳導物質dopamine、serotonin平衡失調，稱為tetrahydrobiopterin缺乏症（tetrahydrobiopterin deficiency，BH4 deficiency）。Tetrahydrobiopterin缺乏症為國內衛生署公告罕見疾病之一，臨床上和幼童發育遲緩有密切之關係，早期的診斷與治療對病患有相當大的助益。目前國內臨床上尚無常規直接檢測腦脊髓液中pterins相關物質含量之措施，本研究擬以高效液相層析法及液相層析串聯質譜分析方法，期建立適當的分析條件，以應用於病患腦脊髓液檢體之檢測。 本研究探討不同緩衝溶液、pH 值及流速對高液相層析方法分離效果之影響；並將電化學提供電位由低至高尋找出最適宜偵測BH4之電位。液相層析串聯質譜方法則依據化合物之分子量，設定適當之參數測量。 本研究所建立分析pterins之方法可分為三個部份： 第一部份以高效液相層析法同時測定neopterin與biopterin：分析使用5 μm逆相碳18管柱（150 × 4.6 mm I.D.），移動相為5 mM檸檬酸緩衝溶液，pH調整為5.2，螢光偵測設定激發波長350 nm，放射波長448 nm，流速為0.5 mL/min。偵測極限可達200 pg（20 nM，5 ng/mL，40 μL）。 第二部分仍以高效液相層析法測定BH4：分析使用5 μm逆相碳18管柱（150 × 4.6 mm I.D.），移動相為10 mM磷酸緩衝溶液，pH調整為2.5，電化學偵測設定700 mV，流速0.4 mL/min。偵測極限可達80 pg（6.4 nM，2 ng/mL，40 μL）。 第三部份為以液相層析串聯質譜儀方法同時測定neopterin、biopterin、BH2與BH4：分析使用5 μm逆相碳18管柱（250 × 4.6 mm I.D.），移動相為5％甲醇，pH調整為2.5。偵測極限最低可達1 pg（0.4 nM，0.1 ng/mL，10 μL）。 本研究進一步以上述建立之方法應用於病患檢體檢測。在西元2004年11月10日至2005年12月31日期間，共納入台大醫院小兒科門診或住院病患中有發育遲緩現象病童54名，收集其腦脊髓液檢體保存以待測量。 病患檢體以高效液相層析串聯質譜分析，皆無可測得之neopterin、biopteirn、BH4或BH2波峰，或許是由於大部份國人腦脊髓液中這些pterins的含量較低，導致測不出pterins含量。因此未來宜進行人數更多、規模更大之檢測，以期建立國內小兒腦脊髓液中pterins相關物質之參考範圍及病患之致病範圍，藉以做為診斷BH4缺乏症之依據，並可進一步用於監測其治療控制之情形。

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. Tetrahydrobiopterin 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 fluororescence 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 10 mM 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 (pH 2.5), 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 trials with both healthy and diseased subjects are needed for setting up reference values for our population in the future.