- 學位論文
利用液相層析質譜儀檢測人類血液之變腎上腺素、去甲變腎上腺素和其二次代謝物
Determination of Metanephrine, Normetanephrine and Phase II Metabolites in Human Plasma Using LC-MS
摘要
嗜鉻細胞癌（pheochromocytomas）為一種罕見的嗜鉻細胞腫瘤，其主要症狀為分泌過多兒茶酚胺（catecholamine）導致之高血壓和心搏過速，若沒有及時診斷治療可能致命。患者經常因為其臨床表現和許多內分泌疾病類似而延後就醫，目前以偵測兒茶酚胺之甲基化代謝物：變腎上腺素（metanephrine, MN）和去甲基變腎上腺素（normetanephrine, NMN）做為嗜鉻細胞瘤的重要標的物，然而變腎上腺素和去甲基變腎上腺素在正常人和發病初期血漿含量極低，因此建立一套高靈敏的檢測方法做為嗜鉻細胞瘤的診斷十分重要。本篇研究使用少量血漿樣品以弱鹼型離子交換管柱進行固相萃取去除多餘蛋白質，再以液相層析串聯質譜儀進行定性及定量分析，並將進一步確認此方法之線性、偵測極限、準確性、回收率及基質效應。除此之外，本研究利用人類肝細胞成分模擬體內代謝反應，以三段式四極桿柱和高解析液相層析質譜儀鑑定其二次代謝產物，並在真實檢體中檢驗此代謝物，判斷其做為嗜鉻細胞瘤之標記物的可能性。結果顯示此方法表現出良好的準確性和穩定性，在病人檢體中亦能檢出；經體外培養合成的變腎上腺素和去甲基變腎上腺素之硫酸化產物，分別為metanephrine-sulfate (MN-S) 和normetanephrine (NMN-S )也藉由液相層析質譜儀鑑出，確認精確質量以驗證其結構，MN-S的訊號值在正常人體(n=11)和確診病患(n=6)體內之p值小於0.05，未來可應用於嗜鉻細胞瘤之診斷。
並列摘要
Pheochromocytomas are rare chromaffin cell tumors. The development of the disease commonly induces hypertension and tachycardia by producing excess catecholamines, which shows similar symptoms to endocrine diseases. This makes it hard to be diagnosed at early stage. In addition, previous studies indicated that it is associated with specific germline mutation. In clinical diagnosis, the methylated metabolites of catecholamines, metanephrine (MN) and normetanephrine (NMN) are used as specific biomarkers for diagnosis of pheochromocytomas. However, MN and NMN in plasma are at trace level both in healthy people or in patients with early-stage illness. In this study, we aim at establishing a highly sensitive assay to determine MN and NMN in human plasma. The samples were extracted and analyzed using weak cation exchange solid phase extraction couple to liquid chromatography mass spectrometry (LC-MS). The method is validated for linearity, limit of quantitation, recovery and matrix effect. Besides, we synthesized the phase II metabolites of MN and NMN in vitro with human liver microsomes and cytosols and determined the sulfo-metabolites, MN-S from MN and NMN-S from NMN, using LC-MS/MS and liquid chromatography-high resolution mass spectrometry (LC-HRMS) to confirm the formulas by their fragment pattern and accurate mass. The sulfate-conjugated metabolites were detected in 17 human plasma specimens (11 normal people and 6 patients) and showed significant positive correlation with their parent substances (p<0.001). MN-S showed significant difference between the normal and patients group (p<0.05) The sulfation metabolites may be potential biomarker for pheochromocytomas.