探討長期服用carnitine病人血中Trimethylamine N-oxide的濃度

Abstract

背景 氧化三甲胺，又稱三甲胺氧化物( Trimethylamine N-oxide、TMAO )為腸道菌群代謝的產物，當富含有膽鹼(choline)及肉鹼(carnitine)等食物進入腸道會被腸內道細菌代謝成三甲胺(Trimethylamine、TMA)，三甲胺會再經由肝臟的酵素(FMO3)代謝成氧化三甲胺。以往對氧化三甲胺的認知為體內的代謝物，在體中是無毒性的小分子，對人體也無不好的影響，然而，近年來有研究指出，氧化三甲胺會造成動脈粥狀硬化(Atherosclerosis)，增加心血管疾病的機率。雖然也有一些研究顯示氧化三甲胺反而是細胞遭遇壓力下的保護機轉，所以血液中TMAO上升對人體的確切影響仍然未明。 肉鹼(L-carnitine)是先天代謝異常患者如有機酸血症(organic academia)、原發性肉鹼缺乏症(primary carnitine deficiency)以及脂肪酸代謝異常(fatty acid oxidation defect)所需要使用的藥物，藉以維持體內代謝的穩定。因為有研究指出肉鹼的代謝物氧化三甲胺會造成動脈粥狀硬(Atherosclerosis)，這引起了此肉鹼的使用在先天代謝異常患者的疑慮，到底補充肉鹼對於這類患者心血管的影響如何？目前並沒有解答。 因為氧化三甲胺在人體極為微量，以往用生化儀器檢測不易，近年來因為質譜儀(mass spectrometry, MS)的發展才有許多人體的微量物質能被檢測，目前氣相層析串聯質譜儀(gas chromatography-tandem mass spectrometry，GC-MS)、液相層析串聯質譜儀(liquid chromatography-tandem mass spectrometry, LC-MS/MS)用在人體微量分子的檢測。目前只有鮮少的醫療院所有在檢測氧化三甲胺，我們欲運用質譜儀(MS)去開發一個簡易、快速、精準的氧化三甲胺檢測技術。 因此我們想探討長期使用高劑量口服肉鹼患者其血液中氧化三甲胺的變化，藉以了解這種長期使用高劑量口服肉鹼治療對先天性代謝異常病人的影響。尤其是先天代謝異常患者照護越來越進步的年代，許多以前無法存活的患者都可以長大，我們希望能及早知道目前肉鹼治療對病人可能的影響，並擬定治療措施。

方法 我們收集長期服用肉鹼的先天代謝異常患者(包括有機酸血症與脂肪酸代謝異常)病友16人、與不需服用肉鹼的先天代謝異常患者10人及年齡性別相符的健康人8人作為對照組參加此研究，我們將採10 mL周邊血液，並利用氣相質譜儀及液相質譜儀，分析其血中三甲胺及氧化三甲胺的含量。 結果 結果顯示本分析方法操作簡單且快速，對氧化三甲胺的檢量線線性 (濃度範圍1至500 ng mL-1、R2 > 0.990)、靈敏度(最低可定量濃度為1 ng mL-1) 及最低可偵測濃度為1 ng mL-1，且有良好的精密度(CV < 9.67 % )及準確度(82.84 % - 111.39 % )。長期服用肉鹼的先天代謝異常患者、不需服用肉鹼的先天代謝異常患者與健康人的血中氧化三甲胺濃度分別為2779.67 ng mL-1、464.31 ng mL-1、71.52 ng mL-1。利用此方法我們發現有服用肉鹼的病人血中氧化三甲胺濃度會比沒有服用肉鹼的人高 ( p =0.016 )。 結論 藉由此研究，我們建立了檢測血漿氧化三甲胺的方法，並確認了肉鹼與氧化三甲胺的關聯性，做為日後治療方向擬定的參考。

Background Trimethylamine N-oxide (TMAO) is a gut-derived metabolite which is generated by bacterial conversion of phosphatidylcholine, choline, betaine and carnitine. Carnitine is abundant in red meat. It is subsequently converted to gaseous trimethylamine. For this reason, TMAO has been proposed to constitute a link between diet and cardiovascular disease(CVD). High TMAO concentrations may increase atherosclerosis by suppressing reverse cholesterol transport (RCT) and bile acid synthesis, therefore, it has been suggested that chronic L-carnitine supplementation might be harmful and may accelerate atherosclerosis. On the other hand, carnitine supplementation is an important treatment in certain types of inborn error of metabolism (IEM). In addition to organic acidemias, fatty acid oxidation defect is another group of the IEM and needs long-term carnitine supplementation. The current treatment for the disease is carnitine supplementation to keep intracellular carnitine concentration. To evaluate the correlation between atherosclerosis and cardiovascular events and carnitine supplementation, the study focused on the correlation between long-term oral carnitine supplementation (more than 1 year) and the TMAO in blood. This study has focused on developing a simple, rapid, and cost-effective quantitative assay for TMAO in plasma. We used the gas chromatographic -mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) and in the purpose of comparing which one can detect TMAO more sensitively and specifically. Material and method We collected plasma samples from 16 IEM patients who need long-term carnitine supplementations, 11 IEM patients with normal acylcarnitine profiles and 8 healthy subjects as control. We collected 10 ml peripheral blood and analyze their Trimethylamine (TMA) and TMAO by using GC-MS and LC-MS. Result We have developed an easy and rapid LC-MS assay for quantification of TMAO. The calibration linear range is 0.5 ng mL-1 to 500 ng mL-1. The LOD is 0.5 ng mL-1. The intra- and inter-day precision (CV< 10%) and high accuracy (< 20% error) were observed using this method. The TMAO level in patient with carnitine supplementation, patient without carnitine supplementation and healthy control is 2779.67 ng mL-1、464.31 ng mL-1、71.52 ng mL-1, respectively. Furthermore, the plasma samples collected in our clinical study has demonstrated that the supplement of carnitine resulted in relative high TMAO in plasma. Conclusion Via this study, we established the simple method to detect TMAO, and also established the correlation between oral carnitine supplementation and TMAO. This will help future treatment strategies formulation in IEM patients.