液相層析螢光法對脂肪酸疾病指標及抗癲癇藥物之分析

Fluorimetric Liquid Chromatographic Method for the Analysis of Fatty Acid Biomarkers and Anticonvulsant

鐘堂嘉(Tang-Chia Chung)

指導教授 ： 吳信隆

高雄醫學大學/藥學院/藥學研究所/博士(2009年)

摘要

脂肪酸(fatty acids)之異常代謝與若干遺傳疾病有關，因此脂肪酸可以作為某些疾病之診斷指標；但是脂肪酸之結構基本上含羧基(-COOH)及飽和或不飽和碳氫基團皆不具實用吸光性；其微量存在時，不易以常用的紫外-可見光分析法或螢光分析法加以偵測，本研究針對疾病之生物指標isovaleric acid及非常長鏈脂肪酸(含docosanoic acid, C22; tetracosanoic acid, C24; 及 hexacosanoic acid, C26)，利用衍生反應導入螢光基團，配合液相層析螢光法，建立脂肪酸之微量分析方法，並成功應用於該類脂肪酸與遺傳疾病有關之診斷分析。又利用類似衍生反應配合層析螢光法應用於抗癲癇藥物gabapentin之微量分析，可作為該藥物在血中濃度之監控(drug monitoring)；本研究之綜合結果如下：
1. 疾病指標isovaleric acid及very-long-chain fatty acids之分析
(A) 異戊酸血症(isovaleric acidemia)是一種代謝異常的遺傳疾病，因為在第15對染色體上的基因有缺陷，使得病人無法代謝isovaleric acid而在體內堆積；故病人的血液及尿液中isovaleric acid的含量會明顯高於正常人。本研究利用2-(2-naphthoxy)ethyl-2-(piperidino) ethanesulfonate (NOEPES) 與isovaleric acid進行衍生反應，之後以液相層析螢光法進行分析，建立尿液檢品中isovaleric acid的分析方法，已應用於新生兒遺傳病isovaleric acidemia之篩檢。本法對正常人與isovaleric acidemia病人尿液之分析，顯示正常人的isovaleric acid的含量範圍為0.73 ~ 4.27 μM (n = 5)，而病人的isovaleric acid則高達2.31 mM (n = 1)。

(B) 腎上腺腦白質失養症(adrenoleukodystrophy, ALD)的病人在其第二十三對性染色體的X染色體上ABCD 1基因有缺陷，使得病人無法合成有關酵素以代謝非常長鏈脂肪酸，造成這些脂肪酸在人體內的堆積，造成對中樞神經系統與腎上腺皮質之不可逆損害。本研究利用NOEPES與非常長鏈脂肪酸docosanoic acid (C22)、tetracosanoic acid (C24) 和hexacosanoic acid (C26)進行衍生反應，對該類脂肪酸標識螢光基團，配合液相層析螢光法建立C22、C24 和C26之高靈敏性分析法，並應用於ALD的診斷分析。由正常人(n = 5)及病人(n = 3)血漿中的C22、C24和 C26濃度比較分析，得知正常人的血漿中C22、C24和C26的濃度(μM)範圍分別為0.60 ~ 0.73 ( = 0.66)、0.49 ~ 0.59 ( = 0.52)及0.19 ~ 0.30 ( = 0.24)，又其濃度比C24/C22和C26/C22分別為0.75 ~ 0.85 ( = 0.80)和0.28 ~ 0.47 ( = 0.37)；但ALD病人的血漿中C22、C24和C26的濃度(μM)範圍分別為1.26 ~ 1.32 ( = 1.28)、1.39 ~ 1.58 ( = 1.50)及1.29 ~ 1.41 ( = 1.36)，又其濃度比C24/C22和C26/C22分別為1.09 ~ 1.23 ( = 1.17)和1.01 ~ 1.11 ( = 1.06)。結果顯示ALD病人的指標脂肪酸濃度及濃度比皆明顯大於正常人，此結果與文獻報告相符合。

2. 抗癲癇藥物－gabapentin
Gabapentin (GBP)是臨床上常使用的抗癲癇藥物，近來其另ㄧ療效被應用在疼痛的治療；GBP之療效和血中濃度有密切的關係，因個別病人投藥後的血中濃度差異大，所以必須調整投與劑量及監測其血中濃度。本研究以(2-naphthoxy)acetyl chloride (NAC)對無螢光基團之GBP進行衍生反應，配合液相層析螢光法，建立gabapentin之微量分析法，並應用於病人血漿中GBP濃度之監測，使GBP之劑量達到適當療效。GBP文獻報告之有效治療濃度為11.7 – 116.8 μM，應用本法測定五位病人在投藥ㄧ週後達到藥物穩定期(static state)時，其血漿中GBP濃度分別為136.52、84.41、50.75、47.37與33.51 μM；其中有ㄧ位病人的血中藥物濃度已超過有效治療濃度範圍，但尚無明顯副作用之主訴；顯示個體對GBP之藥物動力學差異大。

關鍵字

Isovaleric acid ； Docosanoic acid ； Tetracosanoic acid ； Hexacosanoic acid ； Gabapentin ； 螢光衍生 ； 液相層析螢光法 ； 異戊酸血症 ； 腎上腺腦白質失養症 ； 疾病指標 ； 藥物監控

並列摘要

Fatty acid in body fluid from abnormal metabolism is relevant to a variety of hereditary diseases that makes fatty acid a potential diagnostic marker for various diseases. In structure, fatty acid is composed of a carboxyl group and a saturated or unsaturated hydrocarbon moiety usually with weak chromophores resulting in low sensitivity of fatty acid detected by conventional absorption spectrophotometry. This study tried to analyze isovaleric acid (a short chain fatty acid) and very long-chain fatty acids (docosanoic acid, C22; tetracosanoic acid, C24; hexacosanoic acid, C26) as disease biomarkers by fluorimetric liquid chromatography. Application of the proposed method to the analysis of isovaleric acid and C22, C24, and C26 in patients proved feasible. On the other hand, a fluorimetric liquid chromatography with similar mechanism was developed to the analysis of gabapentin (an anticonvulsant) in dosed subject for drug monitoring purpose. The studies were briefly summarized as follows:

1. Biomarkers of isovaleric acid and very long chain fatty acids (VLCFA) in diseases
Isovaleric acidemia or aciduria is a metabolic hereditary disease caused by mutation in the long arm of chromosome XV. Patients with the disease were unable to process isovaleric acid properly, resulting in abnormal level of isovaleric acid in blood (isovaleric acidemia) and urine (isovaleric aciduria) causing serious health problems. Whereas, X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder caused by mutation in ABCD 1 gene on the X chromosome. Patient with ALD are unable to metabolize C22, C24 and C26 properly, leading to high levels of VLCFA in body tissues causing impaired functions of adrenal cortex and brain myelin. In this study, analytical derivatization of isovaleric acid or VLCFA with fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate (NOEPES) was designed to give fluorescent derivatives with high sensitivity by fluorimetric liquid chromatography. Application of the method to the analysis of isovaleric acid in urine of a target patient was performed. The results indicate that isovaleric acid level in urine of the patient is 2.31 mM, about 1000-fold higher than that of isovaleric acid in normal urine of volunteers (0.73 – 4.27 ?嵱, n = 5). On the other hand, application of the method to the analysis of VLCFA in plasma of some target patients was studied. The results indicate that the range of the plasma levels (?嵱, n = 5) for C22, C24 and C26 and the ratio range of C24/C22 and C26/C22 in patients are 1.26 – 1.32 ( = 1.28), 1.39 – 1.58 ( = 1.50), 1.29 – 1.41 ( = 1.36), 1.09 – 1.23 ( = 1.17) and 1.10 – 1.1 ( = 1.06) respectively. In parallel, the range of plasma levels (?嵱, n = 5) of C22, C24 and C26, C24/C22 and the ratio range of C26/C22 in normal volunteers are 0.60 – 0.73 ( = 0.66), 0.49 – 0.59 ( = 0.52), 0.19 – 0.30 ( = 0.24), 0.75 – 0.85 ( = 0.80), and 0.28 – 0.47 ( = 0.37), respectively. The results indicate that the values of C22, C24, C26, C24/C22 and C26/C22 from the patients are definitely higher than that from normal volunteers. The analytical results are compatible with available report, indicating that the developed method is feasible for the analysis of VLCFA as diagnostic biomarkers for ALD patient.

2. Drug monitoring of gabapentin
Gabapentin (GBP) is widely used for the adjunctive therapy of refractory partial seizures. GBP has additional indication for treating various neuropathic pains. The oral bioavailability of GBP is reported to be dose-dependent and individually varied. Therefore, monitoring GBP levels in dosed patients is essential for therapeutic purposes. In this study, GBP without chromophore was derivatized with a fluorescent reagent, (2-naphthoxy)acetyl chloride. The resulting naphthoxy derivative was analyzed by fluorimetric liquid chromatography with high sensitivity. The method was applied to the analysis of gabapentin in patient plasma. The results indicate that GBP levels in five patient plasmas are 136.52, 84.41, 50.75, 47.37 and 33.51 μM. The therapeutic range of plasma GBP is reported to be 11.7 – 116.8 μM. It indicates that one of the patients found with higher level of plasma GBP than the reported range, but there is no complaint of side effects from the patient. The results indicate that pharmacokinetic / pharmadynamic of GBP is individually varied and dose optimization is required.

並列關鍵字

Isovaleric acid ； Docosanoic acid ； Tetracosanoic acid ； Hexacosanoic acid ； Gabapentin ； Fluorescent derivatization ； Fluorimetric liquid chromatography ； Biomarkers ； Isovaleric aciduria ； Adrenoleukodystrophy ； Drug monitoring

參考文獻

[1] F. Kanda, E. Yagi, M. Fukuda, K. Nakajima, T. Ohta, O. Nakata, Elucidation of chemical compounds responsible for foot malodour, Br. J. Dermatol. 122 (1990) 771.

[2] A. Yasuhara, K. Fuwa, Isolation and analysis of odorous components in swine manure, J. Chromatogr. 281 (1983) 225.

[3] S. Willig, M. Lacorn, R. Claus, Development of a rapid and accurate method for the determination of key compounds of pig odor, J. Chromatogr. A, 1038 (2004) 11.

[4] J. F. Lues, W. C. Botha, E. J. Smit, Ion-exchange HPLC analysis of a broad spectrum of organic acids from matured Cheddar cheese and assessment of extraction methods, Food Res. Intern. 31 (1998) 441.

[5] M. Aznar, R. López, J. F. Cacho, V. Ferreira, Identification and quantification of impact odorants of aged red wines from Rioja. GC-olfactometry, quantitative GC-MS, and odor evaluation of HPLC fractions, J. Agric. Food Chem. 49 (2001) 2924.