臺灣博碩士論文加值系統

同半胱胺酸(Homocysteine, Hcy)為甲硫胺酸之中間代謝產物，血漿中結合態及游離態Hcy之總和即為總同半胱胺酸濃度(Total homocysteine concentration, tHcy)。研究指出血漿中tHcy濃度為心血管疾病危險指標之一。因此，本研究利用高效液相層析儀配備螢光偵測器進行血漿中tHcy分析方法之建立，由Hcy及其雙硫鍵化合物homocystin之回復率(Recovery rate，96 ~ 103 %) 評估此分析方法的可行性，並進行其在生物體之應用研究。此外，亦建立維生素E之高效液相層析儀配備螢光偵測器分析方法，以進行動物實驗部份抗氧化能力之評估。本研究以國人健康受試者為對象，進行血漿中tHcy之分析定量(n=375)，結果顯示年齡與血漿中tHcy濃度呈顯著正相關之趨勢，且男性顯著高於女性。而心血管疾病患者之血漿tHcy濃度，亦顯著高於同年齡層之健康受試者(13.62 ± 0.91 v.s. 11.02 ± 0.24 mM，p<0.05)。
另以SD品系大白鼠為實驗對象，以靜脈注射方式快速給予Hcy，進行其在血漿中清除能力(clearance)之比較，結果顯示Hcy滯留於13月齡大白鼠體內之時間較長，清除速率亦較慢。此外，為探討Hcy對生物體氧化 ¾ 抗氧化系統平衡之影響，本研究另以腹腔注射方式連續13天給予Hcy後，發現13月齡之大白鼠血漿中維生素C及HDL膽固醇含量，與其腦部、心臟、肝臟組織中維生素C、E之含量，肝臟中還原態麩胱甘胺酸之含量均顯著降低(p<0.05)；而6週齡大白鼠則只有肝臟中維生素C含量顯著降低(p<0.05)。另一方面，腦部脂質過氧化產物-丙二醛(Malondialdehyde, MDA)於13月齡及6週齡大白鼠則均顯著增加(p<0.05)。綜合上述，Hcy於較高月齡大白鼠體內之清除能力較低，且其可能經由降低生物體內抗氧化物質含量，促進過氧化傷害以改變生物體內氧化-抗氧化系統之恆定，進而促進心血管疾病之發生。

Homocysteine (Hcy) is an intermediate compound formed during methionine metabolism. The results of many recent studies have indicated that elevated plasma levels of total homocysteine (tHcy, defined as the sum of all homocysteine species in plasma, including free, bound to proteins and oxidized forms) is an independent risk factor for vascular disease. We have developed a convenient high performance liquid chromatography assay with fluorimetric detection for the accurate determination of tHcy concentration in plasma. The assay was validated by adding authentic Hcy and homocystine to plasma samples with a recovery rate between 96 % and 103 %. In addition, in this study we also developed a convenient HPLC assay with fluorimetric detection for the accurate determination of a-tocopherol, a lipophilic antioxidant system in biological samples. Plasma tHcy level in Chinese male and female normal subjects increases significantly with increasing age. In the same age, it is markedly higher in men than in women. The plasma tHcy levels of patients undergoing cardiac catheterization were significantly higher than normal subjects in the same age(13.62 ± 0.91 vs. 11.02 ± 0.24 mM).
Male SD rats at the age of 6 weeks and 13 months were used in the current study. After intravenous administration of Hcy (50 mg/ kg BW), rats at the age of 6 weeks show higher elimination rate. Vitamin C and high density lipoprotein cholesterol in plasma were decreased significantly by daily Hcy intraperitoneal injection (100 mg/ kg BW) for 13 days. Additionally, vitamin C and vitamin E levels in brain, heart, and liver were also decreased (p<0.05). The levels of malondialdehyde (MDA), an index of lipid peroxidation, were increased significantly in brain of rats at the age of 6 weeks and 13 months. In conclusion, the results implied that Hcy might induce oxidative stress, which can contribute to vascular disease.

第一章 緒論 …………………………………………………………1
第二章 文獻回顧 ……………………………………………………2
第一節 同半胱胺酸之生成來源及其生理代謝 ……………………2
第二節 血漿中同半胱胺酸之含量與多種疾病之相關性探討 ……7
第三節 同半胱胺酸對心血管疾病之可能致病機轉 ………………11
第四節 血漿中同半胱胺酸之分析研究 ……………………………14
第五節 自由基與活性氧物質 …………………………………………21
第六節 生物體之抗氧化防禦系統 ……………………………………22
第七節 脂質過氧化產物 - 丙二醛 ……………………………………26
第八節 生物體內同半胱胺酸清除率之評估 …………………………28
第九節 研究方向 ………………………………………………………29
第三章 材料與方法 …………………………………………………30
第一節 藥品 …………………………………………………………30
第二節 實驗材料 ……………………………………………………32
一、 人體血漿檢體 ………………………………………………32
二、 實驗動物……………………………………………………32
第三節 實驗流程 ……………………………………………………33
第四節 實驗方法 ……………………………………………………38
一、 一般之製備 …………………………………………………38
二、 生物檢體之製備 ……………………………………………38
三、 血漿檢體中同半胱胺酸分析方法之建立 ………………………39
四、 同半胱胺酸分析方法之正確性及精確度實驗 …………………42
五、 生物檢體中麩胱甘胺酸及半胱胺酸之分析方法…………………46
六、 生物檢體中維生素E分析方法 ……………………………………46
七、 維生素E分析方法之正確性及精確度實驗 ………………………49
八、 生物檢體中維生素C之高效液相層析分析條件 …………………51
九、 組織均質液中丙二醛之分光光度計分析條件 ……………………53
十、 血小板凝集試驗之分析方法 ………………………………………56
十一、血漿中三酸甘油脂、總膽固醇、HDL膽固醇、
LDL膽固醇之分析方法 ……………………………………………57
第五節 統計分析 ………………………………………………………58
第四章 結果 ……………………………………………………………59
第一節 標準曲線之建立與正確性、精確性試驗 ……………………59
一、 同半胱胺酸之測定 ……………………………………………59
(一) 標準曲線的建立 ………………………………………………59
(二) 正確性實驗結果 ………………………………………………59
(三) 精確性實驗結果 ………………………………………………65
1. 同半胱胺酸之穩定性試驗 ……………………………………65
2. 人為誤差之測定 ………………………………………………65
(四) 血漿檢體添加N-Ethylmaleimide之實驗結果 …………………65
二、 維生素E之測定 ………………………………………………68
(一) 標準曲線的建立 ………………………………………………68
(二) 正確性實驗結果 ………………………………………………72
(三) 精確性實驗結果 ………………………………………………74
1. 維生素E之穩定性試驗…………………………………………74
2. 人為誤差之測定 ………………………………………………74
第二節 國人血漿檢體同半胱胺酸之偵測分析結果…………………76
第三節 大白鼠同半胱胺酸清除率實驗結果 …………………………82
第四節 同半胱胺酸與生物體內氧化抗氧化恆定系統相關性之研究結果..88
第五章 討論 ……………………………………………………………97
第一節 分析方法之討論 ………………………………………………97
一、 同半胱胺酸分析方法之討論……………………………………97
二、 維生素E分析方法之討論 ………………………………………99
第二節 國人血漿檢體同半胱胺酸分析定量 …………………………101
第三節 大白鼠同半胱胺酸清除率實驗結果之討論……………………103
第四節 同半胱胺酸與生物體內氧化抗氧化恆定系統之相關性探討 104
第六章 結論……………………………………………………………..109
第七章 參考文獻…………………………………………………………110

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