類澱粉蛋白-beta誘導阿茲海默症大鼠不同組織內生性反式脂肪含量之分析

Analysis of endogenous trans-fatty acids in different tissues of beta amyloid peptide induced Alzheimer’s disease Sprague-Dawley rats

藍文安(Wen-Ann Lan)

指導教授 ： 孫璐西

共同指導教授 ： 何其儻

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02835

摘要

反式脂肪酸(Trans fatty acids, TFAs)為包含一或多個不共軛反式雙鍵結構之不飽和脂肪酸，其主要來源為加工食品中部分氫化的油脂，1990年代許多研究均指出攝取過多反式脂肪會升高罹患心血管疾病的風險。
近年的研究指出體內反式脂肪除食物來源之外，尚有部分經自由基誘導脂肪酸順反異構化之反式脂肪酸在體內生成，即內生性 (endogenous)反式脂肪，其生成量與體內自由基含量呈正相關。研究中指出吸煙者、誘導發炎大鼠及正常老化的大鼠體內具有較多內生性反式脂肪。阿茲海默症 (Alzheimer's disease, AD)為目前失智症最主要的一類，患者腦部出現含有類澱粉蛋白(beta-amyloid peptide, Aβ)的老化斑塊沈積 (senile plaques)及神經糾結 (tangles)，且在腦部可觀察到升高的氧化壓力及發炎反應，推測較高之氧化壓力及發炎亦可能引起脂肪酸順反式異構化。本研究乃探討側腦室注射Aβ25-35誘導阿茲海默症之Sprague-Dawley 大鼠 (SD rats)的腦部、肝臟、心臟及脂肪組織中是否有較高量內生性反式脂肪酸之存在，期可應用為AD之預測性指標。
本研究共分為兩部分，其一為分析大鼠組織內氧化壓力指標，另一部份為分析大鼠組織內脂肪酸組成。在氧化壓力指標方面，肝臟脂質過氧化物thiobarbituric acid reactive substances (TBARS)含量與肝臟麩胱甘肽還原酶 (glutathione reductase, GRd)活性分析中發現Aβ注射組之大鼠體內可能有偏高之氧化壓力。而在肝臟麩胱甘肽過氧化酶 (glutathione peroxidase, GPx)活性分析與血漿TBARS含量分析結果方面則無顯著差異。
在脂肪酸組成分析方面，使用氣相層析串聯質譜儀分析生理食鹽水注射組 (控制組)、Aβ注射組與Aβ及resveratrol 同時注射組之大鼠腦部、肝臟、腹部脂肪組織、副睪脂肪組織與皮下脂肪組織之脂肪酸組成時，發現各組皆未檢出反式脂肪酸，或其含量低於偵測下限，僅大鼠心臟組織中有檢出低於定量極限之反式脂肪，而各組之主要脂肪酸組成則無顯著差異。但Aβ組大鼠腹部脂肪總脂肪酸含量則顯著低於控制組；而Aβ及resveratrol 同時注射組大鼠腹部脂肪總脂肪酸含量亦低於控制組。未偵測到反式脂肪的原因推測如後，注射Aβ之劑量及時間所引致之自由基不足以導致內生性反式脂肪酸生成，或因注射Aβ而生成的自由基侷限在腦內小部分區域 (如海馬迴)，或是採集組織的時間已超過發炎的急性期而自由基生成量減少。未來可進一步探討AD導致自由基生成量之測定與其造成內生性反式脂肪生成的能力。
綜合上述結果，由側腦室連續7天注射Aβ25-35誘發AD之大鼠的腦部、肝臟與脂肪組織中並未偵測到內生性反式脂肪，僅在各組的心臟組織內則偵測到微量TFAs，且各組織中之脂肪酸組成與控制組比較並無顯著差異。

關鍵字

阿茲海默症 ； 類澱粉蛋白 (-beta) ； 內生性反式脂肪 ； 自由基 ； S-D大鼠 ； 氣相層析串連質譜儀

並列摘要

Trans fatty acids (TFAs) are defined as “unsaturated fatty acids that contain one or more isolated (i.e., nonconjugated) double bonds in a trans configuration.”TFAs mainly come from partial hydrogenated oil in processed foods and investigations of TFAs in the 90’s indicated that over intake of TFAs may increase the risk of cardiovascular diseases.
Besides from foods, TFAs may also be formed by free radical induced cis/trans isomerization of endogenous fatty acids from recent investigations and the formation of the “endogenous TFAs” was positively related with the amount of free radicals. Higher levels of endogenous TFAs were found in the smokers, lipopolysaccharides (LPS)-induced inflammation rats and normal aged rats. Alzheimer's disease (AD) is the main cause of dementia. The senile plaques with beta-amyloid peptide (Aβ) and the neuron tangles are universal pathological characters of AD brain . We suspected that the higher oxidative stress and inflammation in AD brain might induce cis/trans isomerization of fatty acids. The objective of this study is to investigate if there are more endogenous TFAs in brains, livers and adipose tissues of ventriclely injected Aβ25-35 induced AD rats, and we hope that the endogenous TFAs can be an indicator of AD.
The study was divided into two parts. The first part was to analyze the oxidative biomarkers, and the second part was to analyze the fatty acid compositions of brains, livers, hearts and adipose tissues. In the oxidative biomarker analyses, liver TBARS and liver glutathione reductase (GRd) activities showed that oxidative stress may be higher in Aβ25-35 group, while plasma TBARS and glutathione peroxidase (GPx) activities were similar in all five groups.
We also analyzed fatty acid compositions of brains, livers, hearts, abdominal adipose tissue, epidydimal adipose tissue and subcutaneous adipose tissue from rats of saline (control), Aβ and ‘Aβwith resveratrol’ injection group. No TFAs were detected and fatty acid compositions were not significantly different in brains, livers, and adipose tissues from the three groups, while trace TFAs (lower than the limit of quantification)were detected in hearts. Besides, total fatty acids content in abdominal adipose tissue from Aβand Aβ with resveratrol’groups were significantly lower than the control group. We infer that the absence of endogenous TFAs might due to not sufficient free radicals induced during seven days of Aβ25-35 injection, or Aβ induced free radicals concentrated only in limited area in brain (such as hippocampus), or even the sacrifice time point was after the acute inflammation period and resulted in decreased free radicals. In the future study, we suggest to investigate the amount of free radicals induced during AD and its ability to promote the formation of endogenous TFAs.
In conclusion, no detectable levels of endogenous TFAs were found in brains, livers and adipose tissues or only trace amount of TFAs appeared in hearts from ventricle injection of Aβ25-35 induced AD rats, and the fatty acid compositions were not significantly different from the control group.

並列關鍵字

beta-amyloid peptide ； endogenous trans fatty acids ； free radicals ； S-D rats ； gas chromatography-mass spectrometry

參考文獻

Adlof, R.; Copes, L.; Emken, E. Analysis of the monoenoic fatty acid distribution in hydrogenated vegetable oils by silver-ion high-performance liquid chromatography. J. Am. Oil Chem. Soc. 1995, 72, 571-574.

Adlof, R. O. Analysis of fatty acid mono- and diacylglycerol positional isomers by silver ion high-performance liquid chromatography. Journal of Chromatography A 1996, 741, 135-138.

Antolín, E. M.; Delange, D. M.; Canavaciolo, V. G. Evaluation of five methods for derivatization and gc determination of a mixture of very long chain fatty acids (c24:0-c36:0). J. Pharm. Biomed. Anal. 2008, 46, 194-199.

Asperger, A.; Engewald, W.; Fabian, G. Thermally assisted hydrolysis and methylation - a simple and rapid online derivatization method for the gas chromatographic analysis of natural waxes. J. Anal. Appl. Pyrolysis 2001, 61, 91-109.

Axen, K. V.; Dikeakos, A.; Sclafani, A. High dietary fat promotes syndrome x in nonobese rats. J. Nutr. 2003, 133, 2244-2249.