- 學位論文
丙酮酸鹽膳食補充品中parapyruvate的含量及其促進人類Hs68纖維母細胞衰老的作用
Content of parapyruvate in commercialized pyruvate supplements and the effects of parapyruvate to induce senescence of human fibroblastic Hs68 cells
摘要
丙酮酸鹽是糖解反應終產物，具有抗氧化、抗發炎、減重、降血糖、降低胰島素阻抗等作用，丙酮酸鹽膳食補充品在市場上以鈉鹽(丙酮酸酸鈉; sodium pyruvate)、鉀鹽(丙酮酸酸鉀; potassium pyruvate)、鈣鹽(丙酮酸酸鈣; calcium pyruvate) 的型式供應，另外，亦有混合不同比例之鈉鹽、鉀鹽和鈣鹽而成的混合型丙酮酸鹽膳食補充品(商品名為triple pyruvate)。parapyruvate是由兩分子的丙酮酸進行醛酮縮合反應(aldol condensation)所形成的雙分子聚合物，為α-ketoglutarate dehydrogenase complex (KGDHC)的抑制劑，而已知KGDHC活性降低在阿滋海默症及帕金森氏症等老化相關之神經退化性疾病中扮演著關鍵性的角色。然而，市售丙酮酸鹽膳食補充品中是否含有parapyruvate以及parapyruvate是否因抑制KGDHC而促使人類Hs68纖維母細胞衰老目前並不清楚。在本研究中，由於市面上無法購得parapyruvate作為標準品，我們將丙酮酸經由鹼處理促使其聚合，進一步以溶劑結晶純化parapyruvate，並以液相層析串聯式質譜儀(Liquid Chromatography Tandem Mass Spectrometer, LC-MS/MS)進行parapyruvate的鑑定，及以高效液相層析法(high performance liquid chromatography, HPLC)分析純度。關於KGDHC活性抑制能力的測定分成體外酵素活性抑制能力測定及細胞中KGDHC活性抑制能力測定。我們所產製的parapyruvte晶體，經LC/MS/MS分析未發現其它不純物的波峰，確認其為純品，且經HPLC分析其純度大於99%。體外酵素活性抑制能力測定，其具有顯著抑制KGDHC活性的能力，並且呈現濃度效應。分析市面上丙酮酸鉀和丙酮酸鈣之食品級原料各一種廠牌，結果顯示其parapyruvate的含量在丙酮酸鉀及丙酮酸鈣分別為7%及10.9%，另外，分析市面上數種廠牌的丙酮酸鹽膳食補充品產品，結果亦顯示含有1.4∼10.6%的parapyruvate。進一步探討parapyruvate是否具有細胞毒性及促進細胞衰老的作用，結果發現Hs68纖維母細胞經0~20 mM parapyruvate處理 24小時，並不會產生細胞毒性，但parapyruvate在濃度1mM以上時具有抑制細胞生長、促進細胞衰老及增加細胞老化標記senescence associated-β-galactosidase (SA-βG)活性的作用，顯示parapyruvate具有顯著促進Hs68細胞衰老的作用。Parapyruvate濃度1 mM以上時具有顯著抑制細胞中KGDHC活性的能力，且呈現濃度效應 ; 添加了KGDHC活化劑鈣離子，亦顯著地拮抗parapyruvate抑制細胞內KGDHC活性及促進細胞衰老的效應。本研究成功的開發了合成parapyruvate的方法，並且發現市面上的丙酮酸鹽膳食補充品原料及產品中含有1.4∼10.9%不等量parapyruvate，parapyruvate能夠藉由抑制KGDHC的活性進而造成細胞衰老，此顯示parapyruvate對人體可能有害，而丙酮酸鹽膳食補充品中所含的parapyruvate劑量，是否會引發食品安全問題，未來值得以動物模式深入的研究。
並列摘要
Pyruvate is an end product of glucose metabolism via glycolysis. Several studies have demonstrated that pyruvate possesses multiple functionalities including antioxidant, anti-inflammatory, weight-loss, blood glucose-lowering and insulin resistant-lowering activities. Commercialized pyruvate supplements are supplied by one of forms of sodium, potassium and calcium pyruvate salts (i.e., sodium pyruvate, potassium pyruvate and calcium pyruvate). In addition, a mixture form of pyruvate supplement with a product name “triple pyruvate” is also supplied in the market by mixing with certain proportions of sodium, potassium and calcium pyruvate salts. Parapyruvate is a dimmer formed by polymerization of 2 molecules of pyruvate via aldol condensation. Parapyruvate is an inhibitor of α-ketoglutarate dehydrogenase complex, KGDHC. On the other hand, it is known that decreased KGDHC activity plays a crucial role in the aging-related neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. However, it is unclear whether commercialized pyruvate supplements and the raw material pyruvate salts contain parapyruvate and whether parapyruvate can induce senescence of human fibroblastic Hs68 cells by inhibition of KGDHC activity. In this study, we made parapyruvate by alkalization of pyruvic acid solution and purified parapyruvate by solvent crystallization, since parapyruvate is not commercially available. The synthesized parapyruvate was identified by LC-MS/MS and the purity of synthesized parapyruvate was monitored by HPLC. Inhibition of in vitro KGDHC activity was assessed either by a NADH colorimetric method or by a commercialized kit, whereas inhibition of intracellular KGDHC activity was determined by a nitroblue tetrazolium (NBT) colorimetric method. We found that the synthesized parapyruvate was quite pure and its purity was more than 99% determined by HPLC. The synthesized parapyruvate also significantly inhibited in vitro KGDHC activity in a concentration dependent manner. We obtained two brands of food grade of raw materials including potassium pyruvate and calcium pyruvate, respectively; we also obtained 4 brands of final capsule products of calcium pyruvate and 1 brand of triple pyruvate, and the contents of parapyruvate in these raw materials and final products were found with a range from 1.4∼10.9% . Furthermore, we explored the effects of parapyruvate on the cytotoxicity and replicative senescence of human fibroblastic Hs68 cells. The results showed that there were no effect of 1∼20 mM parapyruvate on cytotoxicity but parapyruvate at concentrations above 1 mM could inhibit cell growth, induce cell senescence and increase the senescence associated-β-galactosidase (SA-βG) activity. We also found that calcium, an activator of KGDHC could antagonize the effect of parapyruvate in decreasing KGDHC activity and inducing senescence of Hs68 cells. In this study, a method of synthesizing parapyruvate was successfully developed and we found that the contents of parapyruvate in commercialized pyruvate supplements was around 1.4∼10.9%. Parapyruvate could significantly inhibit the activity of KGDHC and induce cell senescence in Hs68 cells. These results suggest that parapyruvate may be harmful to the human health, and may lead to food safety concerns, which merits further investigations with animal models.
參考文獻
延伸閱讀
- 林均憶（2019）。丙酮酸鹽膳食補充品中不純物parapyruvate對線蟲壽命的影響與KGDHC的角色〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU201900178
- 施采瑩（2018）。槲皮素和染料木黃酮調控脂多醣誘發C2C12肌肉細胞發炎反應之功效〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0003-2908201816320600
- 林音秀（2005）。天門冬胺酸及麩胺酸的羥?胺酸衍生物其生理活性功能之探討-----抗氧化、抑制血管收縮素轉換酵素和Semicarbazide - Sensitive Amine Oxidase〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-1704200714541730
- 李燕霖（2005）。槲皮酮及其代謝產物對人類肝癌細胞株Hep 3B生長之抑制機轉及槲皮酮對正常大鼠初代肝細胞麩胱甘肽相關之解毒代謝與抗氧化系統之影響〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2005.01748
- Littorin, M., Truedsson, L., Welinder, H., Skarping, G., Mårtensson, U., & Sjöholm, A. G. (1994). Acute Respiratory Disorder, Rhinoconjunctivitis and Fever Associated with the Pyrolysis of Polyurethane Derived from Diphenylmethane Diisocyanate. Scandinavian Journal of Work, Environment & Health, 20(3), 216-222. https://www.airitilibrary.com/Article/Detail?DocID=03553140-199405-201011220036-201011220036-216-222