鼠尿 alpha-CEHC Sulfate 純化鑑定與 Conjugated alpha-CEHC 分析方法之重建及活化 PPAR alpha 傳訊途徑對維生素 E 代謝生成 alpha-CEHC 之影響

Abstract

近年發現 2,5,7,8-tetramethyl-2-(2’-carboxyethyl)-6-hydroxychroman (alpha-CEHC) 為維生素 E 側鏈被切短後的水溶性代謝產物。目前推測其側鏈截短之代謝途徑牽涉到 omega-hydroxylation 及支鏈 beta-oxidation。由於催化 omega-hydroxylation 之酵素 cytochrome P450 4A1 (CYP4A1) 及過氧化體 beta-oxidation 的酵素 acyl-CoA oxidase 1 (ACO1) 均為受 peroxisome proliferator-activated receptor alpha (PPARalpha) 調控之下游基因。為探討 PPARalpha 對維生素 E 代謝生成 alpha-CEHC 之影響效應，本研究以大鼠之動物模式，投予 PPARalpha 活化劑 clofibrate 及 perfluorodecanoic acid (PFDA) 誘發 PPARalpha 標的基因表現，觀察活化 PPARalpha 傳訊途徑對鼠尿中維生素 E 代謝產物 alpha-CEHC 排出量之影響。 alpha-CEHC 於體內會與 glucuronic acid 或 sulfate 等結合，形成alpha-CEHC conjugate，隨尿液排出體外。文獻分析樣品 alpha-CEHC之方法，多用 beta-glucuronidase 酵素水解 alpha-CEHC conjugates，再以 high performance liquid chromatography with electrochemical detector (HPLC-ECD) 定量之。然而，本實驗發現鼠尿經 HCl 強酸處理後，HPLC-ECD 所測得之 alpha-CEHC 含量會明顯竄升，以 beta-glucuronidase 酵素處理者則否。為確認鼠尿 alpha-CEHC conjugate之結構，我們將鼠尿進行純化分離，經化學結構鑑定確認此HCl releasable alpha-CEHC conjugate為 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate)。另外，我們亦重建酸水解萃取分析尿液 conjugated alpha-CEHC 之方法：將含抗壞血酸之樣品，以 6 N HCl 於 60度C 反應 1 小時進行酸加熱水解，之後以乙醚萃取，續以 HPLC-ECD 進行 alpha-CEHC 定量分析。在熱酸水解過程中，以抗壞血酸作為抗氧化劑可以有效地保護 alpha-CEHC不被破壞。此分析方法的優點為快速、靈敏及回收率佳。 動物實驗共分三部分進行。實驗一採雙因子變因設計，分別給予大鼠餵食含 50 mg/kg all-rac-alpha-tocopheryl acetate (alpha-TA) (L 組)、50 mg/kg alpha-TA + 0.5% clofibrate ( LC 組)、500 mg/kg alpha-TA (H 組) 或 500 mg/kg alpha-TA + 0.5% clofibrate ( HC 組) 之試驗飼料為期一週，其間並收集尿液。結果顯示 clofibrate 處理會造成大鼠肝臟及血清 alpha-tocopherol (alpha-TOH) 含量明顯下降及肝臟中 PPARalpha 下游基因 ACO1、CYP4A1 及 D-bifunctional protein (D-BF) 之酵素活性、蛋白質或 m-RNA 表現量顯著增加。尿液 alpha-CEHC 排出量方面，LC 組老鼠之每日 alpha-CEHC 排出量顯著高於 L 組老鼠 (p<0.05)，HC 組卻稍低於 H 組。若將 alpha-CEHC 排出量以佔每日飲食維生素 E 攝入量的比例來表示，可以發現LC 組老鼠之 alpha-CEHC 排出量顯著高於 L 組 (p<0.05)，HC 組老鼠之 alpha-CEHC 排出量略高於 H 組，但組間無統計差異。 延續實驗一之結果，實驗二改以管餵方式提供正常劑量之維生素 E (5 mg alpha-TA/kg B.W./day)，觀察給予含 0 (C 組)、0.1 % (0.1P 組)、0.25% (0.25P 組)、0.5% (0.5P 組) 或 1 % (1P 組) 之 clofibrate 試驗飼料一週，對維生素E代謝之影響。結果顯示，clofibrate 處理會造成 PPARalpha 標的基因 ACO1、CYP4A1及參與支鏈脂肪酸代謝之 ACO2 及 D-BF 等之酵素活性、蛋白質或 mRNA 表現量顯著增加，且具劑量效應之趨勢。尿液中每日 alpha-CEHC 排出量亦隨 clofibrate 處理劑量增加而上升。且 ACO1、CYP4A1、ACO2 及 D-BF之表現與尿液alpha-CEHC 排出量呈顯著之正相關性 (r=0.30-0.46, p<0.05)。 實驗三則在正常維生素 E 劑量 (50 mg alpha-TA/kg diet) 試驗飼料下，每日以腹腔方式給予 0、0.5、1、2.5、5 或 10 mg/kg B.W. 之 PFDA，誘發 PPARalpha 傳訊途徑，觀察不同類型之 PPARalpha 活化劑對維生素E代謝之影響。結果顯示，PFDA 同樣會造成 PPARalpha 下游基因 ACO1、CYP4A1等之酵素活性、蛋白質或 m-RNA 表現量顯著增加，且具劑量效應。尿液中每日 alpha-CEHC 排出量亦隨 PFDA 處理劑量增加而上升。PPARalpha 下游基因 ACO1、CYP4A1之表現與尿液alpha-CEHC 排出量呈顯著之正相關 (r=0.40-0.56, p<0.05)。至於，目前文獻中提及可能參與維生素 E 代謝之相關蛋白質，如 CYP3A、CYP4F等，於本實驗中其 mRNA或蛋白質表現量與尿液 alpha-CEHC 排出量無關聯性，甚至呈負相關。 因此，本研究之結果初步證實利用 PPARalpha 活化劑 clofibrate 及 PFDA 誘發 PPARalpha 標的基因的表現，會促進體內維生素 E代 謝，增加尿液中 alpha-CEHC 的排出量。活化 PPARalpha 傳訊途徑可能會影響體內維生素 E 的代謝。

2,5,7,8-tetramethyl-2-(2’-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the metabolite of alpha-tocopherol (alpha-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in the urine. Pathway of the metabolism involves omega-hydroxylation of phytyl side chain and the following beta-oxidation. omega-Hydroxylation is known to be catalyzed by cytochrome P450 enzymes (CYPs), of which CYP3A and CYP4F is the most likely candidates. The enzymes which are responsible for the omega-oxidation (CYP4A1) and peroxisomal beta-oxdiation (acyl-CoA oxidase 1, ACO1) of fatty acid are transcriptionally regulated by peroxisome proliferator activated receptor alpha (PPARalpha). In order to investigate effects of PPARalpha activation on the vitamin E metabolism, Wistar rats were treated with PPARalpha activators - clofibrate and perfluorodecanoic acid (PFDA) and urinary alpha-CEHC was monitored in this study. alpha-CEHC was known to be conjugated with glucuronic acid or sulfate. Various CEHCs in biological specimen were mostly measured by high performance liquid chromatography with electrochemical detector (HPLC-ECD) preceded by beta-glucuronidase hydrolysis. In an attempt to analyze alpha-CEHC in rat urine accordingly, it observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was therefore isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60。C in the presence of ascorbate, followed by a one-step ether extraction not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. A total of three rat experiments were conducted to examine the effects of PPARalpha activators on urinary alpha-CEHC excretion. In Experiment 1, rats were fed diets containing 50 mg/kg all-rac-alpha-tocopheryl acetate (alpha-TA) (L), 50 mg/kg alpha-TA + 0.5% clofibrate ( LC ), 500 mg/kg alpha-TA (H) or 500 mg/kg alpha-TA + 0.5%clofibrate (HC) for 1 week, and the urine was collected for alpha-CEHC analysis. PPARalpha target genes including CYP4A1, ACO1 and D-BF is induced significantly by clofibrate revealed by the expression of enzyme activity, protein or mRNA. Clofibrate treatment resulted in a significant decrease of the alpha-TOH content in serum and liver. The urinary alpha-CEHC content of LC group is significantly higher than that of the L group (p<0.05). The ratio of urinary alpha-CEHC to dietary vitamin E intake of the LC group is also significantly higher than the L group. However, no significant difference between H and HC group was found. In Experiment 2, rats were fed vitamin E devoid AIN-76 modified diets containing 0 (C), 0.1 (0.1CF), 0.25 (0.25CF), 0.5 (0.5CF), 1 (1CF) % clofibrate and were i.p. injected with 5 mg alpha-TA/kg B.W. daily for 1 week. Expressions of PPARalpha target genes, namely, CYP4A1, ACO1, ACO2 and D-BF that participated in the metabolism of fatty acid were all increased significantly and does-dependently by the clofibrate treatment as revealed by the of enzyme activity, protein or mRNA expression. The urinary alpha-CEHC excretion of all clofibrate treated groups were also increased does-dependently (p<0.05). Again, there were significantly positive correlations between the urinary alpha-CEHC and the expression of CYP450, CYP4A1 and ACO1 (r=0.40-0.56, p<0.05). In Experiment 3, another PPARalpha activator PFDA was used. All of the 6 groups of rats were fed the AIN-76 modified diet containing 50 mg/kg alpha-TA and respectively tube-fed vehicle (C) or 0.5 (0.5P), 1(1P), 2.5 (2.5P), 5 (5P) or 10 (10P) mg/kg body weight of PFDA daily for 1 week. PPARalpha target genes - CYP4A1 and ACO1 expression in the liver also increased significantly and does-dependently by PFDA as revealed by enzyme activity, protein or mRNA expression (p<0.05). The urinary alpha-CEHC content of all PFDA treated groups also increased does-dependently (p<0.05). Positive correlations between the urinary alpha-CEHC and the expression of CYP4A1 and ACO1 were again observed (r=0.42-0.50, p<0.05). However, CYP3A and CYP4F which has been considered to catalyze vitamin E catabolism to alpha-CEHC showed no correlation with urinary alpha-CEHC in this study (p>0.05). In conclusion, this study demonstrates that PPARalpha activation is associated with an increased urinary alpha-CEHC excretion. The activation of PPARalpha signal pathway may enhance the vitamin E catabolism through up-regulation of some of its target genes (ex. CYP4A1 and ACO1).