以維生素 E 代謝物alpha-CEHC於血漿中含量 為評估維生素 E 營養狀況之可行性初探

Abstract

中文摘要 α-CEHC(2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman)為近年來所鑑定出之α-tocopherol代謝產物。本研究的目的在建立血清中α-CEHC的分析方法，並據以分析攝食不同劑量維生素E鼠之血清α-CEHC，比較血清α-tocopherol與α-CEHC對飲食維生素E攝取量之反應性。 Stahl et al於1999年建立人血清的萃取分析法，是將人血清先以β-glucuronidase酵素作用，以水解血清中conjugatedα-CEHC，再經由萃取濃縮，並以HPLC-ECD分析得之。然本研究發現，β-glucuronidase酵素作用並不能使鼠血清分析所得之α-CEHC增多，而是在終濃度為3M的鹽酸處理下，可使鼠血清分析所得之α-CEHC量增加。HPLC-UV分析證實鼠血清中α-CEHC-sulfate conjugate之存在，且以直接定量α-CEHC-sulfate conjugate之分析結果，確定終濃度為3M之鹽酸處理即可將鼠血清中α-CEHC-sulfate conjugate均水解出來。故將鼠血清不經酸解而萃取得之α-CEHC稱之為Free α-CEHC、而經由鹽酸水解後萃取得之α-CEHC稱之為Total α-CEHC、兩者間之差值為Conjugated α-CEHC。 以上述建立之方法分析李氏論文實驗中，給予大鼠0(E0)、50(E50)、500(E500)、1000(E1000)、5000(E5000) mg/kg diet維生素E飲食兩週後所收集之大鼠血清。E0、E500、E1000、與E5000組，血清中α-tocopherol濃度分別為E50組之0.12、1.5、1.9及2.1倍。而血清中Free α-CEHC濃度，E0、E500、E1000、與E5000組分別為E50組之0.45、9.6、78與370倍。血清中Conjugated-α-CEHC濃度，E0、E500、E1000、與E5000組分別為E50組之0.1、10.7、25與127倍。血清中Totalα-CEHC濃度，E0、E500、E1000、與E5000組分別為E50組之0.15、8.4、34.3與221倍。血清中Free、Conjugated與Totalα-CEHC量與飲食中維生素E攝取量之相關係數R值分別為0.84 (p<0.0001) 、0.90 (p<0.0001)與0.78 (p<0.0001)，而血清中α-tocopherol與飲食中維生素E之相關係數R值為0.49 (p=0.0043)，這些結果顯示血清中α-CEHC濃度能反映飲食中維生素E之攝取量。 本研究繼而重複Stahl. et al 所建立之方法，發現經β-glucuronidase酵素水解無法將人血漿Total conjugated α-CEHC均水解釋出，而是須要先以β-glucuronidase酵素水解後續給予酸處理，才可將人血漿中Total conjugatedα-CEHC均水解而釋出。而先以酵素處理再給予酸處理所得人血漿中α-CEHC之分析值，與將人血漿給予最終鹽酸濃度為3M HCl、60℃、60min的加熱酸處理所得之值相當。此一結果顯示人血漿中Total α-CEHC，可經由加熱酸處理水解或先以酵素再以加酸處理後萃取得之。 以此法隨機收集未補充維生素E之成人血漿(5位)，與一位補充維生素E之成人血樣，分析所得血漿中膽固醇、α-tocopherol、以及加熱酸處理所得之α-CEHC含量。結果5個未補充維生素E之人血樣，其熱酸處理所得 α-CEHC濃度分別為12.4、23.5、23.8、13.8、16.0 pmole/mL，而在α-tocopherol濃度分析結果上，有３位分析所得血漿α-tocopherol濃度偏低，但當將所測得之α-tocopherol含量與血漿中膽固醇校正後，五位血漿中α-tocopherol/cholesterol 比例均＞2.8 (2.8為維生素E充足之指標)；同時血漿中α-tocopherol濃度與血膽固醇濃度呈正相關性(r=0.48，p=0.34)，顯示血漿中所測得之α-tocopherol似乎易受血漿膽固醇濃度之影響。然血漿中α-CEHC濃度與血膽固醇濃度則無相關性(r=0.07,p=0.89)。且血漿中α-tocopherol以cholesterol校正後，其比例值與血漿中所測得之α-CEHC也為正相關(r=0.59, p=0.29)；初步結果均可觀察到以血漿中α-CEHC似乎較α-tocopherol不易受到脂質之影響，並且有為維生素Ｅ營養狀況評估指標之前瞻性。 綜之，本論文研究建立了一適用大量樣品分析人血漿中α-CEHC分析方法，希望能應用在大型營養評估上，以觀察人血漿中α-CEHC作為維生素E營養指標之可行性。

Abstract α-CEHC is a urinary metabolite of -tocopherol found in recent years. This study aimed at the development of a procedure for the analysis of serumα-CEHC and compare the response of serumα-CEHC and -tocopherol to various levels of dietary vitamin E intake in rats. Unlike the results of Stahl et al (1999) obtained from human serum, rat serum α-CEHC measured by HPLC-ECD did not increase after β-glucuronidase pretreatment. However, 3N HCl pretreatment can release conjugatedα-CEHC. Direct measurement of free and sulfate conjugate -CEHC in rat serum by HPLC-UV resulted in values of free and conjugated -CEHC that is equivalent to those obtained by extraction without and with 3M HCl pretreatment. It is thus established that the total α-CEHC is the value determined by the procedure in which rat serum was pretreated with 3M HCl before extraction ; while free α-CEHC is the value without 3M HCl pretreatment. Using the α-CEHC analysis method developed for rat serum , serum from rats fed with 0 (E0), 50 (E50), 500(E500), 1000(E1000) and 5000(E5000) mg/kg diet of vitamin E were measured to examine the relationship between vitamin E intake and serumα-CEHC. The serum -tocopherol concentration of the E0, E500, E1000 and E5000 groups were 0.12-,1.5-,1.9- and 2.1- fold that of the E50 group. In contrast, the serum freeα-CEHC of rats fed 0, 500, 1000 and 5000 mg/kg diet of vitamin E were 0.45-, 9.6-,78- and 370- fold that of the E50 group. Similarly, the conjugated and total α-CEHC in serum of the E0, E500, E1000 and E5000 groups were 0.1-,10.7-,25- and 127- fold (conjugated) as well as 0.15-,8.4-,34.3-and 221+ fold (total) of the E50 group. The correlation coefficient R between serum freeα-CEHC and dietary vitamin E intake were 0.84 (p<0.0001), This R values among dietary vitamin E intake and conjugated or totalα-CEHC were 0.90 (p<0.0001) and 0.78 (p<0.0001) respectively. These values were higher than those between dietary vitamin E intake and α-tocopherol (R= 0.49, p=0.0043). These results indicate that the response of serum α-CEHC concentration to changes in vitamin E intake is more sensitive than that of serum -tocopherol. When human serum α-CEHC was analyzed by the reported method, it was found that β-glucuronidase pretreatment could not release all the conjugated α-CEHC. A modified procedure in which human plasma was incubated with 3N HCl at 60℃ for 60 min for the determination of total CEHC were developed and validated. Alternatively, the α-CEHC of glucuronide conjugate in human plasma can be determined by analyzingα-CEHC with and without β-glucuronidase pretreatment; while theα-CEHC of sulfate conjugate can be determined with and without 3N HCl pretreatment. Using the procedure, plasma samples form 5 donors without vitamin E supplementation were examined. The plasma total -CEHC ranged from 12.4 to 23.8 pmol/mL. Three of the samples had low plasma α-tocopherol concentration (< 7μg/mL). However, the plasma α-tocopherol /cholesterol ratio of these three samples were within the normal range of vitamin E status.(> 2.8μg/mg). The correlation between of -CEHC andα-tocopherol /cholesterol ratio(r=0.59) is better than that between -CEHC andα-tocopherol(r=0.27). In conclusion, a better procedure for the analysis of α-CEHC in serum/plasma was developed. Using the procedure, it has been preliminarily shown that the measurement of plasma/serum -CEHC might be more reliable than that of -tocopherol for the assessment of vitamin E nutrition status.