Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51904
標題: 人體血漿中malondialdehyde測定法之比較與改良
Comparison and improvement of assay methods for malondialdehyde levels in human blood plasma.
作者: 洪玉倫
Houng, Yu-lun
關鍵字: Human plasma;人體血漿;lipid peroxidation;malondialdehyde;脂質過氧化;丙二醛
出版社: 食品科學系
摘要: 
中 文 摘 要
人體血漿中脂質過氧化產物MDA含量常用TBA試劑測定,但方法上既不統一,所得之值也往往相差甚大。因此,本研究使用16位健康男女之血漿,探討常用的四種方法之間的相關性,並且進一步改良樣品之製備步驟,以及探討血漿中total MDA, bound MDA和free MDA之間的關係。
本研究以下述四種方法加以比較:(1)測定TCA上清液之TBARS值(方法A),採用Radiguez-Martinez and Ruiz-Torres (1992)之測定法;(2)測定lipid peroxides(方法B),採用Yagi (1989)之測定法;(3)測定total MDA(方法C),採用Chirico et al. (1993)之HPLC測定法;(4)測定KI處理(以還原lipid peroxides)後之血漿中MDA(方法D),採用Chow and Li (1994)之測定法。實驗結果顯示:不同試驗方法所測得之TBARS值有所不同;方法A和方法B之間有顯著正相關(r = 0.740, P<0.02),方法B和方法D之間有顯著負相關(r = 0.548, P<0.05),方法C和方法D之間也有顯著正相關(r = 0.516, P<0.05);其餘者無顯著負相關或正相關。由實驗結果顯示,這四種方法之適用性有待考量。
本研究將上述四種方法分別配合三種偵測器(分光光度計、螢光光度計和HPLC)加以測定TBARS時,結果發現方法A、B、C、D配合三種偵測器測得之值之間有正相關,但其中以方法D之三種偵測值之間相關性較低(r = 0.709, P<0.002),此外,方法B、C、D由分光光度計所測得之值皆與用螢光光度計或HPLC測得之值之間有顯著差異。方法A以分光計測得之值(0.39±0.15μM)與HPLC測得之值(0.11±0.06μM)有良好之相關性(r = 889, P<0.001),因此,方法A配合分光計可作為簡便之free MDA測定法。方法B以螢光光度計和HPLC測得之TBARS亦具正相關(r = 0.918, P<0.001),且兩者測得之值並無顯著差異(P>0.20),因此,用Yagi之螢光法所測得物質應大部分為MDA而非lipid peroxides。
其次,本研究針對A、B、C、D四法之樣品製備步驟加以改良,並分別探討鹼化、酸種類、酸加熱時間、BHT及KI對測定值之影響。結果
發現:鹼化作用對測定血漿中total MDA而言是相當重要的步驟,可將結合在血漿蛋白質上之MDA游離出來。TCA比H3PO4有較強的沈澱蛋白質之能力,同時,TCA-whole plasma在120分鐘加熱過程中,其TBARS含量在30分鐘後即不再增加。當血漿中加入0.2% BHT,最終測得之TBA-MDA adduct減少23%。此外,加入1%KI亦使TBA-MDA adduct減少43%,因此本樣品改良步驟將BHT及KI並用,以增加方法之可靠性。
本研究在改良樣品之製備步驟後,分別配合三種偵測器測定血漿中MDA含量;結果發現無論分光計、螢光計或HPLC所測得之值均無顯著差異(P>0.05),其值分別為1.92±1.04μM,1.81±0.92μM及1.54±0.72μM,亦即利用分光計測定,其TBARS值即能代表TBA-MDA adduct。此種結果說明本改良步驟具高度之專一性。本改良步驟之回收率,再現性和靈敏度皆與其他配合HPLC測定方法相當,也適用於體外氧化試驗。此外,本改良步驟配合HPLC/Vis偵測器可與其他脂質過氧化指標相比擬。
其次,本研究又利用樣品改良步驟來測定血漿中total MDA、free MDA和bound MDA含量。結果顯示:配合HPLC測定時,改良步驟所測得之total MDA含量(1.45±0.17μM)約等於bound MDA含量(1.34±0.07μM)加上free MDA含量(0.07±0.02μM)。此外,改良步驟所測得之free MDA和bound MDA或total MDA之間無相關(r = 0.620, n = 6, P>0.05),而bound MDA和total MDA之間有正相關(r = 0.941, n = 9, P<0.001)。因此在測定血漿中脂質過氧化物中,測定total MDA或bound MDA含量之代表性比測定free MDA為佳。
以上結果顯示:本改良法之適用性極高,未來期望本研究室能能進一步將此改良法應用在疾病之偵測上。

ABSTRACT
A common method for measurement of MDA (malondialdehyde) levels in human plasma is the TBA (thiobarbituric acid) test. Both the protocols of TBA test and the MDA levels measured as TBARS in human plasma vary widely. The specificity of the TBA reaction has been strongly questioned by various laboratories. Hence, we used the plasma of 16 volunteers to study the correlation between four commonly used methods. Furthermore, we improved the procedure of TBA test and examined the adaptability of the modified procedure. In addition, we investigated the relationship of total MDA, bound MDA and free MDA in human plasma.
Th four methods used were: (1) measurement of TBARS of plasma TCA-supernatant (Method A; Radiguez-Martinez and Ruiz-Torres 1992);(2) measurement of lipid peroxides (Method B; Yagi 1989);(3) measurement of total MDA (Method C; Chirico et al. 1993);(4) measurement of MDA in plasma treated with KI prior to TBA reaction (Method D; Chow and Li 1994). The results show that there were positive correlations between TBARS determined by method A and method B (r = 0.740, P<0.02) and by method C and method D (r = 0.516, P<0.05). There was a negative correlation between TBARS by method B and method D (r = 0.548, P<0.05). The results show the need of standardization of the various TBA tests.
Following the TBA test protocols of each of the four methods, TBARS (or TBA-MDA adducts) were detected by spectrophotometry, flourescence and HPLC/Vis. The results show that there were significant correlations among the values obtained by the three detectors within each protocol. The TBARS obtained from the four methods with spectrophotometric detection were higher than those detected with flourescence and HPLC/Vis detection. There was a significant correlation (r = 0.889, P<0.002) between TBARS determined by method A with spectrophotometric detection and HPLC/Vis detection. Hence, method A with spectrophotometric detection could be a simple method to determine free MDA in plasma. There also was a positive correlation in lipid peroxides determined by method B between flourescence and HPLC/Vis detection (r = 0.918, P<0.001). There was no significant difference in lipid peroxides obtained from method B by flourescence detection and HPLC/Vis detection (P>0.20). Thus, most materials measured by method B should be TBA-MDA adducts rather than lipid peroxides.
In order to improve the TBA test procedure, we investigated the effects of alkaline hydrolysis, types of acids, acid-heating time, BHT (butylated hydroxytoluene) and KI on levels of TBA-MDA adduct in human plasma. Results show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma, because this treatment led to release of MDA from plasma proteins. TCA was a better protein precipitating reagent than H3PO4 since the TBA-MDA adduct of TCA-whole plasma did not increase after 30 min. BHT was used to inhibit the formation of lipid peroxidation during TBA test. The levels of TBA-MDA adduct were decreased 23% by inclusion of 0.2% BHT in plasma. The addition of 1% KI resulted in 43% decrease in the TBA-MDA adduct. Therefore, both BHT and KI were included in the modified procedure.
The total MDA obtained from 16 volunteers by the modified procedure and detected by spectrophotometry, flourescence and HPLC/Vis were 1.92±1.04 μM, 1.81±0.92μM and 1.54±0.72μM, respectively. The differences among the three values were not significant (P>0.05). The data shows that TBARS obtained by the modified procedure were essentially TBA-MDA adduct. The recovery (average: 82~106%), precision (within-assay C.V%: 2~4%, between-assay C.V%: 4~8%) and sensitivity of the modified procedure was comparable to other methods detected by HPLC. In addition, the modified procedure with HPLC/Vis detection compared favorably with other indices of lipid peroxidation.
Furthermore, we measured total MDA, bound MDA and free MDA of plasma by the modified procedure with HPLC/Vis detection. The results show that total MDA (1.45±0.17μM) was equal to bound MDA (1.34±0.07μM) plus free MDA (0.07±0.02μM). There was no significant correlation between free MDA and bound MDA (r = 0.620, n=6, P>0.05) or between free MDA and total MDA (r = 0.587, n=6, P>0.05). By contrast, bound MDA and total MDA was highly correlated (r = 0.941, n=9, P<0.001). Thus, the results indicate that the measurement of total MDA or bound MDA is a better index of lipid peroxidation than the measurement of free MDA in plasma.
In conclusion, the applicability of the modified procedure is good. However, the application of the modified procedure for assessing oxidative stress in various diseases remains to be examined.
Keywords
Human plasma, MDA (malondialehye), lipid peroxidation, HPLC
URI: http://hdl.handle.net/11455/51904
Appears in Collections:食品暨應用生物科技學系

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