改良乙醇脫氫酶法測定血清中微量乙醇

中國衛生檢驗雜志2008年1月第18卷第1期 Chinese Journal of Health Laboratory Technology,Jan2008;ld18No1【論著(zhù)】改良乙醇脫氫酶法測定血清中微量乙醇胡云良2,王慧燕,張立2,李艷霞3,王友沛,王賢理3.溫州醫學(xué)院藥學(xué)院浙江溫州35035:4浙江省蒼南縣人民醫院漸江溫州35035(1.溫州醫學(xué)院附屬第二醫院檢驗科,浙江溫州325027;2.溫州醫學(xué)院檢驗醫學(xué)院,浙25800;5.溫州伊利康生物技術(shù)有限公司,浙江溫州325011)[摘要]目的建立一個(gè)簡(jiǎn)便、靈敏特異試劑穩定的乙醇脫氫酶法檢測血清中微量乙醇濃度。方法選擇Tms-hCl沖液作為乙醇脫氫酶法測定乙醇的反應條件通過(guò)優(yōu)化反應體系及自動(dòng)生化分析儀分析參數,采用兩點(diǎn)終點(diǎn)法對乙醇進(jìn)行檢測并評價(jià)了方法學(xué)性能。結果:乙醇脫氫酶(ADH)最適用量為1.2g/L,氧化型輔酶I(NAD·)最適濃度為9g/L。線(xiàn)性范圍可達100mg/L,批內變異系數(CV)小于28%,批間變異系數(CV)小于39%,平均回收率為998%。本法(Y)與氣相色譜法(X)比較,具有良好的相關(guān)性,y=0.993X-9.76,r=0.998。膽紅素159.1μmo/L、甘油三酯74mmo/L、血紅蛋白116gL、乙醛0.3g/L、異丙醇0.2g/L、甲醇0.7g/L、甲醛0.6g/L以下對本法測定乙醇結果無(wú)明顯干擾。結論:本法測定血清中乙醇無(wú)需除蛋白,具有快速、簡(jiǎn)便、靈敏特異等優(yōu)點(diǎn)適合于自動(dòng)生化分析儀檢測。[關(guān)鍵詞]乙醇;乙醇脫氨酶法;兩點(diǎn)終點(diǎn)法;自動(dòng)生化分析[中圖分類(lèi)號]R446.1[文獻標識碼]B[文章編號]1004-8685(2008)01-007-03An improved method for determination of a small quantity of alcohol in serum with thealcohol dehydrogenaseHu Yun-liang, Wang Hui-yan, Zhang L, Li Yan-xia, Wang You-pei, Wang Xian-di(1. Department of Laboratory Medicine of the Second Affiliated Hospital, Wenzhou Medical College, Wenzho2. School of Laboratory Medicine, Wenzhou Medical College, Wenzhou 325027, China; 3. School of PharmacyCollege, Wenzhou 325027, China; 4. The Peoples Hospital of Cangnan, Cangnan 325800, China; 5. Yilik20Co Ltd, Wenzhou 325011, China)Abstract] Objective: To establish a simple, sensitive, specific method of determination of alcohol in serum with stable rea-gents by catalysis of alcohol dehydrogenase. Methods: Tris-HCI buffer was used in this study. In the condition of alkalescencealcohol dehydrogenase(ADH)catalyzed the substance of alcohol to acetaldehyde, with the simultaneous production of deoxidizednicotinamide adenine dinucleotide(NADH). To analyze alcohol by two point end assay after optimizing reaction system and assayparameter of automatic analyzer. Results: The most adaptive quantity of ADH was 1. 2 g/L, and the best concentration of theNAD'was9g/L The linearity was up to 1200 me/L The within run CV was lower than 2.8% and the between run CV was lowerthan 3.9%. The average recovery was 99.8%. Comparing this method (Y) with gas chromatography(X), the regression equationwas obtained, Y=0.993X-9 76, r=0. 998. The assays showed no interference from as much as 159. I umoV/L of bilirubin7. 4 mmol/L of triglyceride, 11. 6 g/L of hemoglobin, 0. 3 g/L of acetaldehyde, 0. 2 g/L of isopropanol, 0. 7 g/L of methanoland 0.6 g/L of maldehyde. Conclusion: The method established for determining alcohol in serum is a rapid, simple, sensitive, speIt isKey words] Alcohol; Alcohol dehydrogenase; Two point end assay; Automatic analysis體液中乙醇含量測定是酒精中毒的重要判斷依據,通過(guò)量衰竭技術(shù)及氣相色譜法等。其中氣相色譜法是測定乙醇的對攝入酒精后血中乙醇含量的測定,可方便迅速地監測和研參考方法,具有較高的靈敏度與特異性但設備昂貴操作復究乙醇在體內的代謝過(guò)程,從而為預防和減輕酒精中毒的研雜而其它方法在穩定性、靈敏度特異性等方面都存在不同究提供相應的指標和依據。測定乙醇的方法主要有化學(xué)比色程度的缺陷。我們對乙醇脫氫酶法測定乙醇進(jìn)行了方法學(xué)探法、呼出氣乙醇分析、滲透壓測定法酶試劑分析法、放射性能索通過(guò)改進(jìn)試劑配方調整反應條件及分析參數使其檢測乙醇在準確度、特異性及穩定性方面有一定程度的改善,現報[基金項目]溫州市科技局課題(C2002055道如下:[作者簡(jiǎn)介]胡云良(1966-),男,碩士,副教授,主要從事臨床生化教學(xué)與醫院檢驗科工作。中國衛生檢驗雜志2008年1月第18卷第1期 Chinese Journal of Health Laboratory Technology,lan2008;vl18Nol1材料與方法7170型自動(dòng)生化分析儀上測定,觀(guān)察反應動(dòng)力學(xué)結果見(jiàn)圖1。1.1原理按 NCCLS EP6-A文件作線(xiàn)性評價(jià)”。在200-1200mg/L范選用三(羥甲基)氨基甲烷~鹽酸(Tns-HC)緩沖液體圍內稀釋變異