裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸

引用本文: 周朗, 李乔婧, 李永生, 高秀峰. 裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸[J]. 分析化学, 2012, 40(11): 1725-1729. doi: 10.3724/SP.J.1096.2012.20271 shu

Citation: ZHOU Lang, LI Qiao-Jing, LI Yong-Sheng, GAO Xiu-Feng. Rapid Determination of Pyruvic Acid in Erythrocytes by Lysing-heating Fluorescence Capillary Method[J]. Chinese Journal of Analytical Chemistry, 2012, 40(11): 1725-1729. doi: 10.3724/SP.J.1096.2012.20271 shu

裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸

周朗 ^1, ,
李乔婧 ^1, ,
李永生 ^{1,
,} ,
高秀峰 ^2,

通讯作者: 李永生

摘要: 在常规红细胞处理方法中,用生理盐水对红/白细胞清洗分离会导致细胞内丙酮酸外渗,造成丙酮酸测定值不能反映细胞内丙酮酸的真实浓度。本研究设计了"氯化铵裂解液特异性裂解红细胞-离心分离白细胞/血小板-加热去除蛋白-丙酮酸酶荧光毛细管分析"方法。最优化的血样预处理条件是:以16000 r/min离心血液1 min,获得红细胞;裂解液裂解红细胞后在100℃下加热5 min。对比实验发现,本方法优于其它红细胞处理法。本研究中丙酮酸测定方法的线性范围为10-120 mmol/L; 检出限为0.98 mmol/L; 灵敏度为5.64 F L/mmol,高于文献方法60倍以上;测定红细胞内丙酮酸值的相对标准偏差小于2.8% (n=11),回收率在98.3%-104.1%之间。

关键词:

English

Rapid Determination of Pyruvic Acid in Erythrocytes by Lysing-heating Fluorescence Capillary Method

1.
¹ School of Chemical Engineering, Sichuan University, Chengdu 610065, China;

Corresponding author: 李永生

Received Date: 09 March 2012

Abstract: In the conventional method of erythrocytes treatment, there was the efflux of pyruvic acid in erythrocytes (PAE) during washing and separation of erythrocytes/leukocytes using the physiological saline. This phenomenon caused the deviation of the measured value of PAE from its real concentration. This research designed a method comprising the steps of employing the erythrocyte lysis buffer to specifically lyse the red blood cells, separating leukocytes and platelets by centrifugation, removing the protein by heating the hemolysate, and detecting PAE using the pyruvate-enzyme fluorescence capillary analysis. The optimized procedures for the blood pretreatment were as follows: at the centrifugal speed of 16000 r/min, the blood sample was centrifuged for 1 min to obtain erythrocytes, after the erythrocytes were lysed by the lysis buffer, the hemolysate was heated for 5 min at 100℃. Through the contrast experiment, it was found that this method was superior to other erythrocyte treatment method. The pyruvate determination method in this research had a good linearity in the range of 10-120 mmol/L, a detection limit of 0.98 mmol/L, and a sensitivity of 5.64 F L/mmol, it was 60 times higher than that of references method. This method offered a relative standard deviation of PAE less than 2.8% (n=11), and a recovery range of 98.3%-104.1%.

Key words:

1. [1]
  1 Lam T K T, Gutierrez-Juarez R, Pocai A, Rossetti L. Science, 2005, 309(5736): 943-9471 Lam T K T, Gutierrez-Juarez R, Pocai A, Rossetti L. Science, 2005, 309(5736): 943-947
2. [2]
  2 Gajovic N, Binyamin G, Warsinke A, Scheller F W, Heller A. Anal. Chem., 2000, 72(13): 2963-29682 Gajovic N, Binyamin G, Warsinke A, Scheller F W, Heller A. Anal. Chem., 2000, 72(13): 2963-2968
3. [3]
  3 Horiguchi N, Horiguchi H. E. U. Patent, 1522852, 20033 Horiguchi N, Horiguchi H. E. U. Patent, 1522852, 2003
4. [4]
  4 Shepotinovskii V I, Barsukova O I, Mikarshinovskii Z I. S. U. Patent, 1770906, 19724 Shepotinovskii V I, Barsukova O I, Mikarshinovskii Z I. S. U. Patent, 1770906, 1972
5. [5]
  5 Darghouth D, Koehl B, Heilieret J F, Madalinski G, Bovee P, Bosman G, Delaunay J, Junot C, Roméo P H. Haematol.-Hematol. J., 2011, 96(12): 1861-18655 Darghouth D, Koehl B, Heilieret J F, Madalinski G, Bovee P, Bosman G, Delaunay J, Junot C, Roméo P H. Haematol.-Hematol. J., 2011, 96(12): 1861-1865
6. [6]
  6 Markuszewski M J, Szczykowska M, Siluk D, Kaliszan R. J. Pharm. Biomed. Anal., 2005, 39 (3-4): 636-6426 Markuszewski M J, Szczykowska M, Siluk D, Kaliszan R. J. Pharm. Biomed. Anal., 2005, 39 (3-4): 636-642
7. [7]
  7 Ogasawara Y, Funakoshi M, Ishii K. Blood Cell Mol. Dis., 2008, 41(3): 237-2437 Ogasawara Y, Funakoshi M, Ishii K. Blood Cell Mol. Dis., 2008, 41(3): 237-243
8. [8]
  8 LI Yong-Sheng, GAO Xiu-Feng. Spectrosc. Spect. Anal., 2007, 27(8): 1565-15698 LI Yong-Sheng, GAO Xiu-Feng. Spectrosc. Spect. Anal., 2007, 27(8): 1565-1569
9. [9]
  李永生, 高秀峰. 光谱学与光谱分析, 2007, 27(8): 1565-1569李永生, 高秀峰. 光谱学与光谱分析, 2007, 27(8): 1565-1569
10. [10]
  9 Li Y S, Gao X F. Anal. Chim. Acta, 2007, 588 (1): 140-1469 Li Y S, Gao X F. Anal. Chim. Acta, 2007, 588 (1): 140-146
11. [11]
  10 GAO Xiu-Feng, LI Yong-Sheng, JIANG Yan-Xia. Chinese J. Anal. Chem., 2006, 34(9): 1220-122210 GAO Xiu-Feng, LI Yong-Sheng, JIANG Yan-Xia. Chinese J. Anal. Chem., 2006, 34(9): 1220-1222
12. [12]
  高秀峰, 李永生, 姜艳霞. 分析化学, 2006, 34(9): 1220-1222高秀峰, 李永生, 姜艳霞. 分析化学, 2006, 34(9): 1220-1222
13. [13]
  11 Zhao Y Y, Gao X F, Li Y S, Ju X, Zhang J, Zheng J. Talanta, 2008, 76(2): 265-27011 Zhao Y Y, Gao X F, Li Y S, Ju X, Zhang J, Zheng J. Talanta, 2008, 76(2): 265-270
14. [14]
  12 Li Y S, Du Y D, Chen T M, Gao X F. Biosens. Bioelectron., 2010, 25(6): 1382-138812 Li Y S, Du Y D, Chen T M, Gao X F. Biosens. Bioelectron., 2010, 25(6): 1382-1388
15. [15]
  13 LI Yong-Sheng, YANG Wei, LI Qiao-Jing, ZHOU Lang, GAO Xiu-Feng. Chinese J. Anal. Chem., 2011, 39(7): 1058-106413 LI Yong-Sheng, YANG Wei, LI Qiao-Jing, ZHOU Lang, GAO Xiu-Feng. Chinese J. Anal. Chem., 2011, 39(7): 1058-1064
16. [16]
  李永生, 杨微, 李乔婧, 周朗, 高秀峰. 分析化学, 2011, 39(7): 1058-1064李永生, 杨微, 李乔婧, 周朗, 高秀峰. 分析化学, 2011, 39(7): 1058-1064
17. [17]
  14 LI Yong-Sheng, GAO Xiu-Feng. China Patent, 1737538, 201214 LI Yong-Sheng, GAO Xiu-Feng. China Patent, 1737538, 2012
18. [18]
  李永生, 高秀峰. 中国专利, 1737538, 2012李永生, 高秀峰. 中国专利, 1737538, 2012
19. [19]
  15 Hawley T S, Hawley R G. Flow Cytometry Protocols. 2nd ed., New York: Humana Press, 2004: 16415 Hawley T S, Hawley R G. Flow Cytometry Protocols. 2nd ed., New York: Humana Press, 2004: 164
20. [20]
  16 Dubinsky W P, Racker E. J. Membr. Biol., 1978, 44(1): 25-3616 Dubinsky W P, Racker E. J. Membr. Biol., 1978, 44(1): 25-36
21. [21]
  17 ZHU Zhong-Yong. Practical Medical Laboratory Sciences. Beijing: People's Military Medical Press, 1992: 436-44017 ZHU Zhong-Yong. Practical Medical Laboratory Sciences. Beijing: People's Military Medical Press, 1992: 436-440
22. [22]
  朱中庸. 实用医学检验学. 北京: 人民军医出版社, 1992, 436-440朱中庸. 实用医学检验学. 北京: 人民军医出版社, 1992, 436-440

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸

通讯作者: 李永生

English

Rapid Determination of Pyruvic Acid in Erythrocytes by Lysing-heating Fluorescence Capillary Method

Corresponding author: 李永生

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸

通讯作者: 李永生

English

Rapid Determination of Pyruvic Acid in Erythrocytes by Lysing-heating Fluorescence Capillary Method

Corresponding author: 李永生

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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