胆固醇氧化物色谱分析方法研究
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摘要
胆固醇氧化物属于甾类化合物，具有很强的细胞毒害性、致突变性、致癌性以及抑制胆固醇合成等多种不良生理功能，可造成主动脉平滑肌细胞严重损伤，进而导致动脉粥样硬化。本文利用毛细管气相色谱法以及薄层色谱法对胆固醇及其氧化产物进行了研究，另外建立了新药那格列奈中残留溶剂的毛细管气相色谱企业标准分析方法。全文共分四章：第一章对近年来的胆固醇氧化物各类分析方法的进展进行了综述。其中包括了对生物样品的预处理方法，以及应用实例的总结，引用文献50篇。第二章利用气相色谱以及薄层色谱法对多种胆固醇氧化物进行分析研究，并对不同柱子的分离能力以及多种衍生试剂的衍生效果作了比较研究，选定Se-30色谱柱作为分离柱，用氢火焰离子化检测器检测，建立了一套快速、可靠的分析方法。利用该法测定了全脂奶粉中微量胆固醇氧化物的含量，同时对样品中粗脂类的提取及净化过程作了研究。第三章研究了在人体体液温度及酸度条件下的Fenton型Haber-Weiss反应体系中，胆固醇与活性氧—羟基自由基的反应情况；为生物体内胆固醇氧化物的内源自生可能性提供了理论依据。第四章应用毛细管气相色谱法对那格列奈中有机溶剂残留量进行了测定。
Cholesterol oxidation products have adverse biological effects, such as cytotoxicity, mutagenicity, carclnogenicity and inhibition of cholesterrol biosynthesis. Because cholesterol oxidation products can injure aortic smooth muscle cells, lead to atherosclerosis, the determination method of cholesterol oxidation products was studied in this paper. The paper consist of four chapters. In chapter 1,the progress of determination methods of cholesterol oxidation products was reviewed, including of the sample pretreatment and method application in a variety samples, fifty references are cited. In chapter 2, a rapid and reliable method for determination of cholesterol oxidation products was investigated by gas chromatography and thin-layer chromatography . Separation ability of capillary chromatographic columns were compared, derivative reagent were elected. The separation and analysis was performed on SE-30 column with a flame ionization detector under isothermal condition. Cholesterol oxidation products are determined in whole milk powder by gas chromatography, including of extraction of lipid and sample purification. In chapter 3, cholesterol reaction with free radical-
·OH in system of fenton- Haber-Weiss was studied ( PH = 7.4, 36.5℃) . In chapter 4, residual organic solvent in Nateglinide was determined by Capillary gas chromatography .
Cholesterol oxidation products have adverse biological effects, such as cytotoxicity, mutagenicity, carclnogenicity and inhibition of cholesterrol biosynthesis. Because cholesterol oxidation products can injure aortic smooth muscle cells, lead to atherosclerosis, the determination method of cholesterol oxidation products was studied in this paper. The paper consist of four chapters. In chapter 1,the progress of determination methods of cholesterol oxidation products was reviewed, including of the sample pretreatment and method application in a variety samples, fifty references are cited. In chapter 2, a rapid and reliable method for determination of cholesterol oxidation products was investigated by gas chromatography and thin-layer chromatography . Separation ability of capillary chromatographic columns were compared, derivative reagent were elected. The separation and analysis was performed on SE-30 column with a flame ionization detector under isothermal condition. Cholesterol oxidation products are determined in whole milk powder by gas chromatography, including of extraction of lipid and sample purification. In chapter 3, cholesterol reaction with free radical-
·OH in system of fenton- Haber-Weiss was studied ( PH = 7.4, 36.5℃) . In chapter 4, residual organic solvent in Nateglinide was determined by Capillary gas chromatography .
引文
1. Naoki Tamasawa, Makoto Hayakari, Hiroshi Murakami, Jun Matsui, Toshihiro Suda, Reduction of oxysterol levels up-regulates HMG-CoA reductase activity in rat liver, Atherosclerosis, 1997, 131:237-242
2. Smith. L. L, cholesterol Autoxidation, plenun press, new york, 1981:52-55
3. Smith. L. L, cholesterol Autoxidation 1981-1986, Chem. Phys. Lipids., 1987, 44:87
4. Higley N. A. and Taylor S. L., The steatotic and cytotxic effect of cholesterol oxides in cultured L cell, Food Chem. Toxic., 1984, 22:983
5. Sevanian A. and Peterson A. R., Cholesterol epoxide is a direct acting mutagen, Proc. Nat. Acad. Sci. USA., 1984, 31:4198
6. Black, H. S. and Douglas D. R., A model system for the evaluation of the role of cholesterol α-oxides in ultraviolet carcinogenesis, Cancer
Res., 1972,32:2630
7. Shi-Kaung peng, C. Bruce Taylor, Jerome C. Hill and Robert J. Morin, Cholesterol Oxidation Derivatives and Arterial Endothelial Damage, Atherosclerosis, 1985,54:121-133
8. Marc S. Jacobson, Martha G. Price, Adil E. Shamoo and Felix P. Heald, Atherogenesis in White Carneau Pigeons Effects of Low-Level Cholestanetriol Feeding, Atherosclerosis, 1985,57:209-217
9. Kandutsch, A. A. and Chen H. W., Inhibition of cholesterol synthesis by oxygenated sterols, Lipid, 1978, 13:704,
10. S. M. Naseem and F. P. Heald, biochem, interal, 1987, 14:71-84
11．董军，陈文祥，李健斋，高效液相色谱测定微量胆固醇氧化产物，生物化学与生物物理进展，1996，23：179-190
12．陈凤贵，杨胜钦，苏正德，市售鸡肉及其制品中胆固醇与胆固醇氧化物含量之调查，食品科学(台湾)，1993，20(3)：277-290
13. R. J. Lake and P. Scholes, Consumption of Cholesterol Oxides from Fast Foods Fried in Beef Fat in New Zealand, JAOCS, 1997,74(9): 1069
14. Jea Eun Pie, Khira Spahis, and Chriatine Seillan, Evaluation of Oxidative Degradation of Cholesterol in Food and Food Ingredients: Identification and Quantification of Cholesterol Oxides, J. Agric. Food Chem.,1990,38:973-979
15. K Granelli, P Faldt, L-A Appelqvist and B Bergenstahl, Infulence of Surface Structure On Cholesterol Oxidation in Model Food Powders, J Sci Food Agric. 1996,71:75-82
16. Cecil B. Johnson, Isolation of cholesterol oxidation products from animal fat using aminopropyl solid-phase extraction, Journal of Chromatogrphy A, 1996,736:205-210
17. Shu-Mei Lai, J. Ian Gray, and Matthew E. Zabik, Evaluation of Solid Phase Extraction and Gas Chromatography for Determination of Cholesterol Oxidation Products in Spray-Dried Whole Egg, J. Agric. Food Chem.
1995,43:1122-1126
18. Jau-Shyong Chen, Gow-Chin Yen, Cholesterol Oxidation Products in small sun-dried fish Food Chemistry, 1994,50:167-170
19. Antonio J. Angulo, Juan M. Romera, Marfa Ramirez, and Angel Gil, Determination of Cholesterol Oxides in Dairy Products. Effect of Storage Conditions, J. Agric. Food Chem. 1997,45:4318-4323
20．高雅敏，孙璐西，鱿鱼干中胆固醇氧化产物之分析，Food Science，1997，24(2)：242-257
21. M. Careri, A. Mangia, M. Music, G. Parolari, Development of Gas Chromatographic Method for Determination of Cholesterol Oxides in Meat Products, Chromatographia, 1998,47(1/2): 42-48
22. D. U. AHN, J. I. LEE, C. JO, and J. L. SELL, Analysis of Cholesterol Oxides in Egg Yolk and Turkey Meat, Poultry Science, 1999,78:1060-1064
23. Francesc Gguardiola, Rafael Codony, Magda Rafecas, Josep Boatella, Comparison of three methods for the determination of oxysterols in spray-dried egg, Journal of Chromatograph, 1995,705:396-304
24. T. Rodriguez-Estrada, Maria F. Caaboni, Arianna Costa, Giovanni Lercker, Gas Chromatographic Analysis of Cholesterol Oxidation Products on a Thermostable Medium Polarity Capillary Column, J. High Resol. Chromatogr.,1998,21(9):509-512
25．卢义发，汤政宜，市售蛋制品及头足类产品之胆固醇氧化物含量调查研究，食品科学(台湾)，1993，20(2)：168-177
26. Csiky S., Trace enrichment and separation of cholesterol oxidation products by adsorption high-performance liquid chromatography, J Chromatogr, 1982,241:381
27. Tsai. L. S. and Hudson C. A., High-performance liquid chromatography of oxygenated cholesterol and related compounds, J
AOCS.,1981,58:931-934
28. Y. C. Chen, C. P. Chiu & B. H. Chen, Determination of chesterol oxides in heated lard by liquid chromatography, Food Chemistry, 1994,50:53-58
29. S. Kermasha, S. Kubow, M. Goetghebeur, Comparative high-perfomance liquid chromatographic analyses of cholesterol and its oxidation products using diode-array ultraviolet and laser light-scattering detection. Journal of Chromatography A, 1994,685:229-235
30. L. Lakritz and K. C. Jones, Separation and Quantitation of Cholesterol Oxides by HPLC With an Evaporative Light Scattering Detector in a Model System. JAOCS, 1997,74(8):1060
31. Yutaka Saisho, Chika Shimada, and Tsuneji Umeda, Determination of 7 α-Hydroxycholesterol in Dog Plasma by High-performance Liquid Chromatography with Fluorescence Detection, ANALYTICAL BIOCHEMISTRY, 1998, 265:361-361
32. J. A. Garcia Regueiro, C. Maraschiello, Procedure for the determination of eight cholesterol oxidea in poultry meatusing on-column and solvent venting capillary gas chromatography, Journal of Cchromatography A, 1997,764:279-293
33. Suechin X. Li, Geetha Cherian, Dong U. Ahn, Robert T. Hardin, and Jeong S. Sim, Storage, Heating, and Tocopherols Affect Cholesterol Oxide Formation in Food Oils, J. Agric. Food Chem.,1996,44:3830
34. S. X. Li, G. CHERIAN, and J. S. SIM, Cholesterol Oxidation in Egg Yolk Powder During Storage and Heating as Affected by Dietary Oil and Tocopherol, Journal of Food Science, 1996,61(4):721-725
35. S. MC. CLUSKEY, J. F. CONNOLLY, R. DEVERY, B. OBRIIEN, J. KELLY, D. HARRINGTON, a nd C. STANTON, Lipid and Cholesterol Oxidation in Whole Milk Powder during Processing and Storage, Journal of Food Science, 1997,62(2):331-337
36. Peter W. Park, Francesc Guardiola, Susan H. Park, and Paul B. Addis, Kinetic
Evaluation of 3 β-Hydroxycholest-5-en-7-one(7-ketocholesterol) Stability During Saponification, JOACS, 1996,73(5):623-629
37. E. Razzazi-Fazeli, S. Kleineisen, W. Luf, Determination of cholesterol oxides in processed food using high performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization, Journal of Chromatography A, 2000,896:321-334
38. A. FONTANA, F. ANTONIAZZI, M. L.-CIAVATTA, E. TRIVELLONE and G. CIMINO, 'H-NMR Study of Cholesterol Autoxidation in Egg Powder and Cookies Exposed to Adverse Storage, Journal of Food Science, 1993,58(6):1286
39. Richmond W., Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum, Clin Chem.,1973,19:1350-1356
40. Vera K Lebovics, Magdolna Antal and Odon Gaal, Enzymatic Determination of Cholesterol Oxides, J Sci Food. Agric, 1996,71:22-26
41. Folch J., Lees M. and Sloan-Stanley G. H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 1957,226:497-507
42. G. Maerker and J. Unruh, Cholesterol Oxides. 1. Isolation and Determination of Some Cholesterol Oxidation Products, J. Am. Oil Chem. Soc.,1986,63:767-773
43. S. bergstrom and O. Wintersteiner, Autoxidation of Sterols in Colloidal Aqueous Solutions. The Nature of the Products Formed from Cholesterol, J. Biol. Chem.,1941,141:597-610
44. F. Gguardiola, Grasas Aaceites, 1994,45:164
45. Kou. I. L. and Holmes R. P., The analysis of 25-hyroxycholesterol in plasma and cholesterol containing foods by high-performance liquid chromatography, J. Chromatogr.,1985,330:339-346
46. H. -G. Schmarr, H. B. Gross, T. Shibamoto, J Aagric. Food Chem.,1996,44:512
47. J. N. Morgan, D. J. Armstrong, Wide-bore capillary gas chromatography method for quantification of cholesterol oxidation products in egg yolk powder, J. Food Sci.,1989,54:427-429
48. Nan Li, Toshiaki Ohshima, Kei-ichi Shozen, Hideki Ushio and Chiaki Koizumi, Effects of the Degree of Unsaturation of Coexisting Triacylglycerols on Cholesterol Oxidation, JAOCS, 1994,71(6):623-627
49. F. J. Monahan et al, J. Agric. Food Chem.,1992,40:1310
50. FrancescGuardiola, Rafael Codony, Magda Rafecas, Josep Boatella, Adsorption of oxysterols on different microtube materials during silanyzation prior to gas chromatographic determination, Journal of Chromatography A, 1995,705:289-399
2. Smith. L. L, cholesterol Autoxidation, plenun press, new york, 1981:52-55
3. Smith. L. L, cholesterol Autoxidation 1981-1986, Chem. Phys. Lipids., 1987, 44:87
4. Higley N. A. and Taylor S. L., The steatotic and cytotxic effect of cholesterol oxides in cultured L cell, Food Chem. Toxic., 1984, 22:983
5. Sevanian A. and Peterson A. R., Cholesterol epoxide is a direct acting mutagen, Proc. Nat. Acad. Sci. USA., 1984, 31:4198
6. Black, H. S. and Douglas D. R., A model system for the evaluation of the role of cholesterol α-oxides in ultraviolet carcinogenesis, Cancer
Res., 1972,32:2630
7. Shi-Kaung peng, C. Bruce Taylor, Jerome C. Hill and Robert J. Morin, Cholesterol Oxidation Derivatives and Arterial Endothelial Damage, Atherosclerosis, 1985,54:121-133
8. Marc S. Jacobson, Martha G. Price, Adil E. Shamoo and Felix P. Heald, Atherogenesis in White Carneau Pigeons Effects of Low-Level Cholestanetriol Feeding, Atherosclerosis, 1985,57:209-217
9. Kandutsch, A. A. and Chen H. W., Inhibition of cholesterol synthesis by oxygenated sterols, Lipid, 1978, 13:704,
10. S. M. Naseem and F. P. Heald, biochem, interal, 1987, 14:71-84
11．董军，陈文祥，李健斋，高效液相色谱测定微量胆固醇氧化产物，生物化学与生物物理进展，1996，23：179-190
12．陈凤贵，杨胜钦，苏正德，市售鸡肉及其制品中胆固醇与胆固醇氧化物含量之调查，食品科学(台湾)，1993，20(3)：277-290
13. R. J. Lake and P. Scholes, Consumption of Cholesterol Oxides from Fast Foods Fried in Beef Fat in New Zealand, JAOCS, 1997,74(9): 1069
14. Jea Eun Pie, Khira Spahis, and Chriatine Seillan, Evaluation of Oxidative Degradation of Cholesterol in Food and Food Ingredients: Identification and Quantification of Cholesterol Oxides, J. Agric. Food Chem.,1990,38:973-979
15. K Granelli, P Faldt, L-A Appelqvist and B Bergenstahl, Infulence of Surface Structure On Cholesterol Oxidation in Model Food Powders, J Sci Food Agric. 1996,71:75-82
16. Cecil B. Johnson, Isolation of cholesterol oxidation products from animal fat using aminopropyl solid-phase extraction, Journal of Chromatogrphy A, 1996,736:205-210
17. Shu-Mei Lai, J. Ian Gray, and Matthew E. Zabik, Evaluation of Solid Phase Extraction and Gas Chromatography for Determination of Cholesterol Oxidation Products in Spray-Dried Whole Egg, J. Agric. Food Chem.
1995,43:1122-1126
18. Jau-Shyong Chen, Gow-Chin Yen, Cholesterol Oxidation Products in small sun-dried fish Food Chemistry, 1994,50:167-170
19. Antonio J. Angulo, Juan M. Romera, Marfa Ramirez, and Angel Gil, Determination of Cholesterol Oxides in Dairy Products. Effect of Storage Conditions, J. Agric. Food Chem. 1997,45:4318-4323
20．高雅敏，孙璐西，鱿鱼干中胆固醇氧化产物之分析，Food Science，1997，24(2)：242-257
21. M. Careri, A. Mangia, M. Music, G. Parolari, Development of Gas Chromatographic Method for Determination of Cholesterol Oxides in Meat Products, Chromatographia, 1998,47(1/2): 42-48
22. D. U. AHN, J. I. LEE, C. JO, and J. L. SELL, Analysis of Cholesterol Oxides in Egg Yolk and Turkey Meat, Poultry Science, 1999,78:1060-1064
23. Francesc Gguardiola, Rafael Codony, Magda Rafecas, Josep Boatella, Comparison of three methods for the determination of oxysterols in spray-dried egg, Journal of Chromatograph, 1995,705:396-304
24. T. Rodriguez-Estrada, Maria F. Caaboni, Arianna Costa, Giovanni Lercker, Gas Chromatographic Analysis of Cholesterol Oxidation Products on a Thermostable Medium Polarity Capillary Column, J. High Resol. Chromatogr.,1998,21(9):509-512
25．卢义发，汤政宜，市售蛋制品及头足类产品之胆固醇氧化物含量调查研究，食品科学(台湾)，1993，20(2)：168-177
26. Csiky S., Trace enrichment and separation of cholesterol oxidation products by adsorption high-performance liquid chromatography, J Chromatogr, 1982,241:381
27. Tsai. L. S. and Hudson C. A., High-performance liquid chromatography of oxygenated cholesterol and related compounds, J
AOCS.,1981,58:931-934
28. Y. C. Chen, C. P. Chiu & B. H. Chen, Determination of chesterol oxides in heated lard by liquid chromatography, Food Chemistry, 1994,50:53-58
29. S. Kermasha, S. Kubow, M. Goetghebeur, Comparative high-perfomance liquid chromatographic analyses of cholesterol and its oxidation products using diode-array ultraviolet and laser light-scattering detection. Journal of Chromatography A, 1994,685:229-235
30. L. Lakritz and K. C. Jones, Separation and Quantitation of Cholesterol Oxides by HPLC With an Evaporative Light Scattering Detector in a Model System. JAOCS, 1997,74(8):1060
31. Yutaka Saisho, Chika Shimada, and Tsuneji Umeda, Determination of 7 α-Hydroxycholesterol in Dog Plasma by High-performance Liquid Chromatography with Fluorescence Detection, ANALYTICAL BIOCHEMISTRY, 1998, 265:361-361
32. J. A. Garcia Regueiro, C. Maraschiello, Procedure for the determination of eight cholesterol oxidea in poultry meatusing on-column and solvent venting capillary gas chromatography, Journal of Cchromatography A, 1997,764:279-293
33. Suechin X. Li, Geetha Cherian, Dong U. Ahn, Robert T. Hardin, and Jeong S. Sim, Storage, Heating, and Tocopherols Affect Cholesterol Oxide Formation in Food Oils, J. Agric. Food Chem.,1996,44:3830
34. S. X. Li, G. CHERIAN, and J. S. SIM, Cholesterol Oxidation in Egg Yolk Powder During Storage and Heating as Affected by Dietary Oil and Tocopherol, Journal of Food Science, 1996,61(4):721-725
35. S. MC. CLUSKEY, J. F. CONNOLLY, R. DEVERY, B. OBRIIEN, J. KELLY, D. HARRINGTON, a nd C. STANTON, Lipid and Cholesterol Oxidation in Whole Milk Powder during Processing and Storage, Journal of Food Science, 1997,62(2):331-337
36. Peter W. Park, Francesc Guardiola, Susan H. Park, and Paul B. Addis, Kinetic
Evaluation of 3 β-Hydroxycholest-5-en-7-one(7-ketocholesterol) Stability During Saponification, JOACS, 1996,73(5):623-629
37. E. Razzazi-Fazeli, S. Kleineisen, W. Luf, Determination of cholesterol oxides in processed food using high performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization, Journal of Chromatography A, 2000,896:321-334
38. A. FONTANA, F. ANTONIAZZI, M. L.-CIAVATTA, E. TRIVELLONE and G. CIMINO, 'H-NMR Study of Cholesterol Autoxidation in Egg Powder and Cookies Exposed to Adverse Storage, Journal of Food Science, 1993,58(6):1286
39. Richmond W., Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum, Clin Chem.,1973,19:1350-1356
40. Vera K Lebovics, Magdolna Antal and Odon Gaal, Enzymatic Determination of Cholesterol Oxides, J Sci Food. Agric, 1996,71:22-26
41. Folch J., Lees M. and Sloan-Stanley G. H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 1957,226:497-507
42. G. Maerker and J. Unruh, Cholesterol Oxides. 1. Isolation and Determination of Some Cholesterol Oxidation Products, J. Am. Oil Chem. Soc.,1986,63:767-773
43. S. bergstrom and O. Wintersteiner, Autoxidation of Sterols in Colloidal Aqueous Solutions. The Nature of the Products Formed from Cholesterol, J. Biol. Chem.,1941,141:597-610
44. F. Gguardiola, Grasas Aaceites, 1994,45:164
45. Kou. I. L. and Holmes R. P., The analysis of 25-hyroxycholesterol in plasma and cholesterol containing foods by high-performance liquid chromatography, J. Chromatogr.,1985,330:339-346
46. H. -G. Schmarr, H. B. Gross, T. Shibamoto, J Aagric. Food Chem.,1996,44:512
47. J. N. Morgan, D. J. Armstrong, Wide-bore capillary gas chromatography method for quantification of cholesterol oxidation products in egg yolk powder, J. Food Sci.,1989,54:427-429
48. Nan Li, Toshiaki Ohshima, Kei-ichi Shozen, Hideki Ushio and Chiaki Koizumi, Effects of the Degree of Unsaturation of Coexisting Triacylglycerols on Cholesterol Oxidation, JAOCS, 1994,71(6):623-627
49. F. J. Monahan et al, J. Agric. Food Chem.,1992,40:1310
50. FrancescGuardiola, Rafael Codony, Magda Rafecas, Josep Boatella, Adsorption of oxysterols on different microtube materials during silanyzation prior to gas chromatographic determination, Journal of Chromatography A, 1995,705:289-399