臺灣博碩士論文加值系統

本研究應用我們已開發之可移除性衍生試劑配合高效能液相層析分析來檢測嬰幼兒奶粉中膽固醇的含量。由市面上選擇九種嬰兒配方奶粉、三種較大嬰兒奶粉、三種幼兒成長奶粉，各別進行膽固醇含量分析。我們測定的結果：嬰兒配方奶粉、較大嬰兒奶粉、幼兒成長奶粉，於每100 g奶粉中膽固醇含量範圍分別為 3.77 ~ 34.15 mg、12.20 ~ 22.70 mg、7.35 ~ 58.87 mg，且本方法的回收率大於 93%。為了要決定嬰幼兒奶粉中膽固醇含量的合宜性，我們亦進行人乳中膽固醇含量之測定。分別由五位授乳母親之奶汁，進行膽固醇含量測定，結果每100 mL人乳含有膽固醇的範圍為 15.22 ~ 39.33 mg。

This study was applied an our established method, reagents removable derivatization method, with high performance liquid chromatography to determine cholesterol in infant, follow-on and toddler formulae. We selected 9 kinds of infant formulae, 3 kinds of follow-on formulae, and 3 kinds of toddler formulae from the market and the contents of cholesterol were determined separately. The range of cholesterol content in infant, follow-on and toddler formulae were 3.77 ~ 34.15, 12.20 ~ 22.70, 7.35 ~ 58.87 mg per 100 g milk powder respectly. And the recovery rates by the method were all above 93%. In order to find out whether cholesterol content in infant, follow-on and toddler formulae were appropriate, we also determined the contents of cholesterol in human milk. The amount of cholesterol in human milk was determined from five lactating mothers severally. The range of cholesterol content in human milk was shown as 15.22 ~ 39.33 mg per 100 mL.

中文摘要 　 　 i
英文摘要　　　　　　 　 　 ii
圖目錄 　　　　　　　　　　　 　 　 v
表目錄 　　 　　　　　　　 vi
壹 緒論 　 　 1
貳 實驗部份 　 6
一、 試藥及材料 　 6
二、 儀器 　 9
三、 試藥溶液之配製 　 10
四、 膽固醇衍生標準品之合成與精製 　 12
五、 高效能液相層析條件 14
六、 微量衍生反應 　 15
七、 市售嬰幼兒奶粉膽固醇含量之分析 17
(1) 檢品之前處理及衍生反應步驟 18
(2) 檢量線之建立與迴歸分析 19
(3) 同日內與異日間分析之精密度與準確度 19
(4) 奶粉中膽固醇萃取回收率之探討 21
八、 人乳膽固醇含量之分析 22
參 結果與討論 23
一、 衍生標準品之氫核核磁共振光譜 23
二、 高效能液相層析條件
(1) 膽固醇衍生物與內部標準品之高效能液相層析圖 24
(2) 檢測極限之層析圖 25
(3) 最低可定量濃度 25
三、 市售嬰幼兒奶粉膽固醇含量之分析
(1) 檢量線之建立及其迴歸分析 26
(2) 同日內與異日間分析之精密度與準確度 27
(3) 奶粉中膽固醇萃取回收率結果 28
(4) 市售嬰兒奶粉膽固醇分析結果 29
四、 人乳膽固醇之分析結果 36
五、 討論 39
肆 結論 41
伍 參考文獻 42






圖目錄
圖1：人體之膽固醇代謝圖 2
圖2：膽固醇生合成中鯊烯的生合成 3
圖3 : 膽固醇的生合成 4
圖4：膽固醇衍生標準品合成示意圖 12
圖5：cholesterol微量衍生反應及其分析示意圖 15
圖6 : cholesterol-NAOB 氫核核磁共振光譜 23
圖7：膽固醇衍生物與內部標準品之高效能液相層析圖譜 24
圖8：檢測極限層析圖譜 25
圖9：膽固醇微量衍生反應之層析圖 26
圖10：嬰幼兒奶粉膽固醇之微量衍生層析圖 29
圖11：嬰兒配方奶粉膽固醇含量之分佈 30
圖12：較大嬰兒奶粉膽固醇含量之分佈 32
圖13：幼兒成長奶粉膽固醇含量之分佈 34
圖14：人乳之層析圖 36
圖15：分析5位授乳母親乳汁所含膽固醇量之分佈 37
圖16：七種有害之膽固醇氧化物 40




表目錄
表1：膽固醇衍生標準品合成反應所需試劑 13
表2：微量衍生反應各試劑容積與濃度 16
表3：I.S.對cholesterol-NAOB的反應因子 24
表4：膽固醇衍生物測量的迴歸分析 26
表5：測量膽固醇衍生物精密度與準確度 27
表6：奶粉添加膽固醇萃取回收率 28
表7：市售嬰兒配方奶粉膽固醇含量 29
表8：嬰兒配方奶粉每天食用建議量 30
表9：九種嬰兒配方奶粉提供之膽固醇累積食用量 31
表10：市售較大嬰兒奶粉膽固醇含量 32
表11：較大嬰兒奶粉每天食用建議量 33
表 12：較大嬰兒奶粉提供之膽固醇累積食用量 33
表13：市售幼兒成長奶粉膽固醇含量 34
表14：幼兒成長奶粉每天飲食用建議量 35
表15：幼兒成長奶粉提供之膽固醇量 35
表16 : 分析人乳中膽固醇的情形 36
表17：嬰兒吸吮人乳量與膽固醇食用累積量 38
表18：不同營養來源所供應膽固醇含量之比較 39

1.Desager JP, Horsmans Y, Clinical pharmacokinetics of 3-hydroxy-3-methyl glutaryl-coenzyme A reductase inhibitors. Cln. Pharmacokinet., 1996, 5, 348–371.


2.Poduslo SE, Jang Y, Myelin development in infant brain. Neurochem. Res. , 1984, 11, 1615-1626.


3.Opitz JM, de la Cruz F, Cholesterol metabolism in the RSH/Smith-Lemli-Opitz syndrome: summary of an NICHD conference. Am. J. Med. Genet. , 1994, 50, 326-338.


4.Lutjohann D, Bjorkhem I, Locatelli S, Dame C, Schmolling J, von Bergmann K, Fahnenstich H, Cholesterol dynamics in the fetal and neonatal brain as reflected by circulatory levels of 24S-hydroxycholesterol. Acta Paediatr., 2001, 90, 652-657.


5.Kinney HC, Karthigasan J, Borenshteyn NI, Flax JD, Kirschner DA, Myelination in the developing human brain: biochemical correlates. Neurochem. Res., 1994, 19, 983-996.


6.Uauy R, Castillo C, Lipid requirements of infants: implications for nutrient composition of fortified complementary foods. J, Nutr., 2003, 133, 2962S-2972S.


7.Van Biervliet JP, Vinaimont N, Vercaemst R, Rosseneu M, Serum cholesterol, cholesteryl ester, and high-density lipoprotein development in newborn infants: response to formulas supplemented with cholesterol and gamma-linolenic acid. J Pediatr. , 1992, 120, S101-S108. ��

8.Kuo CC, Wen YH, Wu HL, Wu SS, A removable and specific alcoholic group derivatization with 4-[N-methyl, N-(1-naphthyl methyl)amino]-4-oxobutanoic acid method of high performance liquid chromatography for alcohol analysis and its applications. Analytical letters, 2003, 36, 813-825.


9.Acosta PB, RSH/SLO (Smith-Lemli-Opitz) syndrome: designing a high cholesterol diet for the SLO syndrome. Am. J. Med. Genet., 1994, 50, 358-363.


10.Liscum L, Niemann–Pick type C mutations cause lipid traffic jam, Traffic., 2000, 1, 218–225.


11.Kirsch C, Eckert GP, Koudinov AR, Muller WE, Brain cholesterol, statins and Alzheimer''s disease. Pharmacopsychiatry, 2003 , 36 Suppl 2, S113-S119. ����


12.Wright RO, Hu H, Silverman EK, Tsaih SW, Schwartz J, Bellinger D, Palazuelos E, Weiss ST, Hernandez-Avila M, Apolipoprotein E genotype predicts 24-month bayley scales infant development score. Pediatr Res., 2003, 20, 1293-1300.


13.Heverin M, Bogdanovic N, Lutjohann D, Bayer T, Pikuleva I, Bretillon L, Diczfalusy U, Winblad B, Bjorkhem I, Changes in the levels of cerebral and extracerebral sterols in the brain of patients with Alzheimer''s disease. J. Lipid Res., 2003, 1, 523-554.


14.Orso GG, Cholesterol bound to high density lipoproteins: critical review of the methods of analysis and personal data. Ann. Osp. Maria Vittoria Torino, 1982, 24, 286-306.

15.Linda JP, Joanne MM, Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone- stimulated phosphatidylinositol turnover. The Journal of Biological Chemistry, 1998, 35, 22298–22304.


16.Zeina EJ, Hwa KY, Michael AW, Cholesterol efflux from larval Manduca sexta fat body in vitro: high-density lipophorin as the acceptor. Journal of Insect Physiology, 2002, 48, 609–618.


17.Lutjohann D, Breuer O, Ahlborg G, Nennesmo I, Siden A, Diczfalusy U, Bjorkhem I, Cholesterol homeostasis in human brain: evidence for an agedependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc. Natl. Acad. Sci. U S A., 1996, 93, 9799-9804.


18.Lee JJ, Xu QW, Firth TA, Nelson MT, Mawe GM, Cholesterol inhibits spontaneous action potentials and calcium currents in guinea pig gallbladder smooth muscle, Am. J. Physiol., 1999, 227, G1017–G1026.


19.Ikonen E, Roles of lipid rafts in membrane transport, Cell Biology, 2001, 13, 470–477.


20.Koudinov AR, Koudinova NV., Cholesterol, synaptic function and Alzheimer''s disease. Pharmacopsychiatry, 2003, 36 Suppl 2, S107-S112.


21.Crowder CM, Westover EJ, Kumar AS, Ostlund RE, Covey DF, Enantiospecificity of cholesterol function in vivo. The Journal of Biological Chemistry, 2001, 276, 44369–44372.


22.Nilsson IM, Henna OR, Slotte JP, Arthur EJ, Heijne GV, Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols. The Journal of Biological Chemistry, 2001, 276, 41748 – 41754.


23.Goldstein JL, Rawson RB, Brown MS, Mutant mammalian cells as tools to delineate the sterol regulatory element binding protein pathway for feedback regulation of lipid synthesis. Archives of Biochemistry and Biophysics, 2002, 45, 139–148.


24.Fazeli ER, Kleineisen S, Luf W, Determination of cholesterol oxides in processed food using high performance liquid chromatography- mass spectrometry with atmospheric pressure chemical ionisation. Journal of Chromatography A, 2000, 896, 321-334.


25.Tai CY, Chen YC, Chen BH, Analysis, formation and inhibition of cholesterol oxidation products in foods: an overview (part I), Journal of Food and Drug Analysis, 1999, 7, 243-257.