Analysis of cholesterol in mouse brain by HPLC with UV detection

Autoři: María A. Paulazo aff001;  Alejandro O. Sodero aff001
Působiště autorů: Institute of Biomedical Research (BIOMED), Pontifical Catholic University of Argentina (UCA) and National Scientific and Technical Research Council (CONICET), C1107AFF Buenos Aires, Argentina aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0228170


We describe a sensitive high performance liquid chromatography (HPLC)-based method for the determination of cholesterol in brain tissue. The method does not require the derivatization of the analyte and uses separation and quantification by reversed-phase HPLC coupled to UV detection. Lipids were methanol/chloroform extracted following the method of Bligh and Dyer, and separated using isopropanol/acetonitrile/water (60/30/10, v/v/v) as mobile phase. We observed lineal detection in a wide range of concentrations, from 62.5 to 2000 ng/μL, and were able to detect a significant increase in the brain cholesterol levels between postnatal days 2 and 10 in C57BL6 mice. Based on our validation parameters, we consider this analytical method a useful tool to assess free cholesterol in rodent brain samples and cell cultures.

Klíčová slova:

Blood-brain barrier – Cell membranes – Gas chromatography-mass spectrometry – High performance liquid chromatography – Chloroform – Cholesterol – Lipids – Ultraviolet spectroscopy


1. DeVries GH, Norton WT. The lipid composition of axons from bovine brain. J. Neurochem. 1974; 22(2): 259–64. doi: 10.1111/j.1471-4159.1974.tb11588.x 4364338

2. Schmitt S, Castelvetri LC, Simons M. Metabolism and functions of lipids in myelin. Biochim. Biophys. Acta 2015; 1851(8): 999–1005. doi: 10.1016/j.bbalip.2014.12.016 25542507

3. Saher G, Simons M. Cholesterol and myelin biogenesis. Subcell. Biochem. 2010; 51: 489–508. doi: 10.1007/978-90-481-8622-8_18 20213556

4. Saher G, Quintes S, Nave KA. Cholesterol: a novel regulatory role in myelin formation. Neuroscientist 2011; 17(1): 79–93. doi: 10.1177/1073858410373835 21343408

5. Saher G, Stumpf SK. Cholesterol in myelin biogenesis and hypomyelinating disorders. Biochim. Biophys. Acta. 2015; 1851(8): 1083–94. doi: 10.1016/j.bbalip.2015.02.010 25724171

6. Dietschy JM, Turley SD. Thematic review series: brain lipids. Cholesterol metabolism in the central nervous system during early development and in the mature animal. J. Lipid Res. 2004; 45(8): 1375–97. doi: 10.1194/jlr.R400004-JLR200 15254070

7. Lütjohann D, Breuer O, Ahlborg G, Nennesmo I, Siden A, Diczfalusy U et al. Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc. Natl. Acad. Sci. USA 1996; 93: 9799–9804. doi: 10.1073/pnas.93.18.9799 8790411

8. Norton WT, Poduslo SE. Myelination in rat brain: changes in myelin composition during brain maturation. J. Neurochem. 1973; 21(4): 759–73. doi: 10.1111/j.1471-4159.1973.tb07520.x 4754856

9. Lund EG, Guileyardo JM, Russell DW. cDNAcloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain. Proc. Natl. Acad. Sci. USA 1999; 96: 7238–7243. doi: 10.1073/pnas.96.13.7238 10377398

10. Tint GS, Yu H, Shang Q, Xu G, Patel SB. The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols. J. Lipid Res. 2006; 47: 1535–1541. doi: 10.1194/jlr.M600141-JLR200 16651660

11. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37(8): 911–7. doi: 10.1139/o59-099 13671378

12. Marcos J, Shackleton CH, Buddhikot MM, Porter FD, Watson GL. Cholesterol biosynthesis from birth to adulthood in a mouse model for 7-dehydrosterol reductase deficiency (Smith-Lemli-Opitz syndrome). Steroids 2007; 72(11–12): 802–8. doi: 10.1016/j.steroids.2007.07.002 17714750

13. Meljon A, Theofilopoulos S, Shackleton CH, Watson GL, Javitt NB, Knölker HJ et al. Analysis of bioactive oxysterols in newborn mouse brain by LC/MS. J. Lipid Res. 2012; 53(11): 2469–83. doi: 10.1194/jlr.D028233 22891291

14. Ghimenti S, Lomonaco T, Onor M, Murgia L, Paolicchi A, Fuoco R et al. Measurement of warfarin in the oral fluid of patients undergoing anticoagulant oral therapy. PLoS One 2011; 6(12): e28182. doi: 10.1371/journal.pone.0028182 22164240

15. Lomonaco T, Ghimenti S, Piga I, Onor M, Melai B, Fuoco R et al. Determination of total and unbound warfarin and warfarin alcohols in human plasma by high performance liquid chromatography with fluorescence detection. J. Chromatogr. A 2013; 1314: 54–62. doi: 10.1016/j.chroma.2013.08.091 24054125

16. Lomonaco T, Ghimenti S, Piga I, Biagini D, Onor M, Fuoco R et al. Influence of sampling on the determination of warfarin and warfarin alcohols in oral fluid. PLoS One 2014; 9(12): e114430.

17. Martín MG, Pfrieger F, Dotti CG. Cholesterol in brain disease: sometimes determinant and frequently implicated. EMBO Rep. 2014; 15(10): 1036–52. doi: 10.15252/embr.201439225 25223281

18. Arenas F, Garcia-Ruiz C, Fernandez-Checa JC. Intracellular cholesterol trafficking and impact in neurodegeneration. Front. Mol. Neurosci. 2017; 10: 382. doi: 10.3389/fnmol.2017.00382 29204109

19. Czuba E, Steliga A, Lietzau G, Kowiański P. Cholesterol as a modifying agent of the neurovascular unit structure and function under physiological and pathological conditions. Metab. Brain Dis. 2017; 32(4): 935–948. doi: 10.1007/s11011-017-0015-3 28432486

20. Chang TY, Yamauchi Y, Hasan MT, Chang C. Cellular cholesterol homeostasis and Alzheimer's disease. J. Lipid Res. 2017; 58(12): 2239–2254. doi: 10.1194/jlr.R075630 28298292

21. Valenza M, Cattaneo E. Emerging roles for cholesterol in Huntington's disease. Trends Neurosci. 2011; 34(9): 474–86. doi: 10.1016/j.tins.2011.06.005 21774998

22. Duncan IW, Culbreth PH, Burtis CA. Determination of free, total, and esterified cholesterol by high-performance liquid chromatography. J. Chromatogr. 1979; 162(3): 281–92. doi: 10.1016/s0378-4347(00)81515-7 528596

23. Li LH, Dutkiewicz EP, Huang YC, Zhou HB, Hsu CC. Analytical methods for cholesterol quantification. J. Food Drug Anal. 2017; 27(2): 375–386.

24. Lu F, Zhu J, Guo S, Wong BJ, Chehab FF, Ferriero DM, et al. Upregulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain. Pediatr. Res. 2018; 83(6): 1218–1227. doi: 10.1038/pr.2018.49 29718007

25. Nunes VS, Cazita PM, Catanozi S, Nakandakare ER, Quintão ECR. Decreased content, rate of synthesis and export of cholesterol in the brain of apoE knockout mice. J. Bioenerg. Biomembr. 2018; 50(4): 283–287. doi: 10.1007/s10863-018-9757-9 29675736

26. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin. Chem. 1974; 20(4): 470–475. 4818200

27. Amundson DM, Zhou M. Fluorometric method for the enzymatic determination of cholesterol. J. Biochem. Biophys. Methods 1999; 38(1): 43–52. doi: 10.1016/s0165-022x(98)00036-0 10078872

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2020 Číslo 1