Methodological Aspects of Phytosterol Measurements in Biological Samples

Author(s): Helena Gylling*, Piia Simonen, Leena Kaipiainen, Ingmar Wester

Journal Name: Current Medicinal Chemistry

Volume 26 , Issue 37 , 2019


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Abstract:

Phytosterol measurement has gained a lot of interest during the last two decades after foods and supplements with added 4-desmethyl phytosterols were recognized and used as effective and safe non-pharmacologic hypocholesterolemic agents, and also after the mechanisms of intestinal absorption and hepatic excretion of sterols were unraveled. In addition, the wide use of serum phytosterols as biomarkers of cholesterol absorption has increased the interest in their measurement. In this review, the basic methods are discussed without going into details of the practical operations. The analysis includes first lipid extraction and saponification from various biologic matrices such as serum/plasma, feces, or tissues, after which the individual sterols are separated by adsorption chromatography (gas-liquid or liquid or high performance liquid chromatography) based on the polarity of the various sterols. We also deal with some specific aspects of phytosterol measurements in biological samples such as the need of harmonization of their analysis in biological samples, the discrepancies in the results of sitosterol and campesterol concentrations between different studies, and what is known about their biological day-to-day fluctuation. Phytosterols have a remarkable role in human health, so that their complicated and time consuming measurements call attention to routine ways of standardization between the sterol research laboratories.

Keywords: Avenasterol, campesterol, campestanol, cholesterol, gas-liquid chromatography, sitosterol, sitostanol, stigmasterol.

[1]
Meijer, G.W.; Bressers, M.A.; de Groot, W.A.; Rudrum, M. Effect of structure and form on the ability of plant sterols to inhibit cholesterol absorption in hamsters. Lipids, 2003, 38(7), 713-721.
[http://dx.doi.org/10.1007/s11745-003-1119-4] [PMID: 14506834]
[2]
Weststrate, J.A.; Meijer, G.W. Plant sterol-enriched margarines and reduction of plasma total- and LDL-cholesterol concentrations in normocholesterolaemic and mildly hypercholesterolaemic subjects. Eur. J. Clin. Nutr., 1998, 52(5), 334-343.
[http://dx.doi.org/10.1038/sj.ejcn.1600559] [PMID: 9630383]
[3]
Racette, S.B.; Lin, X.; Ma, L.; Ostlund, R.E., Jr Natural dietary phytosterols. J. AOAC Int., 2015, 98(3), 679-684.
[http://dx.doi.org/10.5740/jaoacint.SGERacette] [PMID: 26086252]
[4]
Andersson, S.W.; Skinner, J.; Ellegård, L.; Welch, A.A.; Bingham, S.; Mulligan, A.; Andersson, H.; Khaw, K.T. Intake of dietary plant sterols is inversely related to serum cholesterol concentration in men and women in the EPIC Norfolk population: a cross-sectional study. Eur. J. Clin. Nutr., 2004, 58(10), 1378-1385.
[http://dx.doi.org/10.1038/sj.ejcn.1601980] [PMID: 15054420]
[5]
Ras, R.T.; van der Schouw, Y.T.; Trautwein, E.A.; Sioen, I.; Dalmeijer, G.W.; Zock, P.L.; Beulens, J.W.J. Intake of phytosterols from natural sources and risk of cardiovascular disease in the European Prospective Investigation into Cancer and Nutrition-the Netherlands (EPIC-NL) population. Eur. J. Prev. Cardiol., 2015, 22(8), 1067-1075.
[http://dx.doi.org/10.1177/2047487314554864] [PMID: 25305273]
[6]
Piironen, V.; Lampi, A.M. Occurrence and levels of phytosterols in foods. In: Phytosterols as functional food components and nutraceuticals., Dutta, P.C. Marcel Dekker, Inc: New York. 2004, 1-32.
[7]
Klingberg, S.; Andersson, H.; Mulligan, A.; Bhaniani, A.; Welch, A.; Bingham, S.; Khaw, K.T.; Andersson, S.; Ellegård, L. Food sources of plant sterols in the EPIC Norfolk population. Eur. J. Clin. Nutr., 2008, 62(6), 695-703.
[http://dx.doi.org/10.1038/sj.ejcn.1602765] [PMID: 17440516]
[8]
Valsta, L.M.; Lemström, A.; Ovaskainen, M.L.; Lampi, A.M.; Toivo, J.; Korhonen, T.; Piironen, V. Estimation of plant sterol and cholesterol intake in Finland: quality of new values and their effect on intake. Br. J. Nutr., 2004, 92(4), 671-678.
[http://dx.doi.org/10.1079/BJN20041234] [PMID: 15522137]
[9]
Davis, H.R., Jr; Zhu, L.J.; Hoos, L.M.; Tetzloff, G.; Maguire, M.; Liu, J.; Yao, X.; Iyer, S.P.; Lam, M.H.; Lund, E.G.; Detmers, P.A.; Graziano, M.P.; Altmann, S.W. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J. Biol. Chem., 2004, 279(32), 33586-33592.
[http://dx.doi.org/10.1074/jbc.M405817200] [PMID: 15173162]
[10]
Berge, K.E.; Tian, H.; Graf, G.A.; Yu, L.; Grishin, N.V.; Schultz, J.; Kwiterovich, P.; Shan, B.; Barnes, R.; Hobbs, H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science, 2000, 290(5497), 1771-1775.
[http://dx.doi.org/10.1126/science.290.5497.1771] [PMID: 11099417]
[11]
Lee, M.H.; Lu, K.; Hazard, S.; Yu, H.; Shulenin, S.; Hidaka, H.; Kojima, H.; Allikmets, R.; Sakuma, N.; Pegoraro, R.; Srivastava, A.K.; Salen, G.; Dean, M.; Patel, S.B. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nat. Genet., 2001, 27(1), 79-83.
[http://dx.doi.org/10.1038/83799] [PMID: 11138003]
[12]
Bosner, M.S.; Lange, L.G.; Stenson, W.F.; Ostlund, R.E. Jr. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry. J. Lipid Res., 1999, 40(2), 302-308.
[PMID: 9925660]
[13]
Ostlund, R.E., Jr; McGill, J.B.; Zeng, C.M.; Covey, D.F.; Stearns, J.; Stenson, W.F.; Spilburg, C.A. Gastrointestinal absorption and plasma kinetics of soy Δ(5)-phytosterols and phytostanols in humans. Am. J. Physiol. Endocrinol. Metab., 2002, 282(4), E911-E916.
[http://dx.doi.org/10.1152/ajpendo.00328.2001] [PMID: 11882512]
[14]
Simonen, P.P.; Gylling, H.; Miettinen, T.A. The distribution of squalene and non-cholesterol sterols in lipoproteins in type 2 diabetes. Atherosclerosis, 2007, 194(1), 222-229.
[http://dx.doi.org/10.1016/j.atherosclerosis.2006.07.030] [PMID: 16963050]
[15]
Miettinen, T.A.; Railo, M.; Lepäntalo, M.; Gylling, H. Plant sterols in serum and in atherosclerotic plaques of patients undergoing carotid endarterectomy. J. Am. Coll. Cardiol., 2005, 45(11), 1794-1801.
[http://dx.doi.org/10.1016/j.jacc.2005.02.063] [PMID: 15936608]
[16]
Helske, S.; Miettinen, T.; Gylling, H.; Mäyränpää, M.; Lommi, J.; Turto, H.; Werkkala, K.; Kupari, M.; Kovanen, P.T. Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves. J. Lipid Res., 2008, 49(7), 1511-1518.
[http://dx.doi.org/10.1194/jlr.M800058-JLR200] [PMID: 18398220]
[17]
Weingärtner, O.; Lütjohann, D.; Ji, S.; Weisshoff, N.; List, F.; Sudhop, T.; von Bergmann, K.; Gertz, K.; König, J.; Schäfers, H.J.; Endres, M.; Böhm, M.; Laufs, U. Vascular effects of diet supplementation with plant sterols. J. Am. Coll. Cardiol., 2008, 51(16), 1553-1561.
[http://dx.doi.org/10.1016/j.jacc.2007.09.074] [PMID: 18420097]
[18]
Miettinen, T.A.; Nissinen, M.; Lepäntalo, M.; Albäck, A.; Railo, M.; Vikatmaa, P.; Kaste, M.; Mustanoja, S.; Gylling, H. Non-cholesterol sterols in serum and endarterectomized carotid arteries after a short-term plant stanol and sterol ester challenge. Nutr. Metab. Cardiovasc. Dis., 2011, 21(3), 182-188.
[http://dx.doi.org/10.1016/j.numecd.2009.09.006] [PMID: 20096545]
[19]
Schött, H.F.; Luister, A.; Husche, C.; Schäfers, H.J.; Böhm, M.; Plat, J.; Lütjohann, D.; Laufs, U.; Weingärtner, O. The relationships of phytosterols and oxyphytosterols in plasma and aortic valve cusps in patients with severe aortic stenosis. Biochem. Biophys. Res. Commun., 2014, 446(3), 805-810.
[http://dx.doi.org/10.1016/j.bbrc.2014.03.007] [PMID: 24631689]
[20]
Simonen, P.; Lommi, J.; Hallikainen, M.; Helske-Suihko, S.; Werkkala, K.; Kupari, M.; Kovanen, P.T.; Gylling, H. Dietary plant stanols or sterols neither accumulate in stenotic aortic valves nor influence their structure or inflammatory status. Clin. Nutr., 2015, 34(6), 1251-1257.
[http://dx.doi.org/10.1016/j.clnu.2015.01.001] [PMID: 25614126]
[21]
Demonty, I.; Ras, R.T.; van der Knaap, H.C.M.; Duchateau, G.S.M.J.E.; Meijer, L.; Zock, P.L.; Geleijnse, J.M.; Trautwein, E.A. Continuous dose-response relationship of the LDL-cholesterol-lowering effect of phytosterol intake. J. Nutr., 2009, 139(2), 271-284.
[http://dx.doi.org/10.3945/jn.108.095125] [PMID: 19091798]
[22]
Musa-Veloso, K.; Poon, T.H.; Elliot, J.A.; Chung, C. A comparison of the LDL-cholesterol lowering efficacy of plant stanols and plant sterols over a continuous dose range: results of a meta-analysis of randomized, placebo-controlled trials. Prostaglandins Leukot. Essent. Fatty Acids, 2011, 85(1), 9-28.
[http://dx.doi.org/10.1016/j.plefa.2011.02.001] [PMID: 21345662]
[23]
Ras, R.T.; Geleijnse, J.M.; Trautwein, E.A. LDL-cholesterol-lowering effect of plant sterols and stanols across different dose ranges: a meta-analysis of randomised controlled studies. Br. J. Nutr., 2014, 112(2), 214-219.
[http://dx.doi.org/10.1017/S0007114514000750] [PMID: 24780090]
[24]
Gylling, H.; Hallikainen, M.; Nissinen, M.J.; Miettinen, T.A. The effect of a very high daily plant stanol ester intake on serum lipids, carotenoids, and fat-soluble vitamins. Clin. Nutr., 2010, 29(1), 112-118.
[http://dx.doi.org/10.1016/j.clnu.2009.08.005] [PMID: 19709787]
[25]
Mensink, R.P.; de Jong, A.; Lütjohann, D.; Haenen, G.R.; Plat, J. Plant stanols dose-dependently decrease LDL-cholesterol concentrations, but not cholesterol-standardized fat-soluble antioxidant concentrations, at intakes up to 9 g/d. Am. J. Clin. Nutr., 2010, 92(1), 24-33.
[http://dx.doi.org/10.3945/ajcn.2009.29143] [PMID: 20504972]
[26]
LaRosa, J.C. Low-density lipoprotein cholesterol reduction: the end is more important than the means. Am. J. Cardiol., 2007, 100(2), 240-242.
[http://dx.doi.org/10.1016/j.amjcard.2007.02.089] [PMID: 17631077]
[27]
Devaraj, S.; Autret, B.C.; Jialal, I. Reduced-calorie orange juice beverage with plant sterols lowers C-reactive protein concentrations and improves the lipid profile in human volunteers. Am. J. Clin. Nutr., 2006, 84(4), 756-761.
[http://dx.doi.org/10.1093/ajcn/84.4.756] [PMID: 17023701]
[28]
Miettinen, T.A.; Tilvis, R.S.; Kesäniemi, Y.A. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am. J. Epidemiol., 1990, 131(1), 20-31.
[http://dx.doi.org/10.1093/oxfordjournals.aje.a115479] [PMID: 2293749]
[29]
Grundy, S.M. Plasma noncholesterol sterols as indicators of cholesterol absorption. J. Lipid Res., 2013, 54(4), 873-875.
[http://dx.doi.org/10.1194/jlr.E036806] [PMID: 23402986]
[30]
Vuoristo, M.; Tilvis, R.; Miettinen, T.A. Serum plant sterols and lathosterol related to cholesterol absorption in coeliac disease. Clin. Chim. Acta, 1988, 174(2), 213-224.
[http://dx.doi.org/10.1016/0009-8981(88)90388-9] [PMID: 3383445]
[31]
Andrade, I.; Santos, L.; Ramos, F. Advances in analytical methods to study cholesterol metabolism: the determination of serum noncholesterol sterols. Biomed. Chromatogr., 2013, 27(10), 1234-1242.
[http://dx.doi.org/10.1002/bmc.2840] [PMID: 23165328]
[32]
Mackay, D.S.; Jones, P.J.H.; Myrie, S.B.; Plat, J.; Lütjohann, D. Methodological considerations for the harmonization of non-cholesterol sterol bio-analysis. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2014, 957, 116-122.
[http://dx.doi.org/10.1016/j.jchromb.2014.02.052] [PMID: 24674990]
[33]
Lütjohann, D. Methodological aspects of plant sterol and stanol measurement. J. AOAC Int., 2015, 98(3), 674-676.
[http://dx.doi.org/10.5740/jaoacint.SGELutjohann] [PMID: 25942061]
[34]
Lembcke, J.; Ceglarek, U.; Fiedler, G.M.; Baumann, S.; Leichtle, A.; Thiery, J. Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS. J. Lipid Res., 2005, 46(1), 21-26.
[http://dx.doi.org/10.1194/jlr.C400004-JLR200] [PMID: 15489546]
[35]
McDonald, J.G.; Smith, D.D.; Stiles, A.R.; Russell, D.W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res., 2012, 53(7), 1399-1409.
[http://dx.doi.org/10.1194/jlr.D022285] [PMID: 22517925]
[36]
Phillips, K.M.; Ruggio, D.M.; Bailey, J.A. Precise quantitative determination of phytosterols, stanols, and cholesterol metabolites in human serum by capillary gas-liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl., 1999, 732(1), 17-29.
[http://dx.doi.org/10.1016/S0378-4347(99)00257-1] [PMID: 10517218]
[37]
Matysik, S.; Klünemann, H.H.; Schmitz, G. Gas chromatography-tandem mass spectrometry method for the simultaneous determination of oxysterols, plant sterols, and cholesterol precursors. Clin. Chem., 2012, 58(11), 1557-1564.
[http://dx.doi.org/10.1373/clinchem.2012.189605] [PMID: 22997279]
[38]
Gylling, H.; Plat, J.; Turley, S.; Ginsberg, H.N.; Ellegård, L.; Jessup, W.; Jones, P.J.; Lütjohann, D.; Maerz, W.; Masana, L.; Silbernagel, G.; Staels, B.; Borén, J.; Catapano, A.L.; De Backer, G.; Deanfield, J.; Descamps, O.S.; Kovanen, P.T.; Riccardi, G.; Tokgözoglu, L.; Chapman, M.J. Plant sterols and plant stanols in the management of dyslipidaemia and prevention of cardiovascular disease. Atherosclerosis, 2014, 232(2), 346-360.
[http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.043] [PMID: 24468148]
[39]
Gylling, H.; Halonen, J.; Lindholm, H.; Konttinen, J.; Simonen, P.; Nissinen, M.J.; Savolainen, A.; Talvi, A.; Hallikainen, M. The effects of plant stanol ester consumption on arterial stiffness and endothelial function in adults: a randomised controlled clinical trial. BMC Cardiovasc. Disord., 2013, 13, 50.
[http://dx.doi.org/10.1186/1471-2261-13-50] [PMID: 23841572]
[40]
Matthan, N.R.; Pencina, M.; LaRocque, J.M.; Jacques, P.F.; D’Agostino, R.B.; Schaefer, E.J.; Lichtenstein, A.H. Alterations in cholesterol absorption/synthesis markers characterize Framingham offspring study participants with CHD. J. Lipid Res., 2009, 50(9), 1927-1935.
[http://dx.doi.org/10.1194/jlr.P900039-JLR200] [PMID: 19436064]
[41]
Krawczyk, M.; Lütjohann, D.; Schirin-Sokhan, R.; Villarroel, L.; Nervi, F.; Pimentel, F.; Lammert, F.; Miquel, J.F. Phytosterol and cholesterol precursor levels indicate increased cholesterol excretion and biosynthesis in gallstone disease. Hepatology, 2012, 55(5), 1507-1517.
[http://dx.doi.org/10.1002/hep.25563] [PMID: 22213168]
[42]
Lupattelli, G.; Pirro, M.; Siepi, D.; Mannarino, M.R.; Roscini, A.R.; Vaudo, G.; Pasqualini, L.; Schillaci, G.; Mannarino, E. Non-cholesterol sterols in different forms of primary hyperlipemias. Nutr. Metab. Cardiovasc. Dis., 2012, 22(3), 231-236.
[http://dx.doi.org/10.1016/j.numecd.2010.05.010] [PMID: 20708389]
[43]
Escurriol, V.; Cofán, M.; Moreno-Iribas, C.; Larrañaga, N.; Martínez, C.; Navarro, C.; Rodríguez, L.; González, C.A.; Corella, D.; Ros, E. Phytosterol plasma concentrations and coronary heart disease in the prospective Spanish EPIC cohort. J. Lipid Res., 2010, 51(3), 618-624.
[http://dx.doi.org/10.1194/jlr.P000471] [PMID: 19786566]
[44]
Jaceldo-Siegl, K.; Lütjohann, D.; Sirirat, R.; Mashchak, A.; Fraser, G.E.; Haddad, E. Variations in dietary intake and plasma concentrations of plant sterols across plant-based diets among North American adults. Mol. Nutr. Food Res., 2017, 61(8)
[http://dx.doi.org/10.1002/mnfr.201600828] [PMID: 28130879]
[45]
Ikeda, I. Factors affecting intestinal absorption of cholesterol and plant sterols and stanols. J. Oleo Sci., 2015, 64(1), 9-18.
[http://dx.doi.org/10.5650/jos.ess14221] [PMID: 25742922]
[46]
Nikkilä, K.; Höckerstedt, K.; Miettinen, T.A. High serum cholestanol and low campesterol/sitosterol ratio indicate severe liver damage and liver transplantation in primary biliary cirrhosis. Transplant. Proc., 1992, 24(1), 383-386.
[PMID: 1539326]
[47]
Escurriol, V.; Cofán, M.; Serra, M.; Bulló, M.; Basora, J.; Salas-Salvadó, J.; Corella, D.; Zazpe, I.; Martínez-González, M.A.; Ruiz-Gutiérrez, V.; Estruch, R.; Ros, E. Serum sterol responses to increasing plant sterol intake from natural foods in the Mediterranean diet. Eur. J. Nutr., 2009, 48(6), 373-382.
[http://dx.doi.org/10.1007/s00394-009-0024-z] [PMID: 19412676]
[48]
Correani, A.; Visentin, S.; Cosmi, E.; Ponchia, E.; D’Aronco, S.; Simonato, M.; Vedovelli, L.; Cogo, P.; Carnielli, V.P. The maternal-fetal gradient of free and esterified phytosterols at the time of delivery in humans. Clin. Nutr., 2018, 37(6 Pt A), 2107-2112.
[http://dx.doi.org/10.1016/j.clnu.2017.10.004] [PMID: 29102321]
[49]
Pereira, M.A.; Weggemans, R.M.; Jacobs, D.R., Jr; Hannan, P.J.; Zock, P.L.; Ordovas, J.M.; Katan, M.B. Within-person variation in serum lipids: implications for clinical trials. Int. J. Epidemiol., 2004, 33(3), 534-541.
[http://dx.doi.org/10.1093/ije/dyh057] [PMID: 15020568]
[50]
Smith, S.J.; Cooper, G.R.; Myers, G.L.; Sampson, E.J. Biological variability in concentrations of serum lipids: sources of variation among results from published studies and composite predicted values. Clin. Chem., 1993, 39(6), 1012-1022.
[PMID: 8504530]
[51]
Wu, A.H.B.; Ruan, W.; Todd, J.; Lynch, K.L. Biological variation of β-sitosterol, campesterol, and lathosterol as cholesterol absorption and synthesis biomarkers. Clin. Chim. Acta, 2014, 430, 43-47.
[http://dx.doi.org/10.1016/j.cca.2013.12.040] [PMID: 24394292]
[52]
Miettinen, T.A.; Gylling, H.; Nissinen, M.J. The role of serum non-cholesterol sterols as surrogate markers of absolute cholesterol synthesis and absorption. Nutr. Metab. Cardiovasc. Dis., 2011, 21(10), 765-769.
[http://dx.doi.org/10.1016/j.numecd.2011.05.005] [PMID: 21899991]


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Article Details

VOLUME: 26
ISSUE: 37
Year: 2019
Published on: 17 December, 2019
Page: [6776 - 6785]
Pages: 10
DOI: 10.2174/0929867325666180713160330
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