A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma
2012; Elsevier BV; Volume: 53; Issue: 7 Linguagem: Inglês
10.1194/jlr.d022285
ISSN1539-7262
AutoresJeffrey G. McDonald, Daniel Smith, Ashlee R. Stiles, David W. Russell,
Tópico(s)Steroid Chemistry and Biochemistry
ResumoWe describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 μl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ∪ 1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7α- and 4β-hydroxycholesterol. Intermediates from the cholesterol biosynthetic pathway were also detected, including zymosterol, desmosterol, and lanosterol. This method also allowed the quantification of six secosteroids, including the 25-hydroxylated species of vitamins D2 and D3. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day. We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 μl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ∪ 1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7α- and 4β-hydroxycholesterol. Intermediates from the cholesterol biosynthetic pathway were also detected, including zymosterol, desmosterol, and lanosterol. This method also allowed the quantification of six secosteroids, including the 25-hydroxylated species of vitamins D2 and D3. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day. Sterols play essential roles in the physiological processes of virtually all living organisms. Members of this lipid class are integral building blocks in the cellular membranes of animals and have important functions in signaling, regulation, and metabolism (1.Brown M.S. Goldstein J.L. Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.J. Lipid Res. 2009; 50: S15-S27Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). To date, the majority of studies have focused on cholesterol, the most abundant sterol in mammals that serves as both a precursor and product to a host of important molecules, including steroid hormones, bile acids, oxysterols, and intermediates in the cholesterol biosynthetic pathway (2.Myant N.B. The Biology of Cholesterol and Related Steroids. William Heinemann Medical Books, London1981Google Scholar, 3.Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis.Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1400) Google Scholar–4.Dietschy J.M. Turley S.D. 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: 1375-1397Abstract Full Text Full Text PDF PubMed Scopus (774) Google Scholar). Although cholesterol has gained significant notoriety due to the compound's negative impact on health, other sterols, such as plant-derived phytosterols, are thought to offer potential human health benefits by lowering circulating cholesterol levels (5.Ostlund Jr, R.E. Racette S.B. Stenson W.F. Inhibition of cholesterol absorption by phytosterol-replete wheat germ compared with phytosterol-depleted wheat germ.Am. J. Clin. Nutr. 2003; 77: 1385-1389Crossref PubMed Scopus (110) Google Scholar, 6.Moruisi K.G. Oosthuizen W. Opperman A.M. Phytosterols/stanols lower cholesterol concentrations in familial hypercholesterolemic subjects: a systematic review with meta-analysis.J. Am. Coll. Nutr. 2006; 25: 41-48Crossref PubMed Scopus (115) Google Scholar). Novel functions for sterols and secosteroids continue to be identified. For example, the cholesterol metabolites 25-hydroxycholesterol and 7α,25-dihydroxycholesterol have recently been shown to play important signaling roles in the immune system (7.Bauman D.R. Bitmansour A.D. McDonald J.G. Thompson B.M. Liang G. Russell D.W. 25-Hydroxycholesterol secreted by macrophages in response to Toll-like receptor activation suppresses immunoglobulin A production.Proc. Natl. Acad. Sci. USA. 2009; 106: 16764-16769Crossref PubMed Scopus (234) Google Scholar, 8.Zhang J. Yi T. Shen W. Nguyen D. Pereira J.P. Guerini D. Baumgarten B.U. Roggo S. Wen B. et al.HannedoucheOxysterols direct immune cell migration via EBI2.Nature. 2011; 475: 524-527Crossref PubMed Scopus (314) Google Scholar–9.Liu C. Yang X.V. Wu J. Kuei C. Mani N.S. Zhang L. Yu J. Sutton S.W. Qin N. Banie H. et al.Oxysterols direct B-cell migration through EBI2.Nature. 2011; 475: 519-523Crossref PubMed Scopus (251) Google Scholar). 24-hydroxycholesterol has been shown to be involved in cholesterol turnover in the brain, and it plays a role in memory (10.Kotti T.J. Ramirez D.M.O. Pfeiffer B.E. Huber K.M. Russell D.W. Brain cholesterol turnover required for geranylgeraniol production and learning in mice.Proc. Natl. Acad. Sci. USA. 2006; 103: 3869-3874Crossref PubMed Scopus (193) Google Scholar) and glucose metabolism (11.Suzuki R. Lee K. Jing E. Biddinger S.B. McDonald J.G. Montine T.J. Craft S. Kahn C.R. Diabetes and insulin in regulation of brain cholesterol metabolism.Cell Metab. 2010; 12: 567-579Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Alterations in vitamin D levels arising from the lack of sun exposure and/or use of sunscreen are postulated to have a negative impact on health (12.Ross C.A. Taylor C.L. Yaktine A.L. Del Valle H.B. Dietary Reference Intakes for Calcium and Vitamin D. National Academies Press, Washington, DC2011Google Scholar, 13.Kulie T. Groff A. Redmer J. Hounshell J. Schrager S. Vitamin D: an evidence-based review.J. Am. Board Fam. Med. 2009; 22: 698-706Crossref PubMed Scopus (221) Google Scholar). These findings together with the large number of sterols suggest that there may be undiscovered roles for sterols in biology, and they highlight the need for continued research into the biochemical pathways associated with these compounds. The extraction and analysis of sterols in human plasma present a unique challenge due to their virtual insolubility, sequestration within lipoproteins, and dramatic differences in the levels of individual sterols. Cholesterol is the most abundant sterol, with circulating levels on the order of 1 to 3 mg/ml, whereas 25-hydroxycholesterol is a million-fold less abundant at 1 to 3 ng/ml (14.Lund E.G. Diczfalusy U. Quantitation of receptor ligands by mass spectrometry.in: Russell D.W. Mangelsdorf D.J. Methods in Enzymology. Academic Press, San Diego, CA2003: 24-37Google Scholar, 15.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). The circulating form of sterols in humans is primarily as steryl esters, in which a fatty acid is esterified to carbon 3 of the sterol; however, a small variable percentage of free sterols also exist. Enzymatically formed oxysterols are typically between 59% and 91% esterified (15.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). This duality poses additional analytical challenges because free versus esterified sterols must be isolated or measured separately, or steryl esters must first be converted to free sterols. Representative structures of a sterol, steryl ester, and secosteroid are shown in Fig. 1. Other lipids, such as triglycerides, phospholipids, and sphingolipids that are present in the plasma at concentrations similar to cholesterol, further confound the analysis of sterols. Traditionally, the extraction of sterols has relied on one of two classic methods: Bligh/Dyer method or Folch method (16.Bligh E.G. Dyer W.J. A rapid method of total lipide extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar, 17.Folch J. Lees M. Stanley G.H.S. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Both make use of a mixture of chloroform and methanol to simultaneously disrupt lipoproteins and solubilize lipids. An alkaline hydrolysis step is employed to cleave sterol-fatty acid conjugates; a strong base in alcohol is added to the extract, which is then often incubated at room temperature or elevated temperature (60–100°C) for 1–2 h (18.Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (475) Google Scholar, 19.Lütjohann D. Breuer O. Ahlborg G. Nennesmo I. Siden A. Diczfalusy U. Björkhem I. 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: 9799-9804Crossref PubMed Scopus (568) Google Scholar–20.Yu L. von Bergmann K. Lutjohann D. Hobbs H.H. Cohen J.C. Selective sterol accumulation in ABCG5/ABCG8-deficient mice.J. Lipid Res. 2004; 45: 301-307Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Hydrolysis also serves to degrade other abundant lipid classes, such as triglycerides and phospholipids, which reduces sample complexity. Alternatively, if the goal is to measure only free, unesterified sterols, the alkaline hydrolysis step is eliminated. Lastly, solid-phase extraction (SPE) is utilized to isolate sterols from other components. Ideally, cholesterol could be isolated from the sample because it would simplify subsequent instrumental analysis of other less abundant sterol species; however, SPE does not yet have the inherent resolution or reproducibility to quantitatively isolate cholesterol from all other sterols (18.Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (475) Google Scholar, 21.McDonald J.G. Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.in: Brown H.A. Methods in Enzymology. Academic Press, San Diego, CA2007: 145-170Google Scholar). Although many variations of these extraction procedures have been reported in the literature, the fundamental steps of the various methods are the same. Instrumental analysis of sterols and related compounds in plasma has typically focused on either a single analyte or a limited group of analytes. Historically, these methods have employed GC and GC-MS for the analysis of select sterols (18.Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (475) Google Scholar, 22.Axelson M. Mörk B. Sjövall J. Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood.J. Lipid Res. 1988; 29: 629-641Abstract Full Text PDF PubMed Google Scholar, 23.Miettinen T.A. Ahrens E.H. Grundy S.M. Quantitative isolation and gas–liquid chromatographic analysis of total dietary and fecal neutral steroids.J. Lipid Res. 1965; 6: 411-424Abstract Full Text PDF PubMed Google Scholar). GC and GC-MS have limitations with regard to sample composition, injection volume, sensitivity, and mass spectral scanning functions. Despite these limitations, GC and GC-MS are still widely used for sterol analysis due to their chromatographic resolving capacity, ease of use, and relative low cost of the acquisition and operation of the instruments. Recently, methods have been developed for sterol analysis using LC-MS (24.DeBarber A.E. Connor W.E. Pappu A.S. Merkens L.S. Steiner R.D. ESI-MS/MS quantification of 7[alpha]-hydroxy-4-cholesten-3-one facilitates rapid, convenient diagnostic testing for cerebrotendinous xanthomatosis.Clin. Chim. Acta. 2010; 411: 43-48Crossref PubMed Scopus (29) Google Scholar, 25.DeBarber A.E. Lütjohann D. Merkens L. Steiner R.D. Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol.Anal. Biochem. 2008; 381: 151-153Crossref PubMed Scopus (41) Google Scholar–26.Pulfer M.K. Taube C. Gelfand E. Murphy R.C. Ozone exposure in vivo and formation of biologically active oxysterols in the lung.J. Pharmacol. Exp. Ther. 2005; 312: 256-264Crossref PubMed Scopus (51) Google Scholar). Like GC and GC-MS, these methods are typically optimized for the detection of one or two analytes. In 2007, our laboratory described a method for analyzing a diverse group of sterols and oxysterols using LC-MS (21.McDonald J.G. Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.in: Brown H.A. Methods in Enzymology. Academic Press, San Diego, CA2007: 145-170Google Scholar). The method was optimized for detection of 12 common sterols and oxysterols in a single extraction. Since then, high performance liquid chromatography (HPLC) has become more widely used in the analysis of sterols and is often coupled to triple quadrupole mass spectrometers for quantitative analysis to enhance sensitivity and selectivity. Similarly, improvements in ionization and ion transfer into the mass spectrometer have enhanced the ability to measure low-level metabolites in biological systems. Here we report a method for the quantitative analysis of sterols, oxysterols, and secosteroids in plasma. Using a streamlined extraction procedure and a combination of LC-MS (electrospray ionization and atmospheric pressure chemical ionization), this method allows for the analysis of approximately 60 sterols from a single extraction of 200 μl of plasma. The extraction procedure is flexible and can be tailored to the sample type and information sought. As many sterols are positional isomers, chromatographic resolution remains crucial for the analysis of sterols and related compounds because the MS cannot differentiate between isobaric compounds. The instrumental analysis used in this method employs recent advances in HPLC column technology that provide increased resolution and sensitivity, and the throughput is such that 50 samples can be readily assayed per day. The method lends itself to translational research involving large cohorts of clinically well-characterized patients and can be adapted to serum, tissue, or other biological matrices. Human plasma samples were obtained from 200 subjects of the Cooper Institute (Dallas, TX) following protocols approved by the Institutional Review Board of the University of Texas Southwestern Medical Center. Written consent was obtained from each patient prior to sample collection. Pooled normal human plasma was purchased from Innovative Research (Novi, MI) for use as a control sample. Primary and deuterated sterol and secosteroid standards were obtained from Avanti Polar Lipids (Alabaster, AL), unless otherwise noted. A complete list of analytes is given in Table 1. Dichloromethane (DCM) and hexane were from Burdick and Jackson (Honeywell; Morristown, NJ), methanol, chloroform (CHCl3), acetonitrile (ACN), and water were from Fisher Scientific (Fair Lawn, NJ). Dulbecco's phosphate-buffered saline (DPBS) was from Mediatech (Manassas, VA). Ammonium acetate (NH4OAc) and butylated hydroxytoluene (BHT) were from Sigma-Aldrich (St. Louis, MO), 10N potassium hydroxide (KOH) was from Fisher Scientific, ammonium iodide (NH4I) was from Acros Organics (Geel, Belgium), and N-methyl-N[tert-butyldimethylsilyl trifluoroacetamide with 1% tert-butyldimethylchlorosilane (MTBSTFA with 1% TBDMCS) was from Restek (Bellefonte, PA). All solvents were HPLC-grade or better; all chemicals were ACS-grade or better.TABLE 1Sterols, oxysterols, and secosteroidsCommon NameLIPID MAPS NumberElemental FormulaMWIdentifierRRISourceDehydroergosterolLMST01031023C28H42O394.32A0.77*Relative to cholestenonoe (Δ4) (Identifier I).AVTVitamin D2LMST03010001C28H44O396.34B0.80*Relative to cholestenonoe (Δ4) (Identifier I).AVTVitamin D3LMST03020001C27H46O384.34C0.83*Relative to cholestenonoe (Δ4) (Identifier I).AVTZymosterol†Deuterated analog available from Avanti Polar Lipids.LMST01010066C27H44O384.34D0.87*Relative to cholestenonoe (Δ4) (Identifier I).AVTDesmosterol†Deuterated analog available from Avanti Polar Lipids.LMST01010016C27H44O384.34E0.92*Relative to cholestenonoe (Δ4) (Identifier I).AVT8(9)-DehydrocholesterolLMST01010242C27H44O384.34F0.97*Relative to cholestenonoe (Δ4) (Identifier I).AVTErgosterolLMST01030093C28H44O396.34G0.98*Relative to cholestenonoe (Δ4) (Identifier I).SIG7-DehydrocholesterolLMST01010069C27H44O384.34H1.00*Relative to cholestenonoe (Δ4) (Identifier I).AVT8(14)-Dehydrocholesterol1.00*Relative to cholestenonoe (Δ4) (Identifier I).AVTCholestenone (Δ4)LMST01010015C27H44O384.34I1.00*Relative to cholestenonoe (Δ4) (Identifier I).AVTCholestenone (Δ5)LMST01010248C27H44O384.34J1.03*Relative to cholestenonoe (Δ4) (Identifier I).STERBrassicasterolLMST01030098C28H46O398.35K1.05*Relative to cholestenonoe (Δ4) (Identifier I).SIGLathosterolLMST01010089C27H46O386.35L1.05*Relative to cholestenonoe (Δ4) (Identifier I).§Lathosterol and cholesterol coelute in an actual plasma extract.SIGCholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010001C27H46O386.35M1.06*Relative to cholestenonoe (Δ4) (Identifier I).§Lathosterol and cholesterol coelute in an actual plasma extract.**Cholesterol signal shown 0.1× scale.AVT14-Demethyl-lanosterol†Deuterated analog available from Avanti Polar Lipids.LMST01010176C29H48O412.37N1.07*Relative to cholestenonoe (Δ4) (Identifier I).AVTLanosterolLMST01010017C30H50O426.39O1.09*Relative to cholestenonoe (Δ4) (Identifier I).AVTDihydrocholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010077C27H48O388.37P1.12*Relative to cholestenonoe (Δ4) (Identifier I).SIGCampesterolLMST01030097C28H48O400.37Q1.13*Relative to cholestenonoe (Δ4) (Identifier I).SIGStigmasterolLMST01040124C29H48OR1.13*Relative to cholestenonoe (Δ4) (Identifier I).AVTCycloartenolLMST01100008C38H50O426.39S1.13*Relative to cholestenonoe (Δ4) (Identifier I).STERβ-Sitosterol†Deuterated analog available from Avanti Polar Lipids.LMST01040129C29H50O414.39T1.20*Relative to cholestenonoe (Δ4) (Identifier I).SIG24, 25-DihydrolanosterolLMST01010087C30H52O428.40U1.22*Relative to cholestenonoe (Δ4) (Identifier I).AVTStigmastanolLMST01040128C29H52O412.37V1.27*Relative to cholestenonoe (Δ4) (Identifier I).SIG1α, 25-Dihydroxyvitamin D2†Deuterated analog available from Avanti Polar Lipids.LMST03020660C27H44O3416.33b0.35Relative to 27-hydroxycholesterol (o).SIG7 α, 25-Dihydroxycholesterol†Deuterated analog available from Avanti Polar Lipids.LMST04030166C27H46O34.18.34a0.33Relative to 27-hydroxycholesterol (o).AVT7 α, 27-Dihydroxycholesterol†Deuterated analog available from Avanti Polar Lipids.LMST04030178C27H46O3418.34c0.38Relative to 27-hydroxycholesterol (o).AVT1α, 25-Dihydroxyvitamin D2LMST03010040C27H44O3428.33d0.41Relative to 27-hydroxycholesterol (o).CRO3β, 27-Dihydroxy-5-cholesten-7-oneLMST04030180C27H44O3416.33e0.44Relative to 27-hydroxycholesterol (o).AVT7α, 26-Dihydroxycholest-4-en-3-oneLMST04030157C27H44O3416.33f0.44Relative to 27-hydroxycholesterol (o).AVT16α, 27-DihydroxycholesterolLMST04030179C27H46O3418.34g0.44Relative to 27-hydroxycholesterol (o).STER(20R)-17α, 20-DihydroxycholesterolLMST04030176C27H46O3418.34h0.56Relative to 27-hydroxycholesterol (o).STER25-Hydroxyvitamin D2†Deuterated analog available from Avanti Polar Lipids.LMST03020246C27H44O2400.33i0.77Relative to 27-hydroxycholesterol (o).AVT25-Hydroxyvitamin D3†Deuterated analog available from Avanti Polar Lipids.LMST03010030C28H46O2412.33j0.83Relative to 27-hydroxycholesterol (o).AVT22R-Hydroxycholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010086C27H46O2402.35k0.89Relative to 27-hydroxycholesterol (o).AVT25-Hydroxy-cholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010018C27H46O2402.35l0.93Relative to 27-hydroxycholesterol (o).AVT24S-Hydroxy-cholesterolLMST01010019C27H46O2402.35m0.95Relative to 27-hydroxycholesterol (o).AVT24-OxocholesterolLMST01010133C27H44O2400.33n0.98Relative to 27-hydroxycholesterol (o).STER27-Hydroxy-cholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010057C27H46O2402.35o1.00Relative to 27-hydroxycholesterol (o).AVT15α-HydroxycholesteneLMST01010267C27H46O2402.35p1.00Relative to 27-hydroxycholesterol (o).AVT20-HydroxycholesterolLMST01010201C27H46O2402.35q1.00Relative to 27-hydroxycholesterol (o).STER24(S), 25-EpoxycholesterolLMST01010012C27H44O2400.33r1.02Relative to 27-hydroxycholesterol (o).AVT5α, 6β-DihydroxycholestanolLMST01010052C27H48O3420.36s1.02Relative to 27-hydroxycholesterol (o).STER15-KetocholesteneLMST01010269C27H44O2400.33t1.08Relative to 27-hydroxycholesterol (o).AVT15-KetocholestaneLMST01010270C27H46O2402.35u1.12Relative to 27-hydroxycholesterol (o).AVT3-Oxo-7α-hydroxycholesterolLMST01010271C27H44O2400.33v1.16Relative to 27-hydroxycholesterol (o).SIG7-Oxo-cholestenoneLMST01010272C27H42O2398.32w1.19Relative to 27-hydroxycholesterol (o).STER7α-Hydroxy-cholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010013C27H46O2402.35x1.18Relative to 27-hydroxycholesterol (o).AVT7β-Hydroxy-cholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010047C27H46O2402.351.18Relative to 27-hydroxycholesterol (o).AVT1α-Hydroxyvitamin D2LMST03010028C27H44O2412.33y1.19Relative to 27-hydroxycholesterol (o).EMD1α-Hydroxyvitamin D3LMST03020231C27H44O2400.33z1.20Relative to 27-hydroxycholesterol (o).EMD7-Oxocholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010049C27H44O2400.33a1.20Relative to 27-hydroxycholesterol (o).AVTCholestan-6-oxo-3, 5-diolLMST01010126C27H46O3418.34b1.21Relative to 27-hydroxycholesterol (o).STER15β-HydroxycholestaneLMST01010268C27H46O2402.35c1.21Relative to 27-hydroxycholesterol (o).AVT15α-HydroxycholestaneLMST01010273C27H48O2404.37d1.21Relative to 27-hydroxycholesterol (o).AVT6-KetocholestanolLMST01010276C27H46O2402.35e1.29Relative to 27-hydroxycholesterol (o).SIG6α-Hydroxycholestanol†Deuterated analog available from Avanti Polar Lipids.LMST01010135C27H48O2402.35f1.27Relative to 27-hydroxycholesterol (o).AVT19-HydroxycholesterolLMST01010274C27H46O2402.35g1.29Relative to 27-hydroxycholesterol (o).STER5, 6β-Epoxy-cholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010011C27H46O2402.35h1.33Relative to 27-hydroxycholesterol (o).AVT5α-HydroxycholesterolLMST01010275C27H48O2402.35i1.33Relative to 27-hydroxycholesterol (o).STER5, 6α-Epoxy-cholesterolLMST01010010C27H46O2402.35j1.36Relative to 27-hydroxycholesterol (o).AVT4β-Hydroxy-cholesterol†Deuterated analog available from Avanti Polar Lipids.LMST01010014C27H46O2402.35k1.42Relative to 27-hydroxycholesterol (o).AVTLIPID MAPS numbers can be used to access additional information at lipidmaps.org. The alphabetical identifier corresponds to the chromatographs in Fig. 1. AVT, Avanti Polar Lipids (Alabaster, AL); EMD, EMD Chemicals (Rockland, MA); SIG, Sigma-Aldrich (St. Louis, MO); STER, Seraloids (Newport, RI). MW, molecular weight; RRI, relative retention index.* Relative to cholestenonoe (Δ4) (Identifier I).† Deuterated analog available from Avanti Polar Lipids.§ Lathosterol and cholesterol coelute in an actual plasma extract.** Cholesterol signal shown 0.1× scale.†† Relative to 27-hydroxycholesterol (o). Open table in a new tab LIPID MAPS numbers can be used to access additional information at lipidmaps.org. The alphabetical identifier corresponds to the chromatographs in Fig. 1. AVT, Avanti Polar Lipids (Alabaster, AL); EMD, EMD Chemicals (Rockland, MA); SIG, Sigma-Aldrich (St. Louis, MO); STER, Seraloids (Newport, RI). MW, molecular weight; RRI, relative retention index. Frozen plasma samples (stored at −80°C) were equilibrated to room temperature for approximately 30 min. To ensure homogeneity, each sample was pipetted three times with a 1 ml air-displacement pipette (fitted with a disposable 1 ml barrier tip). Then 200 μl of plasma were added drop-wise to 3 ml of 1:1 DCM:methanol in a 16 × 100 glass tube placed in a 30°C ultrasonic bath. The tube also contained 20 μl of a deuterated sterol and secosteroid standard cocktail and BHT at 50 μg/ml (see supplementary data for standard amounts). The tube was flushed with N2 for several seconds to displace oxygen, sealed with a PTFE-lined screw cap, and incubated at 30°C in the ultrasonic bath for 10 min. Following incubation, the sample was centrifuged at 3,500 rpm for 5 min at 25°C to pellet protein and other insoluble material. The supernatant from each sample was decanted into a 16 × 100 mm glass screw-cap tube and set aside. A 1:1 DCM:methanol solution (3 ml) was added to the pelleted material, and then the tube was capped and vigorously agitated until the pellet was dislodged and disrupted. The sample was centrifuged at 3,500 rpm for 5 min at 25°C, and the resulting supernatant was decanted back into the tube containing the supernatant from the initial extraction. Hydrolysis was performed by adding 300 μl of 10N KOH to each tube and flushing with N2 for several seconds, followed by placement in a water bath at 35°C for 1.5 h. Following hydrolysis, 3 ml of DPBS was added to each sample, the tubes were capped, and the samples were agitated for several seconds. Samples were centrifuged at 3,500 rpm for 5 min at 25°C. The organic (lower) layer was removed with a 9″ Pasteur pipette and transferred to a 16 × 100 glass tube and set aside. DCM (3 ml) was added to the remaining sample, and then the tube was capped, vortexed for several seconds, and centrifuged at 3,500 rpm for 5 min at 25°C. Using a 9″ Pasteur pipette, the organic layer (lower layer) was removed and transferred to the 16 × 100 glass culture tube containing the initial sample. To maximize extraction efficiency, the same pipette was used for each liquid-liquid step, and the pipette was placed into a separate glass culture tube between steps. Hydrolyzed samples were then dried under N2 using a 27-port drying manifold (Pierce; Fisher Scientific, Fair Lawn, NJ). Sterols and secosteroids were isolated using 200 mg, 3 ml aminopropyl SPE columns (Biotage; Charlotte, NC). The column was rinsed and conditioned with 2 × 3 ml of hexane. The extracted and dried plasma sample was dissolved in approximately 1 ml of hexane and gently swirled for several seconds. The sample (and any insoluble material) was then transferred to the SPE column using a 6″ Pasteur pipette and eluted to waste. The extract tube was rinsed with 1 ml of hexane and gently swirled, and the rinse was transferred to the column and eluted to waste. Again, to minimize sample loss, the same Pasteur pipette was used at each step. Following the addition of sample, the column was rinsed with 1 ml of hexane to elute nonpolar compounds. Sterols were then eluted from the column with 4.5 ml (1.5 column volumes) of 23:1 CHCl3:methanol into a new 16 × 100 glass culture tube. The eluted sample was dried under N2. To prepare the sample for instrumental analysis, 400 μl of warm (37°C) 90% methanol was added, and the tube was placed in an ultrasonic bath for 5 min at 30°C. The sample was then transferred to an autosampler vial containing a 500 μl deactivated insert (Restek, Bellefonte, PA) and 20 μl of d6-6α-hydroxycholestanol as an internal standard. If particulate matter was observed in the sample during dissolution, then the vial insert was carefully transferred to a standard microcentrigure tube using fine-point tweezers and centrifuged at room temperature for 5 min at 6,000 rpm in a microcentrifuge. The vial insert was then carefully transferred back to the autosampler vial and stored at room temperature until analysis. A 50 μl aliquot of the sample was removed and transferred to a new autosampler vial for analysis by GC-MS. The sample was dried under N2. Derivatization of sterols was performed by adding 100 μl of 1:1 pyridine:MTBSTFA (1%TBDMCS) with 2 mg/ml NH4I (dissolved first in pyridine), flushing the sample with N2, cappin
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