The purpose of this work was to investigate whether changes in oxysterol and apolipoprotein levels over 5 years are associated with disease course and disability progression in multiple sclerosis (MS). This study included 139 subjects [39 healthy controls (HCs), 61 relapsing-remitting MS (RR-MS) patients, and 39 progressive MS (P-MS) patients]. Oxysterols [24-hydroxycholesterol (24HC), 25-hydroxycholesterol (25HC), 27-hydroxycholesterol (27HC), 7α-hydroxycholesterol (7αHC), and 7-ketocholesterol (7KC)] were measured at baseline and 5 years using a novel mass spectrometric method, and apolipoproteins were measured using immunoturbidometric diagnostic kits. Levels of 24HC (P = 0.004), 25HC (P = 0.029), and 27HC (P = 0.026) increased in P-MS patients. 7KC (P = 0.047) and 7αHC (P = 0.001) levels decreased in RR-MS patients, and there were no changes in any oxysterols in HCs. In MS patients, ApoC-II (all P ≤ 0.01) and ApoE (all P ≤ 0.01) changes were positively associated with all oxysterol levels. Increases in 24HC (P = 0.038) and ApoB (P = 0.038) and decreases in 7KC (P = 0.020) were observed in RR-MS patients who converted to secondary P-MS (SP-MS) at follow-up and in SP-MS patients compared with RR-MS patients. Oxysterols and their associations with apolipoproteins differed between MS patients and HCs over 5 years. Oxysterol and apolipoprotein changes were associated with conversion to SP-MS. The purpose of this work was to investigate whether changes in oxysterol and apolipoprotein levels over 5 years are associated with disease course and disability progression in multiple sclerosis (MS). This study included 139 subjects [39 healthy controls (HCs), 61 relapsing-remitting MS (RR-MS) patients, and 39 progressive MS (P-MS) patients]. Oxysterols [24-hydroxycholesterol (24HC), 25-hydroxycholesterol (25HC), 27-hydroxycholesterol (27HC), 7α-hydroxycholesterol (7αHC), and 7-ketocholesterol (7KC)] were measured at baseline and 5 years using a novel mass spectrometric method, and apolipoproteins were measured using immunoturbidometric diagnostic kits. Levels of 24HC (P = 0.004), 25HC (P = 0.029), and 27HC (P = 0.026) increased in P-MS patients. 7KC (P = 0.047) and 7αHC (P = 0.001) levels decreased in RR-MS patients, and there were no changes in any oxysterols in HCs. In MS patients, ApoC-II (all P ≤ 0.01) and ApoE (all P ≤ 0.01) changes were positively associated with all oxysterol levels. Increases in 24HC (P = 0.038) and ApoB (P = 0.038) and decreases in 7KC (P = 0.020) were observed in RR-MS patients who converted to secondary P-MS (SP-MS) at follow-up and in SP-MS patients compared with RR-MS patients. Oxysterols and their associations with apolipoproteins differed between MS patients and HCs over 5 years. Oxysterol and apolipoprotein changes were associated with conversion to SP-MS. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS that results in blood-brain barrier (BBB) breakdown, inflammation, and neurodegeneration, and causes physical and cognitive disability. There is extensive evidence suggesting that higher levels of total cholesterol (TC) and LDL cholesterol are associated with increasing disability in MS (1Tettey P. Simpson Jr., S. Taylor B.V. van der Mei I.A. Vascular comorbidities in the onset and progression of multiple sclerosis.J. Neurol. Sci. 2014; 347: 23-33Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 2Weinstock-Guttman B. Zivadinov R. Mahfooz N. Carl E. Drake A. Schneider J. Teter B. Hussein S. Mehta B. Weiskopf M. et al.Serum lipid profiles are associated with disability and MRI outcomes in multiple sclerosis.J. Neuroinflammation. 2011; 8: 127Crossref PubMed Scopus (155) Google Scholar, 3Mandoj C. Renna R. Plantone D. Sperduti I. Cigliana G. Conti L. Koudriavtseva T. Anti-annexin antibodies, cholesterol levels and disability in multiple sclerosis.Neurosci. Lett. 2015; 606: 156-160Crossref PubMed Scopus (20) Google Scholar, 4Tettey P. Simpson Jr., S. Taylor B. Blizzard L. Ponsonby A.L. Dwyer T. Kostner K. van der Mei I. An adverse lipid profile is associated with disability and progression in disability, in people with MS.Mult. Scler. 2014; 20: 1737-1744Crossref PubMed Scopus (94) Google Scholar). Cholesterol is required for myelin structure and proper functioning of neuronal, vascular, and immune cells in the CNS. Cholesterol and lipoproteins do not cross the BBB, so the brain is dependent on de novo cholesterol synthesis. Oxysterols are oxygenated cholesterol metabolites that traverse the BBB rapidly (5Heverin M. Meaney S. Lutjohann D. Diczfalusy U. Wahren J. Bjorkhem I. Crossing the barrier: net flux of 27-hydroxycholesterol into the human brain.J. Lipid Res. 2005; 46: 1047-1052Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) and act as signaling mediators between the periphery and the CNS (6Jeitner T.M. Voloshyna I. Reiss A.B. Oxysterol derivatives of cholesterol in neurodegenerative disorders.Curr. Med. Chem. 2011; 18: 1515-1525Crossref PubMed Scopus (47) Google Scholar) to enable the maintenance of cholesterol homeostasis in the brain. Oxysterols are ligands for LXR (7Abildayeva K. Jansen P.J. Hirsch-Reinshagen V. Bloks V.W. Bakker A.H. Ramaekers F.C. de Vente J. Groen A.K. Wellington C.L. Kuipers F. et al.24(S)-hydroxycholesterol participates in a liver X receptor-controlled pathway in astrocytes that regulates apolipoprotein E-mediated cholesterol efflux.J. Biol. Chem. 2006; 281: 12799-12808Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 8Leoni V. Caccia C. 24S-hydroxycholesterol in plasma: a marker of cholesterol turnover in neurodegenerative diseases.Biochimie. 2013; 95: 595-612Crossref PubMed Scopus (83) Google Scholar) and are involved in regulating the biosynthesis, cellular efflux, and elimination of cholesterol (9Björkhem I. Meaney S. Brain cholesterol: long secret life behind a barrier.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 806-815Crossref PubMed Scopus (723) Google Scholar, 10Diczfalusy U. Lund E. Lutjohann D. Bjorkhem I. Novel pathways for elimination of cholesterol by extrahepatic formation of side-chain oxidized oxysterols.Scand. J. Clin. Lab. Invest. Suppl. 1996; 226: 9-17Crossref PubMed Google Scholar, 11Dietschy J.M. Turley S.D. 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 (770) Google Scholar, 12Meaney S. Heverin M. Panzenboeck U. Ekstrom L. Axelsson M. Andersson U. Diczfalusy U. Pikuleva I. Wahren J. Sattler W. et al.Novel route for elimination of brain oxysterols across the blood-brain barrier: conversion into 7alpha-hydroxy-3-oxo-4-cholestenoic acid.J. Lipid Res. 2007; 48: 944-951Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Schwarz M. Lund E.G. Setchell K.D. Kayden H.J. Zerwekh J.E. Bjorkhem I. Herz J. Russell D.W. Disruption of cholesterol 7alpha-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7alpha-hydroxylase.J. Biol. Chem. 1996; 271: 18024-18031Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). We investigated three side-chain oxysterols [24-hydroxycholesterol (24HC), 25-hydroxycholesterol (25HC), and 27-hydroxycholesterol (27HC)] and two B-ring oxysterols [7α-hydroxycholesterol (7αHC) and 7-ketocholesterol (7KC)]. 24HC is produced exclusively in the brain (11Dietschy J.M. Turley S.D. 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 (770) Google Scholar) and is the primary regulator of cholesterol synthesis and the principal mechanism for cholesterol elimination in the brain (14Lund E.G. Xie C. Kotti T. Turley S.D. Dietschy J.M. Russell D.W. Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover.J. Biol. Chem. 2003; 278: 22980-22988Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 15Björkhem I. Lütjohann D. Breuer O. Sakinis A. Wennmalm A. Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro.J. Biol. Chem. 1997; 272: 30178-30184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 16Lütjohann D. Breuer O. Ahlborg G. Nennesmo I. Sidén 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 (566) Google Scholar). 25HC is produced in activated macrophages and mediates inflammatory signaling. 7αHC is the product of the rate-limiting step in the bile acid pathway of cholesterol elimination (17Crosignani A. Zuin M. Allocca M. Del Puppo M. Oxysterols in bile acid metabolism.Clin. Chim. Acta. 2011; 412: 2037-2045Crossref PubMed Scopus (22) Google Scholar), and it is a surrogate marker for bile acid synthesis and cholesterol excretion (17Crosignani A. Zuin M. Allocca M. Del Puppo M. Oxysterols in bile acid metabolism.Clin. Chim. Acta. 2011; 412: 2037-2045Crossref PubMed Scopus (22) Google Scholar). 27HC is produced by CYP27A1 in the acidic pathway (18Chawla A. Saez E. Evans R.M. Don't know much bile-ology.Cell. 2000; 103: 1-4Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). 7KC results from oxidative stress and can induce apoptosis (19Terasaka N. Wang N. Yvan-Charvet L. Tall A.R. High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1.Proc. Natl. Acad. Sci. USA. 2007; 104: 15093-15098Crossref PubMed Scopus (214) Google Scholar), inflammation in endothelial cells, and neuronal injury in the brain (20Diestel A. Aktas O. Hackel D. Hake I. Meier S. Raine C.S. Nitsch R. Zipp F. Ullrich O. Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage.J. Exp. Med. 2003; 198: 1729-1740Crossref PubMed Scopus (128) Google Scholar). van de Kraats et al. (21van de Kraats C. Killestein J. Popescu V. Rijkers E. Vrenken H. Lutjohann D. Barkhof F. Polman C.H. Teunissen C.E. Oxysterols and cholesterol precursors correlate to magnetic resonance imaging measures of neurodegeneration in multiple sclerosis.Mult. Scler. 2014; 20: 412-417Crossref PubMed Scopus (55) Google Scholar) found that MS patients had lower levels of serum 24HC and 27HC compared with healthy controls (HCs), and higher 24HC levels were associated with decreased normalized brain volume measures. Our group also found that 24HC, 27HC, and 7αHC (all P < 0.015) were lower in MS patients compared with HCs, and 7KC was higher in progressive MS (P-MS) compared with relapsing-remitting MS (RR-MS) (P < 0.001) (22Mukhopadhyay S. Fellows K. Browne R.W. Khare P. Krishnan Radhakrishnan S. Hagemeier J. Weinstock-Guttman B. Zivadinov R. Ramanathan M. Interdependence of oxysterols with cholesterol profiles in multiple sclerosis.Mult. Scler. 2017; 23: 792-801Crossref PubMed Scopus (34) Google Scholar). Apolipoproteins contribute to maintaining peripheral cholesterol homeostasis and are surrogates for oxysterol signaling. ApoA-I acts as an acceptor of cholesterol following efflux from cells (23Schwartz K. Lawn R.M. Wade D.P. ABC1 gene expression and ApoA-I-mediated cholesterol efflux are regulated by LXR.Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar). Serum ApoB is strongly associated with multiple oxysterols, including 24HC, 25HC, 27HC, 7αHC, and 7KC (22Mukhopadhyay S. Fellows K. Browne R.W. Khare P. Krishnan Radhakrishnan S. Hagemeier J. Weinstock-Guttman B. Zivadinov R. Ramanathan M. Interdependence of oxysterols with cholesterol profiles in multiple sclerosis.Mult. Scler. 2017; 23: 792-801Crossref PubMed Scopus (34) Google Scholar, 24Alkazemi D. Egeland G. Vaya J. Meltzer S. Kubow S. Oxysterol as a marker of atherogenic dyslipidemia in adolescence.J. Clin. Endocrinol. Metab. 2008; 93: 4282-4289Crossref PubMed Scopus (49) Google Scholar), and is associated with inflammatory markers (25Sniderman A.D. Faraj M. Apolipoprotein B, apolipoprotein A-I, insulin resistance and the metabolic syndrome.Curr. Opin. Lipidol. 2007; 18: 633-637Crossref PubMed Scopus (59) Google Scholar). In this study, we will focus on ApoE and ApoC-II as the primary biomarkers for LXR signaling. ApoC-II shuttles between lipoproteins and is important for the removal of cholesterol from tissues, and its levels are unaffected by inflammation (26Isshiki M. Hirayama S. Ueno T. Ito M. Furuta A. Yano K. Yamatani K. Sugihara M. Idei M. Miida T. Apolipoproteins C-II and C-III as nutritional markers unaffected by inflammation.Clin. Chim. Acta. 2018; 481: 225-230Crossref PubMed Scopus (6) Google Scholar). ApoE, which is modulated by 24HC, plays an important role in maintaining cholesterol homeostasis in the CNS by accepting cholesterol from astrocytes and shuttling it to neurons (7Abildayeva K. Jansen P.J. Hirsch-Reinshagen V. Bloks V.W. Bakker A.H. Ramaekers F.C. de Vente J. Groen A.K. Wellington C.L. Kuipers F. et al.24(S)-hydroxycholesterol participates in a liver X receptor-controlled pathway in astrocytes that regulates apolipoprotein E-mediated cholesterol efflux.J. Biol. Chem. 2006; 281: 12799-12808Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The associations of oxysterol changes with MS disease progression have not been extensively investigated in longitudinal studies. The goals of this study were to investigate oxysterol and apolipoprotein levels in 5 year follow-up samples and assess whether changes in these lipid mediators were associated with MS disease course conversion and disability progression. This study included samples and clinical data from a single-center longitudinal prospective study of clinical, genetic, and environmental risk factors in MS at the MS Center of the State University of New York at Buffalo. The study includes patients with MS and clinically isolated syndrome, HCs, and controls with other neurological diseases. Patients and controls provided blood samples, underwent neurological examination, and responded to a structured questionnaire administered in-person at baseline and at 5 year follow-up visits. The University at Buffalo Human Subjects Institutional Review Board approved the study protocol, and all participants provided written informed consent. The study was conducted in accordance with the principles of the Declaration of Helsinki. This substudy was limited to HCs and MS patients 18–65 years of age with oxysterol and apolipoprotein measures available at baseline and 5 years. MS disease course at baseline and follow-up were reported by MS specialists based on patients' clinical characteristics and published disease course classification (27Lublin F.D. Reingold S.C. Cohen J.A. Cutter G.R. Sorensen P.S. Thompson A.J. Wolinsky J.S. Balcer L.J. Banwell B. Barkhof F. et al.Defining the clinical course of multiple sclerosis: the 2013 revisions.Neurology. 2014; 83: 278-286Crossref PubMed Scopus (1800) Google Scholar). HCs needed to meet the health-screen requirements and had to have a normal physical and neurological examination. They were recruited from respondents to a local advertisement. Children younger than 18 years of age, adults over 65 years of age, clinically isolated syndrome, neuromyelitis optica, or other neurological diseases were excluded from this substudy. P-MS consisted of patients with secondary P-MS (SP-MS) or primary P-MS. A conversion to SP-MS categorical variable was defined to classify MS patients into the three groups: i) RR-MS at baseline and remained RR-MS at follow-up (RR-MS); ii) RR-MS at baseline and converted to SP-MS by the 5 year follow-up visit (RR-MS to SP-MS); and iii) SP-MS at baseline and follow-up (SP-MS). The plasma total plasma oxysterol assay (inclusive of free and esterified oxysterols) included room temperature saponification, solid phase extraction, and a modification of the LC-mass spectrometry analysis conditions previously described (28Narayanaswamy R. Iyer V. Khare P. Bodziak M.L. Badgett D. Zivadinov R. Weinstock-Guttman B. Rideout T.C. Ramanathan M. Browne R.W. Simultaneous determination of oxysterols, cholesterol and 25-hydroxy-vitamin D3 in human plasma by LC-UV-MS.PLoS One. 2015; 10: e0123771Crossref PubMed Scopus (24) Google Scholar). Plasma samples were stored at −80°C until use without prior freeze thaw cycles. Baseline and 5 year follow-up samples were grouped together by a technician separate from the analytical laboratory and analysts were blind to case/control and baseline/follow-up status. In-house quality control materials consisting of −80°C frozen aliquots of pooled human plasma and lipid-stripped human blood plasma spiked with authentic oxysterol standards demonstrated −80°C stability (<15% deviation from nominal) for 4.5 years. Samples, matrix calibrators, and quality control materials were prepared as previously described (28Narayanaswamy R. Iyer V. Khare P. Bodziak M.L. Badgett D. Zivadinov R. Weinstock-Guttman B. Rideout T.C. Ramanathan M. Browne R.W. Simultaneous determination of oxysterols, cholesterol and 25-hydroxy-vitamin D3 in human plasma by LC-UV-MS.PLoS One. 2015; 10: e0123771Crossref PubMed Scopus (24) Google Scholar). In brief, 200 μl of sample were vortex-mixed with 100 μl of deuterated internal standard mix [150 ng/ml each of vitamin D3 (d3), 22HC (d7), 7αHC (d7), 7KC (d7)] and 10 μl of 50 mg/dl ethanolic butylated hydroxytoluene and saponified using 875 μl of 0.5 M ethanolic KOH for 3 h at room temperature under argon. The pH was neutralized using 20 μl of 85% phosphoric acid; 1 ml of water was added, the vial centrifuged, and the supernatant was loaded under gravity onto a HyperSep C18 solid phase extraction cartridge (200 mg, 3 ml) that had been conditioned with 1 ml of hexane:isopropanol (50:50 v/v), 1 ml of methanol, and 2 ml of water, sequentially. Polar lipids were washed off the SPE column using 4 ml of methanol:water (75:25 v/v). Nonpolar sterols (including cholesterol and oxysterols) were eluted using 2 ml of hexane:isopropanol (50:50 v/v). The eluate was evaporated under nitrogen, reconstituted in 300 μl of methanol:water (90:10, v/v), and 75 μl injected for LC-mass spectrometry analysis. Oxysterols were analyzed on a Shimadzu Scientific (Columbia, MD) LCMS-2010A mass spectrometer system with atmospheric pressure chemical ionization interface in positive ion mode. Mobile phase composition was 100% methanol to pump A and methanol:water (50:50 v/v) to pump B, both containing 0.1% formic acid. The chromatographic conditions were optimized from our originally reported method (28Narayanaswamy R. Iyer V. Khare P. Bodziak M.L. Badgett D. Zivadinov R. Weinstock-Guttman B. Rideout T.C. Ramanathan M. Browne R.W. Simultaneous determination of oxysterols, cholesterol and 25-hydroxy-vitamin D3 in human plasma by LC-UV-MS.PLoS One. 2015; 10: e0123771Crossref PubMed Scopus (24) Google Scholar). Oxysterols were separated on a Supelcosil LC-18-S, 10 cm × 3.0 mm, 3 μm column (Sigma-Aldrich, St. Louis, MO). Column oven temperature was set at 10°C and flow rate was at 0.75 ml/min. The mobile phase gradient was as follows: 80% A for 10 min, linear increase to 100% A over 5 min and held for 13 min followed by re-equilibration at 80% A for 6 min; total run time was 34 min. Temperature settings for the mass spectrometer were: interface at 400°C, CDL at 230°C, and heat block at 200°C. Nitrogen was used as a nebulizing gas for the ion source at a flow rate of 2.5 l/min. Data were acquired in a time segmented-single ion monitoring manner to achieve maximum sensitivity. The m/z ratios used for quantifying oxysterols were: m/z 367.30 for 24HC, 25HC, and 7αHC; m/z 385.30 for 27-OHC; m/z 401.40 for 7KC; m/z 374.30 for 22-OHC(d7); m/z 374.30 for 7αHC(d7); and m/z 408.40 for 7KC(d7). Calibrator standards were included in every run and the oxysterol concentrations (in picograms per milliliter) were calculated using calibrator standards. EDTA plasma samples for lipid and apolipoprotein analyses were obtained in the nonfasted state. The methods used for lipid and apolipoprotein analyses were previously published (29Browne R.W. Weinstock-Guttman B. Horakova D. Zivadinov R. Bodziak M.L. Tamano-Blanco M. Badgett D. Tyblova M. Vaneckova M. Seidl Z. et al.Apolipoproteins are associated with new MRI lesions and deep grey matter atrophy in clinically isolated syndromes.J. Neurol. Neurosurg. Psychiatry. 2014; 85: 859-864Crossref PubMed Scopus (32) Google Scholar). Analysts were blinded to the clinical status of samples. Immunoturbidometric diagnostic reagent kits, calibrators, and controls (Kamiya Biomedical, Thousand Oaks, CA) were used for the apolipoprotein (ApoA-I, ApoA-II, ApoB, ApoC-II, and ApoE) assays. The coefficient of variation of these assays is <5%. The SPSS (IBM Inc., Armonk, NY, version 24.0) statistical program was used. Oxysterol concentrations were log-transformed to reduce skew. Apolipoprotein concentrations were normally distributed and not log-transformed. Paired t-tests were used to investigate changes in 24HC, 25HC, 27HC, 7αHC, 7KC, ApoA-I, ApoA-II, ApoB, ApoC-II, and ApoE levels from baseline to follow-up in RR-MS, P-MS, and HC groups alone. Additionally, we used repeated measures analyses to investigate associations between change in oxysterol or apolipoprotein level over 5 years with disease course at baseline (either HC vs. MS or RR-MS vs. P-MS) following adjustment for age, gender, and BMI. The independent samples t-test was used to assess differences in baseline and follow-up levels of 24HC, 25HC, 27HC, 7αHC, 7KC, ApoA-I, ApoA-II, ApoB, ApoC-II , and ApoE between HCs and MS patients and between RR-MS and P-MS patients. Linear regression was used to investigate the associations between change in TC and change in each of the apolipoproteins (ApoA-I, ApoA-II, ApoB, ApoC-II, or ApoE) with the change in each of the oxysterol levels (24HC, 25HC, 27HC, 7αHC, or 7KC) over 5 years. The change in TC or change in the apolipoprotein level of interest was the dependent variable; and age, gender, BMI, and the change in the oxysterol level of interest were treated as covariates. Linear regression was used to investigate associations between changes in oxysterol and apolipoprotein levels from baseline to 5 years with the conversion to SP-MS variable and SP-MS subgroups variable. In these analyses, the change in oxysterol or apolipoprotein variable of interest from baseline to 5 years was the dependent variable; and the predictor variables included age, gender, baseline BMI, baseline oxysterol, or apolipoprotein of interest and either the conversion to SP-MS variable or the SP-MS subgroups variable. Baseline and follow-up clinical and demographic characteristics of the entire study sample and for HC, RR-MS, and P-MS groups are summarized in Table 1. The higher average age and median Expanded Disability Status Scale (EDSS) scores observed in P-MS patients are representative of the progressive disease course. The majority of subjects in the HC (55%), RR-MS (53%), and P-MS (54%) groups had a BMI of 25 kg/m3 or more at baseline. At baseline, the percentages of RR and P-MS patients on disease-modifying therapies (DMTs) were 80% and 77%, respectively. At baseline, the percentages of HC, RR-MS, and P-MS patients on statins were 8, 12, and 13%, respectively.TABLE 1Demographic and clinical characteristics by disease course at baseline visitHCRR-MSP-MSSample size, number396139Percent female28 (71.8%)43 (70.5%)29 (74.4%)Age, yearsBaseline46.4 ± 12.744.7 ± 11.156.0 ± 6.1Follow-up52.2 ± 12.750.7 ± 11.262.2 ± 6.0Disease duration, yearsBaselineμ13.2 ± 8.721.3 ± 10.4Follow-upμ19.2 ± 8.927.3 ± 10.2BMI, kg/m2aEDSS in median [interquartile range (IQR)].Baseline BMI27.0 ± 6.128.1 ± 6.326.0 ± 3.9Normal/overweight/obese45%/31%/24%47%/25%/28%46%/44%/10%Follow-up BMI26.6 ± 6.728.8 ± 7.926.5 ± 4.7Normal/overweight/obese55%/24%/21%33%/35%/32%39%/41%/21%EDSS, median (IQR)bThe BMI category variable was defined according to National Institutes of Health guidelines (https://www.nhlbi.nih.gov/health/educational/lose_wt/BMI/bmi_dis.htm): normal weight (BMI <25 kg/m2), overweight (BMI ≥25 to <30 kg/m2), and obese (BMI ≥30 kg/m2). BMI is missing for one HC and one RR-MS subject at baseline.Baselineμ2.5 (2.0)5.0 (3.0)Follow-upμ2.5 (1.5)6.0 (2.8)TC, mg/dlBaseline207 ± 42210 ± 39214 ± 35Follow-up235 ± 43227 ± 38244 ± 41DMT at baselinecDMT data for two RR-MS subjects are missing at baseline and follow-up. DMT data for one P-MS subject is missing at baseline. The interferon category includes the products AVONEX®, REBIF®, BETASERON®, EXTAVIA®, and PLEGRIDY™, whereas orals include fingolimod, dimethyl fumarate, and teriflunomide.Interferonμ19 (32%)14 (36%)Glatiramer acetateμ13 (22%)10 (26%)Natalizumabμ14 (23%)2 (5%)Otherμ1 (2%)3 (8%)Noneμ12 (20%)9 (23%)DMT at follow-upInterferonμ16 (27%)13 (33%)Glatiramer acetateμ18 (30%)13 (33%)Natalizumabμ2 (3%)1 (3%)Oralsμ10 (17%)2 (5%)Otherμ7 (12%)2 (5%)Noneμ6 (10%)8 (21%)StatinsBaseline8%12%13%Follow-up10%20%13%All continuous variables (age, BMI, disease duration) are mean ± standard deviation.a EDSS in median [interquartile range (IQR)].b The BMI category variable was defined according to National Institutes of Health guidelines (https://www.nhlbi.nih.gov/health/educational/lose_wt/BMI/bmi_dis.htm): normal weight (BMI <25 kg/m2), overweight (BMI ≥25 to <30 kg/m2), and obese (BMI ≥30 kg/m2). BMI is missing for one HC and one RR-MS subject at baseline.c DMT data for two RR-MS subjects are missing at baseline and follow-up. DMT data for one P-MS subject is missing at baseline. The interferon category includes the products AVONEX®, REBIF®, BETASERON®, EXTAVIA®, and PLEGRIDY™, whereas orals include fingolimod, dimethyl fumarate, and teriflunomide. Open table in a new tab All continuous variables (age, BMI, disease duration) are mean ± standard deviation. Figure 1 summarizes the changes in oxysterol (24HC, 25HC, 27HC, 7KC, and 7αHC) levels from baseline to 5 years in HC, RR-MS, and P-MS patients. There were no significant changes in any oxysterol levels between baseline and 5 years in HCs. We compared oxysterol levels in the entire MS patient group to the HC group and found that follow-up 7αHC levels were 14.5 ng/ml lower in MS patients compared with HCs (P = 0.012). No other differences were observed between baseline or follow-up levels of oxysterols in the MS patient group compared with HCs. In RR-MS patients, 7KC levels decreased by 1.2 ng/ml (P = 0.047), and 7αHC levels decreased by 15.8 ng/ml (P = 0.001) from baseline to 5 years. No significant changes in 24HC, 25HC, or 27HC were observed in the RR-MS patients. In P-MS patients, 24HC levels increased by 7.9 ng/ml (P = 0.004), 25HC levels increased by 1.3 ng/ml (P = 0.029), and 27HC levels increased by 33.2 ng/ml (P = 0.026) from baseline to 5 years. There were no significant changes in 7KC or 7αHC from baseline to 5 years in the P-MS group. Follow-up levels of 25HC were 1.4 ng/ml higher in P-MS patients compared with RR-MS patients (P = 0.008). Follow-up levels of 7KC were 2.6 ng/ml higher in P-MS patients compared with RR-MS patients (P = 0.023). There were no other differences in baseline or follow-up oxysterols between RR-MS and P-MS patients. In repeated measures analyses that adjusted for age, gender, BMI, and RR-MS versus P-MS diagnosis at baseline, the greater decreases in 7KC and 7αHC over time in RR-MS compared with P-MS patients, and the greater increases in 25HC over time in P-MS patients compared with RR-MS patients remained significant (data not shown). These results suggest that MS disease course at baseline (RR-MS or P-MS) is associated with changes in oxysterol levels over 5 years. Figure 2 summarizes the changes in apolipoproteins (ApoA-I, ApoA-II, ApoB, ApoC-II, and ApoE) levels from baseline to 5 years in the HCs, RR-MS patients, and P-MS patients. In HCs, ApoA-I levels increased by 10.9 mg/ml (P = 0.012), ApoA-II levels increased by 2.0 mg/ml (P = 0.041), ApoB levels increased by 7.1 mg/ml (P = 0.049), ApoC-II levels increased by 0.55 mg/ml (P = 0.001), and ApoE levels increased by 0.37 mg/ml (P = 0.044) over 5 years. In the RR-MS group, only ApoA-I levels increased by 12.5 mg/ml (P < 0.001) from baseline to 5 years. No significant changes from baseline to follow-up were observed for ApoA-II, ApoB, ApoC-II, or ApoE levels in the RR-MS group. In the P-MS group, ApoC-II levels increased by 0.6 mg/ml (P = 0.014), and ApoB levels increased by 6.2 mg/ml (P = 0.011) from baseline to 5 years. No significant changes were observed in ApoA-I, ApoA-II, and ApoE levels in the P-MS group. In repeated measures analyses that corrected for age, gender, BMI, and HC versus MS diagnosis at baseline, we found that the increases in ApoC-II levels from baseline to 5 years in HCs compared with MS patients remained significant (data not shown). We investigated the associations, if any, between apolipoprotein, cholesterol, and oxysterol changes, because oxysterols are known to induce ApoE and ApoC-II biosynthesis via transcriptional activation of LXR (30Mak P.A. Laffitte B.A. Desrumaux C. Joseph S.B. Curtiss L.K. Mangelsdorf D.J. Tontonoz P. Edwards P.A. Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta.J. Biol. Chem. 2002; 277: 31900-31908Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In the HC group, greater in