Plasma oxysterols and angiographically determined coronary atherosclerosis: a case control study

Several in vitro and in vivo experiments have implicated oxysterols in the aetiology and progression of atherosclerosis. Oxysterols may be formed endogenously by oxidation of cholesterol and thus may form a marker of LDL oxidation. They may also be obtained exogenously through dietary intake. We investigated the association of oxysterols with the degree of coronary stenosis in patients undergoing coronary angiography. Cases with severe coronary atherosclerosis 80 stenosis in one of the major coronary vessels, n =80 were compared with controls with no or minor stenosis 50 stenosis in all three major coronary vessels, n =79 . Cases and controls were prestratified on age, gender and smoking habits. Evaluated were plasma levels of unesterified 7 hydroxycholesterol, 7 hydroxycholesterol, 25 hydroxycholesterol, 7 ketocholesterol, cholestane triol and 5,6 epoxycholestanol. 7 Hydroxycholesterol made up 67 of the total amount of plasma oxysterol concentration and was the only one significantly higher in cases 1.53 mu g per 100 ml vs 1.27 mu g per 100 ml, p 0.05 . Further, cases had somewhat higher LDL cholesterol levels and significantly lower HDL cholesterol levels than controls. After multivariate adjustment to account for this difference in lipid levels and for the prestratification factors the mean difference between cases and controls for 7 hydroxycholesterol 0.14 mu g per 100 ml was no longer significant. Also the other oxysterols showed no significant association with the degree of coronary stenosis. Multiple logistic regression analyses showed an adjusted odds ratio of 1.07 95 CI, 0.45-2.59 in the highest tertile of total plasma oxysterol level. We conclude, that this study does not support the hypothesis that plasma oxysterols form an additional risk factor for coronary atherosclerosis.     

Relevance and mechanism of oxysterol stereospecifity in coronary artery disease

Cholesterol oxidation products (oxysterols) are markers for in vitro LDL oxidation. They are potent inducers of programmed cell death and are also found in high concentrations inside atherosclerotic lesions. Among physiologically occurring oxysterols, 7beta-OH-cholesterol suggests an increase of lipid peroxidation in vivo. In the underlying study, we quantified free plasma oxysterols by means of gas chromatography in patients with stable coronary artery disease (CAD). Total free plasma oxysterols were elevated more than 2-fold in patients with stable CAD (233 +/- 49 vs 108 +/- 19 ng/ml, n = 22, P < 0.05) compared to a control group (n = 20) with similar atherogenic risk profile and angiographically normal coronary arteries. We found that 7-ketocholesterol, as well as the beta-isomers of epoxide (25.7 +/- 10.0 vs 7.3 +/- 1.4 ng/ml, P = 0.07) and 7beta-OH-cholesterol (65.1 +/- 15.7 vs 19.4 +/- 8.9 ng/ml, P < 0.01), was mainly responsible for this increase. To elucidate a potential relevance of oxysterol stereospecificity in regard to endothelial damage, we further conducted in vitro experiments using human arterial endothelial cells (HAECs). Surprisingly, beta-isomers exerted an up to 10-fold higher amount of cell death in equivalent doses when compared to alpha-isomers. The greater cytotoxic potential of beta-isomers was due to increased apoptosis, preceded by mitochondrial release of cytochrome c with subsequent caspase-3 activation. Stereospecific release of cytochrome c depended on the presence of an intact cytoplasmic membrane, hinting at the existence of a putative oxysterol receptor or a direct stereospecific effect on membrane biology. Finally, both isoforms of oxysterols directly released cytochrome c only in conjunction with protein containing cytosol and endoplasmatic reticulum. Free plasma oxysterol levels, particularly 7-ketocholesterol, beta-epoxide and 7beta-OH-cholesterol, are elevated in patients with stable CAD, independent of their LDL cholesterol levels. Due to the highly increased cytotoxicity of oxysterol beta-isomers in vitro, they may represent important atherogenic risk factors.     

Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E° mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 – 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7β-hydroxycholesterol (7β-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E° mice, whereas the level of 7α-hydroxycholesterol (7α-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E° mice.     

Changes in the phospholipid composition of the arterial cell can result in severe atherosclerotic lesions

The oxysterol concentration in the plasma and the phospholipid composition of vascular tissue obtained by coronary artery bypass grafting (CABG) were compared with plasma and vascular tissue from age and sex matched controls. The plasma from CABG patients had a higher concentration of oxysterols than was present in the controls. Human endothelial cells were cultured for 72 hours in a medium containing plasma obtained from CABG patients, from controls or from the same controls to which 5 oxysterols were added to make the total oxysterol level equivalent to that in the CABG plasma and then pulsed with calcium (45Ca(2+)) for one hr. A significantly higher influx of 45Ca(2+) was noted in the endothelial cells cultured in the plasma obtained from CABG patients and from the controls with 5 added oxysterols, but not in those cultured without added oxysterols indicating that oxysterols increased calcium influx into endothelial cells. A phospholipid analysis indicated that the arterial tissue from CABG patients had 48.2% sphingomyelin in its phospholipid fraction compared to 10% in arterial tissue from umbilical cords. The saphenous vein obtained during CABG surgery from the same patient had only 24% sphingomyelin in its phospholipid fraction and unlike the coronary arteries had no atherosclerotic lesions. The higher level of oxysterol in the plasma of patients suffering from severe atherosclerosis could increase the concentration of sphingomyelin in the arterial cell membrane and thereby increase calcium influx required for producing the calcific type VII lesions in the coronary arteries.     

24S-hydroxycholesterol alters activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel through intercalation into plasma membrane

Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20S‑hydroxycholesterol, 22R‑hydroxycholesterol, 24S‑hydroxycholestero, 25‑hydroxycholesterol, and 27‑hydroxycholesterol, induced current reduction of large-conductance Ca²⁺- and voltage-activated K⁺ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7α‑hydroxycholesterol and 7‑ketocholesterol were without effect. By using 24S‑hydroxycholesterol (24S‑HC), the major brain oxysterol, we explored the inhibition mechanism. 24S‑HC inhibited Slo1 BK channels with an IC₅₀ of ~2 μM, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γ‑cyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.     

Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E degrees mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 - 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7beta-hydroxycholesterol (7beta-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E degrees mice, whereas the level of 7alpha-hydroxycholesterol (7alpha-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E degrees mice.     

Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status

Oxidant stress seems to play a role in several setting of human pathology, such as atherosclerosis, cancer, and aging. The study of oxidant stress in human disease should be based on the evaluation of either sensitive and specific markers of enhanced oxidant stress, such as oxysterols, or antioxidant defense, by measuring alpha-tocopherol. We have developed a rapid method to measure the oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol in plasma (50 healthy subjects) and tissue as an index of oxidant stress in vivo, and from the same sample alpha-tocopherol content. The mean plasma concentration of 7beta-hydroxycholesterol and 7-ketocholesterol was 4.6+/-1.1 and 13.4+/-7.6 ng/mL, respectively. Plasma alpha-tocopherol concentration was 5.8+/-1.0 micromol/mol cholesterol. Samples from atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more alpha-tocopherol compared to normal arteries. No significant difference in cholesterol and oxysterol content was observed between cirrhotic and normal liver. However, cirrhotic liver contained significantly smaller concentration of alpha-tocopherol compared to normal liver. In conclusion, we have developed a rapid and reliable method for the assay of cholesterol oxidation products and alpha-tocopherol in plasma and tissue useful for estimation of oxidant stress/antioxidant balance.     

Bile acid synthesis precursors in familial combined hyperlipidemia: The oxysterols 24S-hydroxycholesterol and 27-hydroxycholesterol

Familial combined hyperlipidemia (FCHL), the most common inherited disorder of lipid metabolism is characterized by increasing cholesterol synthesis precursors due to hepatic overproduction of cholesterol. The bile acids synthesis pathway has not been previously studied in FCHL. The aim of this work was to study the oxysterol levels which are involved in the bile acids synthesis from cholesterol in FCHL. Clinical parameters and subclinical atherosclerosis were studied in a total of 107 FCHL patients and 126 normolipidemic controls. Non cholesterol sterols (desmosterol and lanosterol) and oxysterols (27-hydroxycholesterol and 24S-hydroxycholesterol) were measured by high performance liquid chromatography tandem mass spectrometry. Desmosterol and lanosterol, markers of cholesterol synthesis, had a positive correlation with BMI and apo B. However, no correlation was found for 24S-hydroxycholesterol and 27-hydroxycholesterol, precursors of bile acids, with these clinical parameters. Only 27-hydroxycholesterol had a positive correlation with apo B, ρ = 0.204 (P = 0.037). All oxysterol levels were higher in FHCL as compared to normal controls. A total of 59 FCHL subjects (59%) presented values of 24S-hydroxycholesterol above the 95th percentile of this oxysterol in the control population. All oxysterols showed no association with fat mass in contrast with non-cholesterol sterols. FCHL subjects with oxysterol overproduction had less carotid intima media thickness (cIMT), which suggests less atherosclerosis in these subjects. In summary, our data indicate that high oxysterol levels might be good markers of FCHL, unrelated to fat mass, and may exert a protective mechanism for cholesterol accumulation.     

The role of oxysterols in control of endothelial stiffness

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE^−/− mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.     

High levels of 15-oxygenated steroids in circulation of patients with multiple sclerosis: fact or fiction?

15-Oxygenated cholesterol species such as 5α-cholest-8(14)ene-3β,15α-diol (15HC) and 3β-hydroxy-5α-cholest-8(14)-en-15-one (15KC) are commercially available synthetic products unlikely to occur in biological systems. Surprisingly, Farez et al. recently reported that these two steroids occur in human circulation at levels considerably higher than those of any other endogenous oxysterol [Farez, M. et al. 2009. Toll-like receptor 2 and poly(ADP-ribose) polymerase 1 promote central nervous system neuroinflammation in progressive EAE. Nat. Immunol. 10: 958-964]. The levels were reported to be increased in patients with multiple sclerosis in a progressive phase and the authors suggested that this could be utilized diagnostically. Based on extensive in vitro experiments exposing cells to the same high levels of 15HC as found in vivo (1000 ng/ml) the authors concluded that 15HC may be an important pathogenetic factor in multiple sclerosis. Using combined gas chromatography-mass spectrometry we fail to detect significant plasma levels of 15HC either in healthy controls or in patients with multiple sclerosis (levels < 2 ng/ml). If 15KC is present in these plasma samples, the concentration of it must be <10 ng/ml. Our failure to detect significant levels of the above steroids could not be due to loss during hydrolysis and work-up because recovery of the added two oxysterols was close to 100%. Autoxidation of lipoprotein-bound cholesterol resulted in extensive conversion of cholesterol into 7-oxygenated but not 15-oxygenated sterols. We conclude that if present there are trace amounts only of the above 15-oxygenated steroids in human circulation and that the role of such oxysterols as pathogenetic factors and biomarkers must be reconsidered.     

Increased plasma levels of oxysterols, in vivo markers of oxidative stress, in patients with familial combined hyperlipidemia: reduction during atorvastatin and fenofibrate therapy

Familial combined hyperlipidemia (FCHL), the most common inherited disorder of lipid metabolism, is associated with an increased risk of atherosclerosis that is not fully explained by the metabolic disturbances of these patients. Oxidative damage to lipid components accumulating in the plasma of FCHL patients might contribute to explaining this lack of evidence. Cholesterol is one of the preferential targets of oxidation in LDL and this may contribute to setting a proatherogenetic phenotype in FCHL. We investigated plasma oxysterols (7-ketocholesterol and 7beta-hydroxycholesterol) and alpha-tocopherol as in vivo hallmarks of lipid-related oxidative stress. Oxidative stress hallmarks were measured in 45 FCHL patients and 54 sex- and age-matched healthy controls; in FCHL patients, oxidative stress and lipid profile parameters were also assessed in response to lipid-lowering drugs in a 24-week randomized, open-label trial with atorvastatin or fenofibrate. FCHL patients showed markedly increased levels of oxysterols (p < 0.001) and reduced alpha-tocopherol/total lipids (p < 0.001) compared to controls. These differences were independent of the presence of clinical atherosclerosis and persisted after correction for hyperlipidemia. Atorvastatin and fenofibrate significantly improved the lipid profile and caused a comparable decrease in plasma oxysterols, with the normalization of 7-ketocholesterol and a significant reduction of 7beta-hydroxycholesterol (p < 0.001). These drugs also decreased the ratio of alpha-tocopherol/total lipids by more than 30% (p < 0.001). In conclusion, FCHL patients showed increased hallmarks of cholesterol oxidation and decreased levels of alpha-tocopherol/total lipids. Atorvastatin and fenofibrate displayed comparable efficiency in decreasing oxysterols, but they further decreased lipid-corrected alpha-tocopherol levels in plasma. More research work is needed to understand the clinical meaning of these findings, which may help to understand the role of oxidative stress in FCHL and lipid-lowering therapy.     

Identification of autoantibodies in human plasma recognizing an apoB-100 LDL receptor binding site peptide

The aim of this study was to test the hypothesis that autoantibodies recognize amino acid sequences in the LDL receptor binding region of apolipoprotein B-100 (apoB-100). Autoantibodies against an unmodified or malondialdehyde (MDA)-modified LDL receptor binding site peptide were determined by ELISA in baseline plasma samples of 78 cases with coronary events and 149 matched controls recruited from the prospective Malm? Diet Cancer Study. IgG and IgM recognizing this peptide were detected in all subjects but did not differ between cases and controls. Inverse associations were observed between IgG against the native binding site and plasma oxidized LDL (r = -0.21, P < 0.005), but there were no significant associations with total or LDL cholesterol levels. In univariate analyses, inverse associations were found between baseline carotid intima-media thickness and IgG against the MDA-modified binding site (r = -0.14, P < 0.05), but this association was lost when controlling for other major cardiovascular risk factors. Specificity studies demonstrated that the binding of autoantibodies to these sequences could be inhibited by oxidized but not by native LDL. Autoantibodies recognizing the LDL receptor binding site in apoB-100 are frequently expressed. Their association with plasma oxidized LDL suggests that they have been generated in response to breakdown products of LDL oxidation, but their influence on cholesterol metabolism and the development of atherosclerosis appears limited.     

The hypocholesterolemic agent LY295427 reverses suppression of sterol regulatory element-binding protein processing mediated by oxysterols

The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4-6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action.     

Serum ferritin concentration is associated with plasma levels of cholesterol oxidation products in man

Cholesterol oxidation products, oxysterols, are thought to play a part in the initiation and development of human atherosclerotic lesions. Excessive body iron has been suggested to promote atherosclerosis and coronary heart disease through its pro-oxidative properties. In the present study, the associations between serum ferritin and plasma oxysterol concentrations were examined in 669 eastern Finnish men. Serum ferritin concentration had statistically significant (p <.05) direct correlations with most of the measured oxysterols. In multivariate adjusted regression models, serum ferritin concentration predicted significantly the levels of 27-hydroxycholesterol (beta = 0.13, p <.001), 7alpha-hydroxycholesterol (beta = 0.11, p =.005), 25-hydroxycholesterol (beta = 0.10, p =.007), 7-ketocholesterol (beta = 0.10, p =.009), and 7beta-hydroxycholesterol (beta = 0.10, p =.02). In conclusion, excess body iron, as assessed by serum ferritin, is associated with increased levels of circulating oxysterols, both of enzymatic and nonenzymatic origin, in man.     

Plasma Pentraxin 3 Levels Do Not Predict Coronary Events but Reflect Metabolic Disorders in Patients with Coronary Artery Disease in the CARE Trial

Chronic inflammation closely associates with obesity, metabolic syndrome, diabetes mellitus, and atherosclerosis. Evidence indicates that the immunomodulator pentraxin 3 (PTX3) may serve as a biomarker of these cardiometabolic disorders, but whether PTX3 predicts cardiovascular complications is unknown. We examined the association of plasma PTX3 levels with recurrent coronary events via a prospective, nested, case-control design in the CARE trial. Among 4159 patients who had a prior myocardial infarction 3 to 20 months before enrollment and also had total cholesterol levels <240 mg/dL and LDL cholesterol levels between 115 and 175 mg/dL, we measured plasma PTX3 levels at baseline by high-sensitivity ELISA in 413 cases with recurrent myocardial infarction or coronary death during a 5-year follow-up period, and in 366 sex- and age-matched controls. Cases with recurrent coronary events and controls had similar PTX3 levels, and PTX3 did not predict recurrent coronary events — a finding that contrasts with that of C-reactive protein (CRP) and serum amyloid A (SAA) in this cohort. We then associated PTX3 levels with metabolic disorders. Low plasma PTX3 levels correlated with high body-mass index, waist circumference, and triglycerides; and with low HDL cholesterol. Overall, PTX3 levels correlated inversely with the number of metabolic syndrome components. PTX3 levels also correlated inversely with apoCIII and tissue plasminogen activator, but did not associate with CRP. Although the study further links low PTX3 levels with various features associated with metabolic syndrome, the results do not indicate that PTX3 can predict recurrent coronary events among MI survivors.     

Simultaneous Determination of Oxysterols,Cholesterol and 25-Hydroxy-Vitamin D3 in Human Plasma by LC-UV-MS

Background

Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.

Methods

A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.

Results

Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.

Conclusion

The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.