Plasma oxysterols are associated with serum lipids and dementia risk in older women.

INTRODUCTION: Apolipoprotein E4 (APOE4) carriers' tendency toward hypercholesterolemia may contribute to Alzheimer's disease (AD) risk through oxysterols, which traverse the blood-brain barrier. METHODS: Relationships between baseline plasma oxysterols, APOE status, serum lipids, and cognitive impairment risk were examined in 328 postmenopausal women from the Women's Health Initiative Memory Study. Women were followed for 25 years or until incident dementia or cognitive impairment. RESULTS: Levels of 24(S)-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), and 24-OHC/27-OHC ratio did not differ by APOE status (p's > 0.05). Higher 24-OHC and 27-OHC were associated with higher total, low density lipoprotein (LDL), non-high density lipoprotein (HDL), remnant, LDL/HDL, and total/HDL cholesterol and triglycerides (p's < 0.05). Higher 24-OHC/27-OHC was associated with greater dementia risk (hazard ratio = 1.51, 95% confidence interval:1.02-2.22), which interaction analyses revealed as significant for APOE3 and APOE4+, but not APOE2+ carriers. DISCUSSION: Less favorable lipid profiles were associated with higher oxysterol levels. A higher ratio of 24-OHC/27-OHC may contribute to dementia risk in APOE3 and APOE4+ carriers.

A Retrospective Multicentric Study of 34 Patients with Niemann-Pick Type C Disease and Early Liver Involvement in France.

OBJECTIVE: To describe the clinical features and course of liver involvement in a cohort of patients with Niemann-Pick type C disease (NP-C), a severe lysosomal storage disorder. STUDY DESIGN: Patients with genetically confirmed NP-C (NPC1, n = 31; NPC2, n = 3) and liver involvement before age 6 months were retrospectively included. Clinical, laboratory test, and imaging data were collected until the last follow-up or death; available liver biopsy specimens were studied using anti-CD68 immunostaining. RESULTS: At initial evaluation (median age, 17 days of life), all patients had hepatomegaly, 33 had splenomegaly, and 30 had neonatal cholestasis. Portal hypertension and liver failure developed in 9 and 4 patients, respectively. Liver biopsy studies, performed in 16 patients, revealed significant fibrosis in all 16 and CD68+ storage cells in 15. Serum alpha-fetoprotein concentration measured in 21 patients was elevated in 17. Plasma oxysterol concentrations were increased in the 16 patients tested. Four patients died within 6 months of life, including 3 from liver involvement. In patients who survived beyond age 6 months (median follow-up, 6.1 years), cholestasis regressed in all, and portal hypertension regressed in all but 1; 25 patients developed neurologic involvement, which was fatal in 16 patients. CONCLUSIONS: Liver involvement in NP-C consisted of transient neonatal cholestasis with hepatosplenomegaly, was associated with liver fibrosis, and was responsible for death in 9% of patients. The combination of liver anti-CD68 immunostaining, serum alpha-fetoprotein measurement, and studies of plasma biomarkers should facilitate early identification of NP-C.

Plasma oxyphytosterols most likely originate from hepatic oxidation and subsequent spill-over in the circulation.

We evaluated oxyphytosterol (OPS) concentrations in plasma and various tissues of two genetically modified mouse models with either increased cholesterol (apoE KO mice) or increased cholesterol and plant sterol (PS) concentrations (apoExABCG8 dKO mice). Sixteen female apoE KO and 16 dKO mice followed the same standard, low OPS-chow diet. Animals were euthanized at 36 weeks to measure PS and OPS concentrations in plasma, brain, liver and aortic tissue. Cholesterol and oxysterol (OS) concentrations were analyzed as reference for sterol oxidation in general. Plasma campesterol (24.1 ± 4.3 vs. 11.8 ± 3.0 mg/dL) and sitosterol (67.4 ± 12.7 vs. 4.9 ± 1.1 mg/dL) concentrations were severely elevated in the dKO compared to the apoE KO mice (p < 0.001). Also, in aortic and brain tissue, PS levels were significantly elevated in dKO. However, plasma, aortic and brain OPS concentrations were comparable or even lower in the dKO mice. In contrast, in liver tissue, both PS and OPS concentrations were severely elevated in the dKO compared to apoE KO mice (sum OPS: 7.4 ± 1.6 vs. 4.1 ± 0.8 ng/mg, p < 0.001). OS concentrations followed cholesterol concentrations in plasma and all tissues suggesting ubiquitous oxidation. Despite severely elevated PS concentrations, OPS concentrations were only elevated in liver tissue, suggesting that OPS are primarily formed in the liver and plasma concentrations originate from hepatic spill-over into the circulation.

Interactions of Oxysterols with Atherosclerosis Biomarkers in Subjects with Moderate Hypercholesterolemia and Effects of a Nutraceutical Combination (Bifidobacterium longum BB536, Red Yeast Rice Extract) (Randomized, Double-Blind, Placebo-Controlled Study).

BACKGROUND: Oxysterol relationship with cardiovascular (CV) risk factors is poorly explored, especially in moderately hypercholesterolaemic subjects. Moreover, the impact of nutraceuticals controlling hypercholesterolaemia on plasma levels of 24-, 25- and 27-hydroxycholesterol (24-OHC, 25-OHC, 27-OHC) is unknown. METHODS: Subjects (n = 33; 18-70 years) with moderate hypercholesterolaemia (low-density lipoprotein cholesterol (LDL-C:): 130-200 mg/dL), in primary CV prevention as well as low CV risk were studied cross-sectionally. Moreover, they were evaluated after treatment with a nutraceutical combination (Bifidobacterium longum BB536, red yeast rice extract (10 mg/dose monacolin K)), following a double-blind, randomized, placebo-controlled design. We evaluated 24-OHC, 25-OHC and 27-OHC levels by gas chromatography/mass spectrometry analysis. RESULTS: 24-OHC and 25-OHC were significantly correlated, 24-OHC was correlated with apoB. 27-OHC and 27-OHC/total cholesterol (TC) were higher in men (median 209 ng/mL and 77 ng/mg, respectively) vs. women (median 168 ng/mL and 56 ng/mg, respectively); 27-OHC/TC was significantly correlated with abdominal circumference, visceral fat and, negatively, with high-density lipoprotein cholesterol (HDL-C). Triglycerides were significantly correlated with 24-OHC, 25-OHC and 27-OHC and with 24-OHC/TC and 25-OHC/TC. After intervention, 27-OHC levels were significantly reduced by 10.4% in the nutraceutical group Levels of 24-OHC, 24-OHC/TC, 25-OHC, 25-OHC/TC and 27-OHC/TC were unchanged. CONCLUSIONS: In this study, conducted in moderate hypercholesterolemic subjects, we observed novel relationships between 24-OHC, 25-OHC and 27-OHC and CV risk biomarkers. In addition, no adverse changes of OHC levels upon nutraceutical treatment were found.

The contribution of plasma oxysterols in the challenging diagnostic work-up of infantile cholestasis.

BACKGROUND: Infantile cholestasis (IC) is defined as an impairment of bile production or flow occurring in the first months of life. The diagnostic approach in IC is challenging since the differential diagnosis is broad. METHODS: We retrospectively evaluated 91 cholestatic infants referred to our department from 2014 to 2019. Patients with cholestasis underwent a complete IC diagnostic work-up including quantification of plasma oxysterols 7-ketocholesterol (7-KC) and cholestan-3ß,5α,6ß-triol (C-Triol). RESULTS: Oxysterols concentrations were mildly elevated in IC compared to control population. 7-KC and C-Triol plasma levels presented a linear relationship between them and with Spleen-Z score. Patients with NP-C showed the highest concentrations of both oxysterols compared with other etiologies of IC. Excluding NP-C patients, oxysterols concentrations were similar among all other etiological groups with no correlations found between them and the levels of cholesterol and bilirubin. ROC analysis identified AUCs of 1.0 for both oxysterols in predicting NP-C. CONCLUSION: Infants with IC should undergo a stepwise evaluation in which detailed clinical and deep analytical assessments are the main crossroads. Plasma oxysterols, a simple, reliable, and convenient diagnostic test should be included in the first steps of the diagnostic process in IC.

Urinary and serum oxysterols in children: developmental pattern and potential biomarker for pediatric liver disease.

Few reports describe oxysterols in healthy children or in children with liver disease. We aimed to determine whether developmental changes in urinary and serum oxysterols occur during childhood, and to assess whether oxysterols might be biomarkers for pediatric liver disease. Healthy children enrolled as subjects (36 and 35 for urine and serum analysis, respectively) included neonates, infants, preschoolers, and school-age children, studied along with 14 healthy adults and 8 children with liver disease. We quantitated 7 oxysterols including 4ß-, 20(S)-, 22(S)-, 22(R)-, 24(S)-, 25-, and 27-hydroxycholesterol using liquid chromatography/electrospray ionization-tandem mass spectrometry. Urinary total oxysterols were significantly greater in neonates than in infants (P < 0.05), preschoolers (P < 0.001), school-age children (P < 0.001), or adults (P < 0.001), declining with age. Serum total oxysterols in neonates were significantly lower than in infants (P < 0.05), preschoolers (P < 0.001), school-age children (P < 0.05), or adults (P < 0.01). Compared with healthy children, total oxysterols and 24(S)-hydroxycholesterol in liver disease were significantly increased in both urine (P < 0.001 and P < 0.001, respectively) and serum (P < 0.001 and P < 0.05, respectively). Oxysterols in liver disease, particularly 24(S)-hydroxycholesterol, were greater in urine than serum. Oxysterols change developmentally and might serve as a biomarker for pediatric liver disease. To our knowledge, this is the first such report.

Cholestane-3ß, 5α, 6ß-triol: Further insights into the performance of this oxysterol in diagnosis of Niemann-Pick disease type C.

In recent years the oxysterol species cholestane-3ß, 5α, 6ß-triol (C-triol) has found application as a diagnostic biomarker for Niemann-Pick disease type C. Other studies have described increased C-triol in patients with Niemann-Pick disease type A/B and milder increases in lysosomal acid lipase deficiency (LALD), whereas they note normal C-triol levels in Smith-Lemli-Opitz syndrome (SLOS) and familial hypercholesterolaemia (FH) patients. Herein, we review data collected in our laboratory during method evaluation along with 5 years of routine analysis and present findings which differ from those reported by other groups with respect to LALD, SLOS and FH in particular, whilst providing further evidence regarding the clinical sensitivity and specificity of this biomarker, which are difficult to accurately assess. All of our Wolman disease (severe LALD) patients have demonstrated gross elevations of C-triol at diagnosis, with reduction to normal levels after induction of enzyme replacement therapy. In diagnostic specimens from SLOS patients we observed very low or undetectable C-triol levels whereas in post-therapeutic SLOS patients demonstrated normalised levels; we also describe a homozygous FH patient in which C-triol is significantly elevated. Upon investigation, we found that C-triol was formed artefactually from cholesterol during our sample preparation, i.e. this is a false positive of analytical origin; at present it is unclear whether similar effects occur during sample preparation in other laboratories. Our data demonstrates clinical sensitivity of 100% during routine application to diagnostic specimens; this is in keeping with other estimates, yet in a small proportion of patients diagnosed prior to C-triol measurement, either by Filipin staining of fibroblasts or molecular genetics, we have observed normal C-triol concentrations. Clinical specificity of C-triol alone is 93.4% and 95.3% when performed in conjunction with lysosomal enzymology. These performance statistics are very similar to those achieved with Filipin staining of cultured fibroblasts in the 5 years preceding introduction of C-triol to routine use in our laboratory. It is increasingly apparent to us that although this analyte is a very useful addition to the diagnostic tools available for NPC, with considerable advantages over more invasive and time-consuming methods, the interpretation of results is complex and should be undertaken only in light of clinical details and results of other analyses including enzymology for lysosomal acid lipase and acid sphingomyelinase.

Impaired bile acid metabolism with defectives of mitochondrial-tRNA taurine modification and bile acid taurine conjugation in the taurine depleted cats.

Taurine that conjugates with bile acid (BA) and mitochondrial-tRNA (mt-tRNA) is a conditional essential amino acid in humans, similarly to cats. To better understand the influence of acquired depletion of taurine on BA metabolism, the profiling of BAs and its intermediates, BA metabolism-enzyme expression, and taurine modified mt-tRNAs were evaluated in the taurine deficient diet-supplemented cats. In the taurine depleted cats, taurine-conjugated bile acids in bile and taurine-modified mt-tRNA in liver were significantly decreased, whereas unconjugated BA in serum was markedly increased. Impaired bile acid metabolism in the liver was induced accompanied with the decreases of mitochondrial cholesterol 27-hydroxylase expression and mitochondrial activity. Consequently, total bile acid concentration in bile was significantly decreased by the low activity of mitochondrial bile acid synthesis. These results implied that the insufficient dietary taurine intake causes impaired bile acid metabolism, and in turn, a risk for the various diseases similar to the mitochondrial diseases would be enhanced.

Evaluation of oxysterol levels of patients with silicosis by LC-MS/MS method.

Silicosis is one of the prolonged and irreversible occupational diseases. Crystalline silica dust, which has been linked with silicosis, occurs in different industrial areas such as constructions, ceramic, quarry, and pottery. There are significant numbers of newly diagnosed cases every year in Turkey. Patients with silicosis suffer from inflammatory respiratory disorders and silicosis-related complications such as rheumatoid arthritis, systemic sclerosis, and vasculitis. Oxysterols are defined as 27-carbon intermediates or end products of cholesterol. They are also implicated in the etiology of disease states such as atherosclerosis, neurodegenerative, and inflammatory diseases. The aim of the study is to evaluate cholesterol oxidation products in the patients with silicosis and determination of sphingosine-1-phosphate (S1P) levels which is a sphingolipid metabolite. In addition to these parameters, it is aimed to determine the possible lipid peroxidation by different parameters. For this purpose, blood samples and urine were collected from 47 patients and 30 healthy individual with their consents. In order to evaluate oxysterols, 7-ketocholesterol and cholestan 3ß,5α,6ß-triol levels were measured by LC-MS/MS method. The measured levels of 7-KC were 0.101 ± 0.005 µmol/l in patient and 0.050 ± 0.003 µmol/l in control plasma samples. Triol levels were measured as 0.038 ± 0.005 µmol/l in patient group and 0.033 ± 0.004 µmol/l in control group (p < 0.001). In addition, lipid peroxidation products were measured by human-8-isoprostane, human-4-hydroxynonenal (4-HNE), and human malondialdehyde (MDA) ELISA kits. The measured levels of HNE in the patient and control groups were 735.14 ± 288.80 pg/ml and 595.72 ± 108.62 pg/ml in plasma and 606.02 + 118.23 pg/ml and 531.84 + 107.18 pg/ml in urine, respectively (p < 0.05). F2-iP results of patients and controls were 450.0 + 101.40 pg/dl and 386.9 + 112.7 pg/ml for urine and 432.7 ± 188,8 pg/dl and 321.9 ± 69.4 pg/dl for plasma, respectively (p < 0.05). MDA levels of plasma were measured as 44.1 ± 14.6 nmol/ml in the patient and 31.9 ± 10.5 nmol/ml in the control (p < 0.05). Levels of MDA for urine samples were 30.15 + 5.06 nmol/ml and 25.15 + 6.07 nmol/ml in patients and controls, respectively (p < 0.05). S1P levels were decreased in patients compared to control group (49.05 ± 10.87 and 67.57 ± 16.25, p < 0.001). The results not only indicate a correlation between cholesterol oxidation, lipid peroxidation, and silicosis, but also provide better understanding of the role of the lipids in the mechanism of this inflammatory disease.

Zinc, copper, and oxysterol levels in patients with type 1 and type 2 diabetes mellitus.

BACKGROUND: The present study has the objective to assess the zinc (Zn), copper (Cu), and oxysterols plasma levels in type 1 (DM1) (n = 26) and type 2 (DM2) (n = 80) diabetes patients, as compared to healthy controls (n = 71), in order to testify whether metal levels may have a significant impact on the association between oxysterols and diabetes. METHODS: Plasma trace elements and plasma oxysterols were assessed using atomic absorption spectrometry and LC-MS/MS, respectively. Lifestyle, smoking status, alcohol intake, and drug usage, as well as microvascular complications, were also monitored and reported. RESULTS: The obtained data demonstrated that both DM1 and DM2 patients were characterized by significantly elevated HbA1c, FBG, TC, LDL-C, VLDL-C, and TG levels as compared to controls. Plasma Zn levels and Zn/Cu ratio in DM1 and DM2 patients were about 3- and 2-fold lower than controls. No significant differences in plasma Cu levels were reported. The 7-ketocholesterol (7-kchol) levels in DM1 and DM2 patients exceeded these values in healthy individuals by 2.5 and 5-fold, respectively. Similarly, cholestan-3ß, 5α, 6ß-triol (chol-triol) levels were more than 3- and 6-fold higher when compared to the respective values in non-diabetic controls. In regression models decreased plasma Zn and elevated oxysterol levels were significantly associated with HbA1c and fasting plasma glucose levels, after adjustment for anthropometric and clinical variables, as well as routine biochemical markers. CONCLUSIONS: Plasma Zn concentration is inversely associated with both 7-kchol and chol-triol levels. Assessment of Zn and oxysterol levels may be used both for risk assessment and as targets for the treatment of diabetes mellitus.

Plasma oxysterol levels in luminal subtype breast cancer patients are associated with clinical data.

Oxygenated metabolites of cholesterol (oxysterols) have been previously demonstrated to contribute to progression of various cancers and to modulate resistance to breast cancer endocrine therapy in vitro. We measured prognostic roles of circulating levels of seven major oxysterols in the progression of luminal subtype breast carcinoma. Liquid chromatography coupled with tandem mass spectrometry was used for determination of levels of non-esterified 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, cholesterol-5ß,6ß-epoxide, and cholestane-3ß,5α,6ß-triol in plasma samples collected from patients (n = 58) before surgical removal of tumors. Oxysterol levels were then associated with clinical data of patients. All oxysterols except cholesterol-5α,6α-epoxide were detected in patient plasma samples. Circulating levels of 7α-hydroxycholesterol and 27-hydroxycholesterol were significantly lower in patients with small tumors (pT1) and cholesterol-5ß,6ß-epoxide and cholestane-3ß,5α,6ß-triol were lower in patients with stage IA disease compared to larger tumors or more advanced stages. Patients with higher than median cholestane-3ß,5α,6ß-triol levels had significantly worse disease-free survival than patients with lower levels (p = 0.037 for all patients and p = 0.015 for subgroup treated only with tamoxifen). In conclusion, this study shows, for the first time, that circulating levels of oxysterols, especially cholestane-3ß,5α,6ß-triol, may have prognostic roles in patients with luminal subtype breast cancer.

Formation and metabolism of oxysterols and cholestenoic acids found in the mouse circulation: Lessons learnt from deuterium-enrichment experiments and the CYP46A1 transgenic mouse.

While the presence and abundance of the major oxysterols and cholestenoic acids in the circulation is well established, minor cholesterol metabolites may also have biological importance and be of value to investigate. In this study by observing the metabolism of deuterium-labelled cholesterol in the pdgfbret/ret mouse, a mouse model with increased vascular permeability in brain, and by studying the sterol content of plasma from the CYP46A1 transgenic mouse overexpressing the human cholesterol 24S-hydroxylase enzyme we have been able to identify a number of minor cholesterol metabolites found in the circulation, make approximate-quantitative measurements and postulate pathways for their formation. These "proof of principle" data may have relevance when using mouse models to mimic human disease and in respect of the increasing possibility of treating human neurodegenerative diseases with pharmaceuticals designed to enhance the activity of CYP46A1 or by adeno-associated virus delivery of CYP46A1.

Antiviral oxysterols are present in human milk at diverse stages of lactation.

Oxysterols are cholesterol oxidation derivatives. Those containing an additional hydroxyl group on the side chain of the cholesterol molecule result from a physiological enzymatic synthesis and include the majority of oxysterols present in the circulation. Among these, 25-hydroxycholesterol (25OHC) and 27-hydroxycholesterol (27OHC) are characterized by a broad antiviral activity and are now considered involved in the innate immune response against viruses. Despite the emerging role of these sterols in the innate antiviral defences, no data are available on their presence in human breast milk (BM) to date. In this study, we investigated the content of oxysterols of enzymatic synthesis in BM of twelve donor mothers at different stages of lactation (i.e. in colostrum, transitional milk, and mature milk) by gas chromatography-mass spectrometry analysis. The side-chain oxysterols 25OHC, 27OHC, and 24S-hydroxycholesterol (24SOHC) were actually present in BM in all stages of lactation, but the concentration of 27OHC showed a remarkable peak in colostrum. Antiviral assays revealed that all the colostrum samples contained 27OHC concentrations that were active in vitro against two relevant pediatric viral pathogens: the human rotavirus and the human rhinovirus. Overall, this study discloses new antiviral components of BM and suggests a passive transfer of these protective factors to the infant via breastfeeding, especially in the first few days of lactation.

Oxysterol/chitotriosidase based selective screening for Niemann-Pick type C in infantile cholestasis syndrome patients.

BACKGROUND: Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disease (1 per 100 000 newborns) caused by NPC proteins impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. To date the NP-C diagnostics is usually based on cholesterol detection in fibroblasts using an invasive and time-consuming Filipin staining and we need more arguments to widely introduce oxysterols as a biomarkers in NP-C. METHODS: Insofar as NP-C represents about 8% of all infant cholestases, in this prospective observational study we tried to re-assess the specificity plasma oxysterol and chitotriosidase as a biochemical screening markers of NP-C in children with cholestasis syndrome of unknown origin. For 108 patients (aged from 2 weeks to 7 years) the levels of cholestane-3ß,5α,6ß-triol (C-triol) and chitotriosidase (ChT) were measured. For patients with elevated C-triol and/or ChT the NPC1 and NPC2 genes were Sanger-sequenced and 47 additional genes (from the custom liver damage panel) were NGS-sequenced. RESULTS: Increased C-triol level (> 50 ng/ml) was detected in 4 (of 108) infants with cholestasis syndrome of unknown origin, with following molecular genetic NP-C diagnosis for one patient. Plasma cholesterol significantly correlates with C-triol (p < 0.05). NGS of high C-triol infants identified three patients with mutations in JAG1 (Alagille syndrome) and ABCB11 (Byler disease) genes. Increased ChT activity was detected in 8 (of 108) patients with various aetiologies, including NP-C, Byler disease and biliary atresia. CONCLUSION: Combined analysis of ChT activity and C-triol levels is an effective method for identifying NP-C.

Oxysterols and apolipoproteins in multiple sclerosis: a 5 year follow-up study.

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.

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.

Quantification of Oxysterol Nuclear Receptor Ligands by LC/MS/MS.

Oxidative derivatives of cholesterol such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and (25S),26-hydroxycholesterol are endogenous ligands for the liver X receptors (LXRα and LXRß). The LXRs are nuclear hormone receptors known as "intracellular cholesterol sensors" because of their ability to bind to and be activated by oxysterols at circulating concentrations. Oxysterols are expressed in a tissue-specific manner and are generally at least 104 to 106-fold less abundant than cholesterol. Thus, the extraction and measurement of oxysterols from plasma and tissues are facilitated by the removal of bulk sterols by solid phase extraction prior to quantitative analysis by mass spectrometry. In this chapter we describe step by step methods for extracting and quantitating oxysterols from biological samples using electrospray ionization LC/MS/MS.

Oxysterol species: reliable markers of oxidative stress in diabetes mellitus.

PURPOSE: To assess the plasma oxysterol species 7-ketocholesterol (7-Kchol) and cholestane-3ß,5α,6ß-triol (chol-triol) as biomarkers of oxidative stress in type 1 and type 2 diabetes mellitus (DM). METHODS: In total, 26 type 1 and 80 type 2 diabetes patients, along with 205 age- and gender-matched healthy controls, were included in this study. Oxysterols were quantified by liquid chromatography coupled with tandem mass spectrometry and N,N-dimethylglycine derivatization. Correlations between oxysterols and clinical/biochemical characteristics of the diabetes patients, and factors affecting 7-Kchol and chol-triol, were also determined. RESULTS: Plasma 7-Kchol and chol-triol levels were significantly higher in type 1 and type 2 diabetes patients compared to healthy controls (P < 0.001). Significant positive correlations were observed between oxysterol levels and levels of glycated hemoglobin (HbA1c), glucose, serum total cholesterol, low-density lipoprotein, very-low-density lipoprotein, and triglycerides, as well as the number of coronary risk factors. Statins, oral hypoglycemic agents, and antihypertensive agents reduced the levels of oxysterols in type 2 diabetes patients. Statin use, HbA1c levels, and the number of coronary risk factors accounted for 98.8% of the changes in 7-Kchol levels, and total cholesterol, smoking status, and the number of coronary risk factors accounted for 77.3% of the changes in chol-triol levels in type 2 diabetes patients. CONCLUSIONS: Plasma oxysterol levels in DM, and particularly type 2 DM, may yield complementary information regarding oxidative stress for the clinical follow-up of diabetes patients, especially those with coronary risk factors.

Colitis Alters Oxysterol Metabolism and is Affected by 4ß-Hydroxycholesterol Administration.

BACKGROUND AND AIMS: Inflammatory bowel diseases [IBD] represent a challenging health issue with a complex aetiology involving genetic and environmental parameters. Although our understanding of the pathophysiology of IBD has improved, much remains to be explored. In this context, bioactive lipids, more specifically oxysterols, i.e. oxygenated derivatives of cholesterol, represent an interesting avenue to investigate. Indeed, oxysterols or their receptors are involved in inflammation and immune regulation. Therefore, we set out to study the oxysterome in IBD. METHODS: We used both high-performance liquid chromatograph/mass spectroscopy and molecular biology tools to quantify oxysterol levels and the expression of their metabolic enzymes in several models of murine colitis [both acute and chronic], as well as in colon biopsies from patients with Crohn's disease and ulcerative colitis. RESULTS: We found that the oxysterome is altered in IBD, in both acute and chronic murine models as well as in human IBD. Two of the oxysterols quantified, 4ß-hydroxycholesterol and 25-hydroxycholesterol, were consistently altered in all our models and therefore could be of interest in this context. Hence, we administered them to mice with colitis. While 25-hydroxycholesterol had no effect, 4ß-hydroxycholesterol worsened colon inflammation. CONCLUSIONS: Our study addresses the potential involvement of oxysterols in colitis and clearly points towards an active role as well as a clinical relevance for these bioactive lipids.

Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort. A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 µL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8-250 ng mL-1 for free hydroxycholesterols, 0.2-10 ng mL-1 for dihydroxycholesterols, 0.2-500 ng mL-1 for bile acids and 16-2000 µg mL-1 for cholesterol with acceptable accuracy and precision. In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained. In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.