Allosteric Modulation of NMDARs Reverses Patients' Autoantibody Effects in Mice.

BACKGROUND AND OBJECTIVES: To demonstrate that an analog (SGE-301) of a brain-derived cholesterol metabolite, 24(S)-hydroxycholesterol, which is a selective positive allosteric modulator (PAM) of NMDA receptors (NMDARs), is able to reverse the memory and synaptic alterations caused by CSF from patients with anti-NMDAR encephalitis in an animal model of passive transfer of antibodies. METHODS: Four groups of mice received (days 1-14) patients' or controls' CSF via osmotic pumps connected to the cerebroventricular system and from day 11 were treated with daily subcutaneous injections of SGE-301 or vehicle (no drug). Visuospatial memory, locomotor activity (LA), synaptic NMDAR cluster density, hippocampal long-term potentiation (LTP), and paired-pulse facilitation (PPF) were assessed on days 10, 13, 18, and 26 using reported techniques. RESULTS: On day 10, mice infused with patients' CSF, but not controls' CSF, presented a significant visuospatial memory deficit, reduction of NMDAR clusters, and impairment of LTP, whereas LA and PPF were unaffected. These alterations persisted until day 18, the time of maximal deficits in this model. In contrast, mice that received patients' CSF but from day 11 were treated with SGE-301 showed memory recovery (day 13), and on day 18, all paradigms (memory, NMDAR clusters, and LTP) had reversed to values similar to those of controls. On day 26, no differences were observed among experimental groups. DISCUSSION: An oxysterol biology-based PAM of NMDARs is able to reverse the synaptic and memory deficits caused by CSF from patients with anti-NMDAR encephalitis. These findings suggest a novel adjuvant treatment approach that deserves future clinical evaluation.

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism.

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2 It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.

Identification of Correlative Shifts in Indices of Brain Cholesterol Metabolism in the C57BL6/Mecp2tm1.1Bird Mouse, a Model for Rett Syndrome.

Rett syndrome (RS) is a pervasive neurodevelopmental disorder resulting from loss-of-function mutations in the X-linked gene methyl-Cpg-binding protein 2 (MECP2). Using a well-defined model for RS, the C57BL6/Mecp2tm1.1Bird mouse, we have previously found a moderate but persistently lower rate of cholesterol synthesis, measured in vivo, in the brains of Mecp2-/y mice, starting from about the third week after birth. There was no genotypic difference in the total cholesterol concentration throughout the brain at any age. This raised the question of whether the lower rate of cholesterol synthesis in the mutants was balanced by a fall in the rate at which cholesterol was converted via cholesterol 24-hydroxylase (Cyp46A1) to 24S-hydroxycholesterol (24S-OHC), the principal route through which cholesterol is ordinarily removed from the brain. Here, we show that while there were no genotypic differences in the concentrations in plasma and liver of three cholesterol precursors (lanosterol, lathosterol, and desmosterol), two plant sterols (sitosterol and campesterol), and two oxysterols (27-hydroxycholesterol [27-OHC] and 24S-OHC), the brains of the Mecp2 -/y mice had significantly lower concentrations of all three cholesterol precursors, campesterol, and both oxysterols, with the level of 24S-OHC being ~20% less than in their Mecp2 +/y controls. Together, these data suggest that coordinated regulation of cholesterol synthesis and catabolism in the central nervous system is maintained in this model for RS. Furthermore, we speculate that the adaptive changes in these two pathways conceivably resulted from a shift in the permeability of the blood-brain barrier as implied by the significantly lower campesterol and 27-OHC concentrations in the brains of the Mecp2-/y mice.

Reproducibility of serum oxysterols and lanosterol among postmenopausal women: Results from EPIC-Heidelberg.

BACKGROUND: Circulating oxysterols have been proposed as biological markers of disease risk. However, within-person reproducibility of circulating oxysterols over time is not well established. METHODS: We evaluated the one-year reproducibility of 11 oxysterols and lanosterol among 30 postmenopausal women with repeat blood samples in the European Prospective Investigation into Cancer and Nutrition (EPIC) - Heidelberg, Germany cohort. Liquid chromatography-mass spectrometry (LC/MS) was performed to quantify serum concentrations of 22R-hydroxycholesterol, 25-hydroxycholesterol, 24S-hydroxycholesterol, 27-hydroxycholesterol, 22S-hydroxycholeterol, 24,25-epoxycholesterol, 5α,6ß-dihydroxycholestanol, 7α-hydroxycholesterol, 5ß,6ß-epoxycholesterol, 5α,6α-epoxycholesterol, 24-dihydrolanosterol, and lanosterol. We evaluated Spearman correlations and intraclass correlation coefficients (ICCs) between quantifiable concentrations measured in repeat samples taken one-year apart to estimate within-person reproducibility. RESULTS: Spearman correlations (ICCs) over one year ranged from 0 (ICC=0.10) for 5ß,6ß-epoxycholesterol and 0.10 (ICC=0.20) for 5α,6α-epoxycholesterol, representing low within-person stability, to 0.81 (ICC=0.75) for 27-hydroxycholesterol and 0.86 (ICC=0.91) for 24S-hydroxycholesterol, representing relatively high within-person stability. Correlations between oxysterols and lanosterol ranged from 0.01 between 24S-hydroxycholesterol and lanosterol to 0.70 between 5α,6α-epoxycholesterol and 5ß,6ß-epoxycholesterol. CONCLUSIONS: Our results demonstrate that for 27-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol and lanosterol, a single serum measurement can reliably estimate average levels over a one-year period. Circulating oxysterols are of increasing interest in epidemiologic studies of chronic disease risk including cancer and cardiovascular disease. Our data suggest that within-person stability of oxysterols differs depending on the individual oxysterol evaluated. We identified four oxysterols and lanosterol as stable over time to inform the use of circulating oxysterols in epidemiologic studies.

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol.

Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.

Breast cancer and (25R)-26-hydroxycholesterol.

(25R)-26-Hydroxycholesterol (27-hydroxycholesterol) has been found to accumulate in breast tissue and to stimulate tumor growth via the estrogen receptor. Although most tissues express CYP27A1, the highest levels are in macrophages and most attention had been given to the production of 27-hydroxycholesterol in sub-endothelial macrophages as part of reverse cholesterol transport. In view of the newly identified biologic activity, it is important to consider the determinants of the levels of 27-hydroxycholesterol in macrophages that infiltrate breast tissue. Among these determinants are the oxysterol binding proteins expressed in macrophages, the level of expression of CYP7B1, the oxysterol 7 alpha hydroxylase that generates an inactive triol, and further oxidation of 27-hydroxycholestrol to the C27 acid by multifunctional CYP27A1. Transport of 27-hydroxycholesterol from macrophages to plasma is HDL-associated. In many tissues the ratio of 27-hydroxycholesterol to cholesterol (ng/µg) is higher than that in plasma. Tamoxifen, an effective estrogen receptor antagonist that prevents breast cancer, also has the biologic property of blocking several steps in the lanosterol to cholesterol metabolic pathway. In genetically disposed women, tamoxifen may increase the amount of 27-hydroxycholesterol in breast tissue.

Lysophosphatidylcholine promotes SREBP-2 activation via rapid cholesterol efflux and SREBP-2-independent cytokine release in human endothelial cells.

Lysophosphatidylcholine (LPC) and oxysterols which are major components in oxidized low-density lipoprotein have been shown to possess an opposite effect on the expression of sterol regulatory element-binding protein-2 (SREBP-2) target genes in endothelial cells. In this study, we aimed at elucidating the mechanisms of activation of SREBP-2 by LPC and evaluating the effects of LPC and 25-hydroxycholesterol (25-HC) on the release of inflammatory cytokines. Human umbilical vein endothelial cells were treated with LPC or oxysterols including 25-HC. LPC activated SREBP-2 within 15 min, resulting in induction of expression of SREBP-2 target genes which were involved in intracellular cholesterol homeostasis. The rapid activation of SREBP-2 was caused by enhanced efflux of intracellular cholesterol, which was evaluated using (14)C-acetate. The LPC-induced activation of SREBP-2 was inhibited by addition of 25-HC. In contrast, both LPC and 25-HC increased release of interleukin-6 (IL-6) and IL-8, respectively and additively. In conclusion, LPC activated SREBP-2 via enhancement of cholesterol efflux, which was suppressed by 25-HC. The release of inflammatory cytokines such as IL-6 and IL-8 in endothelial cells was SREBP-2-independent. LPC and 25-HC may act competitively in cholesterol homeostasis but additively in inflammatory cytokine release.

Pharmacokinetic and pharmacogenomic modelling of the CYP3A activity marker 4ß-hydroxycholesterol during efavirenz treatment and efavirenz/rifampicin co-treatment.

OBJECTIVES: To assess the effect of the major efavirenz metabolizing enzyme (CYP2B6) genotype and the effects of rifampicin co-treatment on induction of CYP3A by efavirenz. PATIENTS AND METHODS: Two study arms (arm 1, n = 41 and arm 2, n = 21) were recruited into this study. In arm 1, cholesterol and 4ß-hydroxycholesterol were measured in HIV treatment-naive patients at baseline and then at 4 and 16 weeks after initiation of efavirenz-based antiretroviral therapy. In arm 2, cholesterol and 4ß-hydroxycholesterol were measured among patients taking efavirenz during rifampicin-based tuberculosis (TB) treatment (efavirenz/rifampicin) just before completion of TB treatment and then serially following completion of TB treatment (efavirenz alone). Non-linear mixed-effect modelling was performed. RESULTS: A one-compartment, enzyme turnover model described 4ß-hydroxycholesterol kinetics adequately. Efavirenz treatment in arm 1 resulted in 1.74 (relative standard error = 15%), 3.3 (relative standard error = 33.1%) and 4.0 (relative standard error = 37.1%) average fold induction of CYP3A for extensive (CYP2B6*1/*1), intermediate (CYP2B6*1/*6) and slow (CYP2B6*6/*6) efavirenz metabolizers, respectively. The rate constant of 4ß-hydroxycholesterol formation [mean (95% CI)] just before completion of TB treatment [efavirenz/rifampicin co-treatment, 7.40 × 10(-7) h(-1) (5.5 × 10(-7)-1.0 × 10(-6))] was significantly higher than that calculated 8 weeks after completion [efavirenz alone, 4.50 × 10(-7) h(-1) (4.40 × 10(-7)-4.52 × 10(-7))]. The CYP3A induction dropped to 62% of its maximum by week 8 of completion. CONCLUSIONS: Our results indicate that efavirenz induction of CYP3A is influenced by CYP2B6 genetic polymorphisms and that efavirenz/rifampicin co-treatment results in higher induction than efavirenz alone.

Identification of novel regulatory cholesterol metabolite, 5-cholesten, 3ß,25-diol, disulfate.

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3ß, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.

Sample preparation: a critical step in the analysis of cholesterol oxidation products.

In recent years, cholesterol oxidation products (COPs) have drawn scientific interest, particularly due to their implications on human health. A big number of these compounds have been demonstrated to be cytotoxic, mutagenic, and carcinogenic. The main source of COPs is through diet, and particularly from the consumption of cholesterol-rich foods. This raises questions about the safety of consumers, and it suggests the necessity for the development of a sensitive and a reliable analytical method in order to identify and quantify these components in food samples. Sample preparation is a necessary step in the analysis of COPs in order to eliminate interferences and increase sensitivity. Numerous publications have, over the years, reported the use of different methods for the extraction and purification of COPs. However, no method has, so far, been established as a routine method for the analysis of COPs in foods. Therefore, it was considered important to overview different sample preparation procedures and evaluate the different preparative parameters, such as time of saponification, the type of organic solvents for fat extraction, the stationary phase in solid phase extraction, etc., according to recovery, precision and simplicity.

Profiling of oxidized lipid products of marine fish under acute oxidative stress.

Free radical products including reactive oxygen species are potent to oxidize lipids and reliable measurements have been established mostly in human and rodent. To date, robust biomarkers were not used to assess the peroxidation in marine fish. The changes of oxidized lipid products from polyunsaturated fatty acids and cholesterol were assessed after exposure of H(2)O(2) to fish (medaka). Oxidized lipid products released by free radical reaction (F(2)-isoprostanes and metabolites, F(3)-isoprostanes, neuroprostanes, 7-ketocholesterol, 7ß-hydroxycholesterol), by lipoxygenase enzymes (5(S)-, 8(S)-, 12(S)- and 15(S)-HETE, and resolvin D1) and by cytochrome P450 (9(S)-, 11(S)- and 20-HETE, and 27-hydroxycholestrol) were measured in fish muscle using LC/MS/MS. Arachidonate, docosahexaenoate, eicosapentaenoate and cholesterol levels, and antioxidant enzymes activity (catalase, SOD and gluthathione reductase) measurement were also determined. Activity of antioxidant enzymes especially catalase were elevated in presence of H(2)O(2) however longer exposure time suppressed the antioxidant activities. Arachidonate, docosahexaenoate, eicosapentaenoate and cholesterol levels were reduced in presence of H(2)O(2) and oxidized lipid products (isoprostanes, neuroprostanes 5(S)-HETE, 20-HETE, 7-ketocholesterol, 27-hydroxycholesterol and resolvin D1) were rapidly released in the fish muscle. This study validates oxidized lipid products, noticeably isoprostanes are measurable in marine fish muscle and should be considered when assessing oxidative stress especially due to exogenous factors.

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS.

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C(21)-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time.

High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry.

Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography-tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (~25 pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC-MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC-MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin- heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.

Increased 25-hydroxycholesterol concentrations in the lungs of patients with chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: 25-Hydroxycholesterol (25-HC) is produced from cholesterol by the enzyme cholesterol 25-hydroxylase and is associated with atherosclerosis of vessels. Recently, 25-HC was reported to cause inflammation in various types of tissues. The aim of this study was to assess the production of 25-HC in the airways and to elucidate the role of 25-HC in neutrophil infiltration in the airways of patients with chronic obstructive pulmonary disease (COPD). METHODS: Eleven control never-smokers, six control ex-smokers without COPD and 13 COPD patients participated in the lung tissue study. The expression of cholesterol 25-hydroxylase in the lung was investigated. Twelve control subjects and 17 patients with COPD also participated in the sputum study. The concentrations of 25-HC in sputum were quantified by liquid chromatography/mass spectrometry/mass spectrometry analysis. To elucidate the role of 25-HC in neutrophilic inflammation of the airways, the correlation between 25-HC levels and neutrophil counts in sputum was investigated. RESULTS: The expression of cholesterol 25-hydroxylase was significantly enhanced in lung tissue from COPD patients compared with that from control subjects. Cholesterol 25-hydroxylase was localized in alveolar macrophages and pneumocytes of COPD patients. The concentration of 25-HC in sputum was significantly increased in COPD patients and was inversely correlated with percent of predicted forced vital capacity, forced expiratory volume in 1 s and diffusing capacity of carbon monoxide. The concentrations of 25-HC in sputum were significantly correlated with sputum interleukin-8 levels and neutrophil counts. CONCLUSIONS: 25-HC production was enhanced in the airways of COPD patients and may play a role in neutrophilic inflammation.

Relative contribution of individual oxidized components in ox-LDL to inhibition on endothelium-dependent relaxation in rat aorta.

BACKGROUND AND AIM: Oxidized low-density lipoprotein (ox-LDL) causes atherosclerosis and endothelial dysfunction. No study up to the present date has examined the relative contribution of all the oxidized components in ox-LDL to inhibition on vascular function. Our aim was to investigate the effects of individual oxidized components at concentrations similar to those in ox-LDL on the impairment of endothelium-dependent relaxation in rat aorta. METHODS AND RESULTS: Rat thoracic aorta was pre-treated with lysophosphatidylcholine (LPC), cholesterol oxidized products (COPs), oxidized linoleic acid (ox-18:2) and oxidized linolenic acid (ox-18:3) at concentrations similar to those in human ox-LDL. Ox-LDL as a whole caused 61% inhibition while LPC, COPs and ox-18:2 at concentrations similar to those in ox-LDL caused 12%, 24% and 19% inhibition, respectively, on endothelium-dependent relaxation, suggesting that COPs produced the most adverse effect followed by ox-18:2 and LPC in an additional way. Three COPs including 7-ketocholesterol, 7α-hydroxycholesterol and 7ß-hydroxycholesterol showed inhibition on endothelium-dependent relaxation with E(max) being reduced to 79-87% compared with the control E(max) (95%). At Western blot analysis phosphorylation of eNOS at Ser1177 site and total eNOS were not altered by ox-LDL treatment, indicating that ox-LDL did not affect nitric oxide (NO) synthesis capacity. Ox-LDL might react directly with NO and lower NO bioavailability. CONCLUSION: The present study demonstrated the relative contribution of individual oxidized components in ox-LDL in the inhibition of endothelium-dependent relaxation in rat aorta. This inhibitory effect could be caused by the reduction of NO bioactivity.

Lipid and cholesterol oxidation in chicken meat are inhibited by sage but not by garlic.

UNLABELLED: The effects of the addition of sage and garlic in chicken meat on lipid and cholesterol oxidation, having as prooxidant factors the addition of salt, thermal treatment, and frozen storage, were evaluated. The content of unsaturated fatty acids did not change in the presence of sage; on the contrary, with garlic, the content of these fatty acids decreased after cooking and storage. Hexanal and pentanal contents were lower in patties containing sage, and higher in those with garlic. The 7-ketocholesterol was the cholesterol oxide found in higher amount in raw chicken on day 0, while the formation of 7ß- and 7α-hydroxycholesterol was verified only from day 30 on. Cooking and storage resulted in increase of total cholesterol oxides and decrease of α- and γ-tocopherol. Sage was effective in controlling lipid and cholesterol oxidation, minimizing the prooxidant effects of salt, cooking, and storage. However, garlic presented no effect as antioxidant and accelerated lipid oxidation. PRACTICAL APPLICATION: The addition of sage to chicken meat (0.1 g/100 g) is a good alternative to prevent and delay the formation of compounds derived from lipid oxidation that are responsible for off-flavors and loss of nutritional quality during long-term frozen storage. Care must be taken when using garlic to seasoning chicken meat products, such as hamburgers and meatballs, especially cooked or precooked due to its potential to promote lipid oxidation and consequently raising the risk of having the product rejected by the consumer.

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using ¹5N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.

7-Ketocholesterol and 7-hydroxycholesterol in pork meat and its gravy thermally treated without additives and in the presence of onion and garlic.

The effect of onion and garlic on the formation of two cholesterol oxidation products (COPs): 7-ketocholesterol and 7-hydroxycholesterol was evaluated by comparing their concentrations in meat and gravy samples obtained from three pork dishes prepared in the presence and absence of these flavourings. The concentration of these compounds in meat samples was between 82.4 and 1331.6 ng/g of cooked meat. Gravies contained lower amounts: from 18.3 to 45.6 ng/g of cooked meat. The addition of onion (30 g/100g of meat) caused a decrease in 7-ketocholesterol and 7-hydroxycholesterol concentrations in all of the investigated pork dishes by 9.5-79%, whilst the addition of 15 g of garlic to 100g of meat lowered the concentration by 17 to 88%. The greatest decrease was found in grilled minced chops. The quantitative assessment of 7-ketocholesterol and 7-hydroxycholesterol was carried out by thin-layer chromatography with densitometric detection.

Determination of oxysterols in oxidatively modified low-density lipoprotein by semi-micro high-performance liquid chromatography with electrochemical detection.

A simple method for the determination of oxysterols was developed by semi-micro high-performance liquid chromatography with electrochemical detection (semi-micro HPLC-ECD). Semi-micro HPLC-ECD was established using a C30 microbore column, acetonitrile containing 50 mmol/L LiClO(4) as a mobile phase, and an applied potential at +2.8V versus Ag/AgCl. The current peak height was linearly related to the amount of sterol injected from 12.5 to 250 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.9% (n=6). This method was applied to the determination of seven oxysterols in oxidatively modified low-density lipoprotein (Ox-LDL). Oxysterols were determined with a recovery of more than 78.0% and an RSD of less than 2.9% (n=6) except for 7-ketocholesterol. 7-Ketocholesterol was determined as a sum of intact 7-ketocholesterol and its degradation product on saponification, cholesta-3,5-dien-7-one, with a recovery of 98.0% and an RSD of 2.5% (n=6). From these results, the current method enabled the simultaneous determination of seven oxysterols without any derivatization, providing a useful tool for the assessment of oxysterol contents in Ox-LDL.