27-Hydroxylation of oncosterone by CYP27A1 switches its activity from pro-tumor to anti-tumor.

Oncosterone (6-oxo-cholestane-3ß,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or ß- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3ß,5α,6ß-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3ß,26-diol), 27H-CT ((25R)-cholestane-3ß,5α,6ß,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3ß,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.

Replacement of soybean oil by Hermetia illucens larvae fat in broiler diets alters the breast muscle lipidome and reduces lipid oxidation of the breast muscle during heat-processing.

Replacement of soybean oil by insect fat from Hermetia illucens (HI) has been reported to increase the proportions of saturated fatty acids (SFA) and decrease those of polyunsaturated fatty acids (PUFA) in total lipids of breast and thigh meat in broilers. Since the susceptibility of meat to oxidation is strongly dependent on its PUFA content, the present study hypothesised that replacement of soybean oil by HI larvae fat in broiler diets reduces the formation of lipid oxidation products, including oxidation products of cholesterol and phytosterols, in heat-processed breast muscle of broilers. To test this hypothesis, 100 male, 1-day-old Cobb 500 broilers were assigned to three groups and fed three different nutrient adequate diets, which varied only in the fat source (group HI-0: 0% HI larvae fat and 5% soybean oil; group HI-2.5: 2.5% HI larvae fat and 2.5% soybean oil; group HI-5.0: 5.0% HI larvae fat and 0% soybean oil), in a three-phase feeding system for 35 days. While the growth performance of the broilers was not different, the absolute and relative breast muscle weights were higher in group HI-5.0 than in group HI-0 (p < 0.05). The proportions of C12:0, C14:0, C14:1, C16:0, C16:1 and total SFA were higher and those of C18:1, C18:2 n-6, C18:3 n-3 and total PUFA were lower in breast muscle total lipids of group HI-5.0 than in groups HI-2.5 and HI-0 (p < 0.05). Lipidomic analysis of breast muscle revealed that the concentration of triacylglycerols was 46% and 53% lower in groups HI-2.5 and HI-5.0, respectively, than in group HI-0 (p < 0.05), whereas all other lipid classes detected did not differ among groups. Concentrations of thiobarbituric acid-reactive substances, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and total cholesterol oxidation products in heat-processed breast muscle were lower in group HI-5.0 than in group HI-0 (p < 0.05). Concentrations of oxidation products of phytosterols in heat-processed breast muscle were generally much lower than those of cholesterol oxidation products and did not differ between the three groups of broilers. In conclusion, complete replacement of soybean oil with HI larvae fat in broiler diets strongly alters the fatty acid composition of breast muscle total lipids and reduce lipid oxidation of the breast muscle during heat-processing.

Increased cholesterol absorption is associated with In-stent-restenosis after stent implantation for stable coronary artery disease.

BACKGROUND AND AIMS: Blood cholesterol levels are regulated by competing mechanisms of cholesterol synthesis, absorption and excretion. Plant sterols are natural constituents of plants, are not synthesized in humans, and serve as markers for cholesterol absorption. Ezetimibe lowers the intestinal absorption of cholesterol and plant sterols. We analyzed the associations of differences in cholesterol metabolism, in particular increased cholesterol absorption, and the occurrence of in-stent restenosis (ISR) in patients with stable coronary artery disease. METHODS: Elective stent implantation of de novo stenosis was conducted in 59 patients (74.6 % males, 67.2 ± 9.6 years). Cholesterol and non-cholesterol sterols were quantified in serum samples by gas chromatography or mass spectrometry. ISR was assessed by optical coherence tomography (OCT) and quantitative angiography (QCA) after six months. RESULTS: Markers for cholesterol absorption (e.g. campesterol-to-cholesterol) were positively associated with ISR measured by QCA (%diameter stenosis, late lumen loss) and OCT (proliferation volume, %area stenosis), whereas markers for cholesterol synthesis (e.g. lathosterol-to-cholesterol) were negatively associated with ISR (%area stenosis: r = -0.271, p = 0.043). There was no association between ISR and total cholesterol, LDL, HDL, triglycerides. Markers for cholesterol absorption (e.g. campesterol-to-cholesterol) were significantly lower in ezetimibe-treated patients compared to patients on a statin only (1.29 ± 0.69 vs. 2.22 ± 1.23; p = 0.007). Combined lipid-lowering with ezetimibe plus statin reduced ISR compared to statin only (13.7 ± 10.4 vs. 22.5 ± 12.1 %diameter stenosis, p = 0.015). CONCLUSIONS: Differences in cholesterol metabolism, more specifically increased cholesterol absorption, are associated with ISR.

24(R, S)-Saringosterol - From artefact to a biological medical agent.

Enhancing the cholesterol turnover in the brain via activation of liver x receptors can restore memory in a mouse model for Alzheimer's disease. The edible Asian brown alga Sargassum fusiforme (Hijiki) contains high amounts of oxysterols such as (3ß, 24ξ)-stigmasta-5, 28-dien-3, 24-diol (24[R, S]-saringosterol) that are a potent liver x receptor agonists. We aimed to find native European seaweed species with contents of 24(R, S)-saringosterol that are comparable to those found in Sargassum fusiforme. Additionally, we hypothesize that seasonal variations modify the amount of 24(R, S)-saringosterol in seaweeds. Sterols and oxysterols were extracted with chloroform/methanol from various seaweed species harvested in the Eastern Scheldt in different seasons between October 2016 and September 2017. Identification and quantification of the lipids was performed by gas chromatography- mass spectrometry and gas chromatography- flame ionization detection. We confirmed that brown algae Undaria pinnatifida harvested in February and Sargassum muticum harvested in October contained the highest amounts of 24(R, S)-saringosterol (32.4 ± 15.25 µg/g, mean ± S.D. and 32.95 ± 2.91 µg/g, respectively) and its precursor fucosterol (1.48 ± 0.11 mg/g), higher than Sargassum fusiforme (20.94 ± 3.00 µg/g, mean ± S.D.), while Ascophyllum nodosum and Fucus vesiculosus and Fucus serratus contained amounts of 24(R, S)-saringosterol (22.09 ± 3.45 µg/g, 18.04 ± 0.52 µg/g and 19.47 ± 9.01 µg/g, mean ± S.D., respectively) comparable to Sargassum fusiforme. In other algae only minor amounts of these sterols were observed. The green algae Ulva lactuca contained only 0.29 mg/g fucosterol and 10.3 µg/g 24 (R, S)-saringosterol, while all investigated red algae did not contain any 24(R, S)-saringosterol or fucosterol. In the Eastern Scheldt algae harvested in September/October delivered the highest yield for 24(R, S)-saringosterol, with the exception of Undaria pinnatifida that showed the highest levels in February. We showed that exposure of lipid extracts of Ulva lactuca to sunlight at room temperature or in the presence of oxygen to UV-C light lead to the quantitative conversion of fucosterol into 24(R, S)-saringosterol. Exposing pure fucosterol to UV-light did not convert any fucosterol into 24(R, S)-saringosterol underscoring the requirement of seaweed constituents in the conversion of fucosterol into 24(R, S)-saringosterol. In conclusion, we showed that brown seaweeds harvested from the Eastern Scheldt contain amounts of 24(R, S)-saringosterol comparable to Sargassum fusiforme, varying per season and showing the highest amounts in spring. In accordance with these observations the amount of 24(R, S)-saringosterol in the brown seaweeds can be modulated by light.

Effects of supplementation of DL-methionine on tissue and plasma antioxidant status during heat-induced oxidative stress in broilers.

Exposure to high ambient temperature has been shown to impair growth performance and to cause oxidative stress in broilers. This study investigated the hypothesis that supplementation with methionine (Met) as DL-Met (DLM) more than the National Research Council recommendations improves growth performance and alleviates oxidative stress in broilers exposed to high ambient temperature. One-day-old male Cobb-500 broilers (n = 68) were allotted to 4 groups and phase-fed 3 basal diets during days 1 to 10, 11 to 21, and 22 to 35. One group was kept under thermoneutral temperature conditions and received the basal diets with Met + cysteine (Cys) concentrations according to recommendations of NRC. The other 3 groups were kept in a room with an increased ambient temperature from week 3 to 5 and were fed either the basal diet or the basal diets supplemented with 2 levels of DLM in which Met + Cys concentrations exceeded NRC recommendations by around 20% (group DLM1) and 40% (group DLM2), respectively. As expected, the broilers exposed to high ambient temperature showed a lower feed intake, lower body weight gains, a higher feed:gain ratio, and biochemical indications of oxidative stress in comparison to broilers kept under thermoneutral temperature conditions. Supplementation of DLM did not improve the growth performance in broilers exposed to high ambient temperature. However, the broilers supplemented with DLM had increased concentrations of glutathione in liver and breast muscle (groups DLM1 and DLM2), increased concentrations of tocopherols in the liver (group DLM2), and reduced concentrations of 7α-hydroxycholesterol and 7-ketocholesterol in heat-processed thigh muscle (groups DLM1 and DLM2) in comparison to the control group exposed to high ambient temperature. Concentrations of thiobarbituric acid-reactive substances and vitamin C in plasma, liver, and muscle were not different between the 3 groups exposed to heat stress. Nevertheless, the study shows that supplementation of DLM in slight excess of the Met concentration required for maximum growth performance improved the antioxidant status in tissues and reduced the susceptibility of muscle toward oxidation in heat-stressed broilers.

Effect of DL-Methionine Supplementation on Tissue and Plasma Antioxidant Status and Concentrations of Oxidation Products of Cholesterol and Phytosterols in Heat-Processed Thigh Muscle of Broilers.

In this study, the hypothesis that supplementation with methionine (Met) as DL-Met (DLM) in excess of the National Research Council (NRC) recommendations improves the antioxidant system in broilers was investigated. Day-old male Cobb-500 broilers (n = 72) were divided into three groups which were fed a control diet or diets supplemented with two levels of DLM in which the concentrations of Met + Cys exceeded the recommendations of NRC by 15-20% (group DLM 1) or 30-40% (group DLM 2), respectively. The three groups of broilers did not show differences in body weight gains, feed intake, and feed conversion ratio. However, broilers of groups DLM 1 and DLM 2 had higher concentrations of glutathione (GSH) in liver and thigh muscle and lower concentrations of cholesterol oxidation products (COPs) in heat-processed thigh muscle than broilers of the control group. Concentrations of several oxidation products of phytosterols in heat-processed thigh muscle were also reduced in groups DLM 1 and DLM 2; however, the concentration of total oxidation products of phytosterols was not different between the three groups. The study shows that DLM supplementation improved the antioxidant status due to an increased formation of GSH and reduced the formation of COPs during heat-processing in thigh muscle.

Serum Concentration of Plant Sterol Oxidation Products (POP) Compared to Cholesterol Oxidation Products (COP) after Intake of Oxidized Plant Sterols: A Randomised, Placebo-Controlled, Double-Blind Dose‒Response Pilot Study.

Plant sterols (PS) are oxidized to PS oxidation products (POP). This study quantified the change in serum POP compared to cholesterol oxidation products (COP) after the intake of increasing POP doses. This was a double-blind, randomized, placebo-controlled, dose‒response pilot study with healthy individuals in four groups (15 per group). The control group received products with no added PS or POP and treatment groups received daily 20-25 g margarine with added PS (mean 3 g/d) and two cookies (~28 g) for six weeks. Cookies delivered 8.7 (low-dose), 15.2 (medium-dose), or 37.2 (high-dose) mg/d POP. Fasting serum POP and COP were measured at the baseline, days 14, 28, and 42 in all participants and days 7, 21, and 35 in a subset. Sixty individuals completed the study; 52 were included in per protocol analysis. Serum POP increased with increasing POP intake and plateaued at dose >15 mg/d. Stabilized POP concentrations were (mean ± SD) 38.9 ± 6.9, 91.0 ± 27.9, 144.4 ± 37.9 and 203.0 ± 63.7 nmol/L, for control, low-, medium-, and high-dose POP groups, respectively. For all groups, the serum COP ranged from 213 to 262 nmol/L and the average POP/COP ratio was <1. Serum POP concentrations increased non-linearly, reaching stabilized concentrations in <7 days, and remained below COP concentrations after the intake of increasing POP doses.

Low serum lathosterol levels associate with fatal cardiovascular disease and excess all-cause mortality: a prospective cohort study.

IMPORTANCE: A more precise identification of patients at "high cardiovascular risk" is preeminent in cardiovascular risk stratification. OBJECTIVE: To investigate the relationships between markers of cholesterol homeostasis, cardiovascular events and all-cause mortality. DESIGN, SETTING AND PARTICIPANTS: We quantified markers of cholesterol homeostasis by gas chromatography-mass spectrometry in 377 subjects with suspected coronary artery disease, who were not on lipid-lowering drugs at baseline. All patients were followed for occurrence of cardiovascular events and mortality over a period of 4.9 +/- 1.7 years. The standardized mortality ratio (SMR) was calculated as the ratio of the observed and the expected deaths based on the death rates of the Regional Databases Germany, and Poisson regression (rate ratio, RR) was used to compare subgroups. The SMR and RR were standardized for sex, age category and calendar period. In addition, Cox regression (Hazard ratio, HR) was used to determine the effect of co-variables on (cardiovascular) mortality within the cohort. MAIN OUTCOMES: Cardiovascular events, cardiovascular mortality and all-cause mortality. RESULTS: A total of 42 deaths were observed in 1818 person-years corresponding with an SMR of 0.99 (95% CI 0.71-1.33; p = 0.556). A fatal cardiovascular event occurred in 26 patients. Lower levels of lathosterol were associated with increased cardiovascular mortality (HR 1.59; 95% CI: 1.16-2.17; p = 0.004) and excess all-cause mortality (HR 1.41; 95% CI: 1.09-1.85; p = 0.011). Lower lathosterol tertile compared to the adjacent higher tertile was associated with 1.6 times higher all-cause mortality risk (RR 1.60; 95% CI 1.07-2.40; p for trend = 0.022). This corresponded with a 2.3 times higher mortality risk of a lathosterol-LDL ratio equal to or below the median (RR 2.29; 95% CI 1.19-4.43; p = 0.013). None of the other cholesterol homeostasis markers were associated with cardiovascular and all-cause mortality. CONCLUSIONS: In patients not on lipid-lowering agents, low serum lathosterol correlated with increased risk of cardiovascular events and excess all-cause mortality.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Plasma levels of the oxyphytosterol 7α-hydroxycampesterol are associated with cardiovascular events.

BACKGROUND AND AIMS: There are safety issues regarding plant sterol ester-enriched functional food. Oxidized plant sterols, also called oxyphytosterols, are supposed to contribute to plant sterol atherogenicity. This study aimed to analyze associations of plasma oxyphytosterol levels with cardiovascular events. METHODS: Plasma cholesterol was measured by gas chromatography-flame ionization detection. Plasma campesterol and sitosterol and their 7-oxygenated metabolites were analyzed by gas chromatography-mass selective detection. RESULTS: In 376 patients admitted for elective coronary angiography, who were not on lipid-lowering drugs, 82 cardiovascular events occurred during a follow-up period of 4.2 ±â€¯1.8 years. Patients with cardiovascular events had significantly higher 7α-hydroxycampesterol plasma levels (median, 0.46; [interquartile range (IQR) 0.22-0.81] nmol/L vs. median, 0.25 [IQR, 0.17-0.61] nmol/L; p = 0.003) and 7α-hydroxycampesterol-to-cholesterol ratios (median 0.08 [IQR, 0.04-0.14] nmol/mmol vs. median, 0.05 [IQR 0.03-0.11] nmol/mmol; p = 0.005) than controls without such events. Patients above the median were characterized by higher cumulative event rates in Kaplan-Meier-analysis (Logrank-test p = 0.084 and p = 0.025) for absolute and cholesterol corrected 7α-hydroxycampesterol, respectively. After adjustment for influencing factors and related lipids, the hazard ratios per one standard deviation of the log-transformed variables (HR) were 1.19 [95% confidence interval (CI), 0.95-1.48], p = 0.132 for 7α-hydroxycampesterol and HR, 1.18 [95% CI, 0.94-1.48], p = 0.154 for 7α-hydroxycampesterol-to-cholesterol ratio. None of the other investigated oxyphytosterols showed an association with cardiovascular events. CONCLUSIONS: In patients not on lipid-lowering drugs, absolute plasma levels of 7α-hydroxycampesterol and their ratios to cholesterol are associated with cardiovascular events. Further research is required to elucidate the role of OPS in cardiovascular diseases.

International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods.

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

Cathepsin D regulates lipid metabolism in murine steatohepatitis.

Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism. CONCLUSION: CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

Thermal stability of plant sterols and formation of their oxidation products in vegetable oils and margarines upon controlled heating.

Fat-based products like vegetable oils and margarines are commonly used for cooking, which may enhance oxidation of plant sterols (PS) present therein, leading to the formation of PS oxidation products (POP). The present study aims to assess the kinetics of POP formation in six different fat-based products. Vegetable oils and margarines without and with added PS (7.5-7.6% w/w) in esterified form were heated in a Petri-dish at temperatures of 150, 180 and 210°C for 8, 12 and 16min. PS and POP were analysed using GC-FID and GC-MS-SIM, respectively. Increasing PS content, temperature and heating time led to higher POP formation in all tested fat-based products. PS (either naturally occurring or added) in margarines were less susceptible to oxidation as compared to PS in vegetable oils. The susceptibility of sitosterol to oxidation was about 20% lower than that of campesterol under all the applied experimental conditions. During heating, the relative abundance of 7-keto-PS (expressed as% of total POP) decreased in all the fat-based products regardless of their PS contents, which was accompanied by an increase in the relative abundance of 7-OH-PS and 5,6-epoxy-PS, while PS-triols were fairly unchanged. In conclusion, heating time, temperature, initial PS content and the matrix of the fat-based products (vegetable oil vs. margarine) showed distinct effects on POP formation and composition of individual POP formed.

Oxidation of sitosterol and transport of its 7-oxygenated products from different tissues in humans and ApoE knockout mice.

The most common phytosterols in the human diet are sitosterol and campesterol, which originate exclusively from plant derived food. These phytosterols are taken up by NPC1L1 transport from the intestine into the enterocytes together with cholesterol and other xenosterols. Phytosterols are selectively pumped back from the enterocytes into the intestinal lumen and on the liver site from hepatocytes into bile by heterodimeric ABCG5/G8 transporters. Like cholesterol, both phytosterols are prone to ring and side chain oxidation. It could be shown that oxyphytosterols, found in atherosclerotic tissue, are most likely of in situ oxidation (Schött et al.; Biochem. Biophys. Res. Commun. 2014 Apr 11;446(3):805-10). However, up to now, the entire mechanism of phytosterol oxidation is not clearly understood. Here, we provide further information about the oxidation of sitosterol and the transport of its oxidation products out of tissue. Our survey includes data of 104 severe aortic stenosis patients that underwent an elective aortic valve cusp replacement. We studied their phytosterol concentrations, as well as absolute and substrate corrected oxyphytosterol levels in plasma and valve cusp tissue. In addition, we also examined phytosterol and oxyphytosterol concentrations in plasma and tissues (from brain and liver) of 10 male ApoE knockout mice. The ratio of 7-oxygenated-sitosterol-to-sitosterol exceeds the ratio for 7-oxygenated-campesterol-to-campesterol in plasma and tissue of both humans and mice. This finding indicates that sitosterol is oxidized to a higher amount than campesterol and that a selective oxidative mechanism might exist which can differentiate between certain phytosterols. Secondly, the concentrations of oxyphytosterols found in plasma and tissue support the idea that oxysitosterols are preferably transported out of individual tissues. Selective oxidation of sitosterol and preferred transport of sitosterol oxidation products out of tissue seem to be a metabolic pathway of forced sitosterol clearance from tissue compartments.

7ß-Hydroxysitosterol crosses the blood-brain barrier more favored than its substrate sitosterol in ApoE-/- mice.

In this study, we compare the distribution of intraperitoneally injected sitosterol, 7ß-hydroxysitosterol or vehicle only (control) for 28days in male ApoE-/- mice. Furthermore we examine its impact on surrogate markers of cholesterol biosynthesis and sterol absorption rate in plasma, brain and liver tissues from these animals. Injection of sitosterol revealed a 32.1% (P=0.013) lower plasma total cholesterol compared with control. Cholesterol corrected plasma and absolute brain and liver levels of sitosterol are 4.1-, 1.7-, and 7.2-fold (P<0.001 for all) higher, respectively. This is in accordance with a reduced plasma campesterol to cholesterol ratio (-16.2%; P=0.018) together with a 24.1% (P=0.047) lower concentration of hepatic lathosterol. After injection of 7ß-hydroxysitosterol the concentrations of 7ß-hydroxysitosterol in plasma, brain and liver are 21.0-, 65.8- and 42.7-fold (P<0.001 for all) higher, respectively, compared with control. Injection of 7ß-hydroxysitosterol revealed significantly lower plasma cholesterol corrected cholestanol and campesterol (-44.2%; P=0.001 and -24.5; P=0.004) as well as lower absolute liver cholestanol levels (-31.9%; P<0.001) compared with control. Intraperitoneally injected sitosterol and 7ß-hydroxysitosterol differently influence cholesterol metabolism in plasma and liver. We conclude that the polar 7ß-hydroxysitosterol compound can pass the blood brain barrier with higher efficacy than its substrate, sitosterol. Though present in higher amounts in the brain, both, sitosterol and 7ß-hydroxysitosterol do not influence cholesterol metabolism in the brain as proven by our surrogate markers.

Postprandial plasma oxyphytosterol concentrations after consumption of plant sterol or stanol enriched mixed meals in healthy subjects.

Epidemiological studies have reported inconsistent results on the relationship between increased plant sterol concentrations with cardiovascular risk, which might be related to the formation of oxyphytosterols (plant sterol oxidation products) from plant sterols. However, determinants of oxyphytosterol formation and metabolism are largely unknown. It is known, however, that serum plant sterol concentrations increase after daily consumption of plant sterol enriched products, while concentrations decrease after plant stanol consumption. Still, we have earlier reported that fasting oxyphytosterol concentrations did not increase after consuming a plant sterol- or a plant stanol enriched margarine (3.0g/d of plant sterols or stanols) for 4weeks. Since humans are in a non-fasting state for most part of the day, we have now investigated effects on oxyphytosterol concentrations during the postprandial state. For this, subjects consumed a shake (50g of fat, 12g of protein, 67g of carbohydrates), containing no, or 3.0g of plant sterols or plant stanols. Blood samples were taken up to 8h and after 4h subjects received a second shake (without plant sterols or plant stanols). Serum oxyphytosterol concentrations were determined in BHT-enriched EDTA plasma via GC-MS/MS. 7ß-OH-campesterol and 7ß-OH-sitosterol concentrations were significantly higher after consumption of a mixed meal enriched with plant sterol esters compared to the control and plant stanol ester meal. These increases were seen only after consumption of the second shake, illustrative for a second meal effect. Non-oxidized campesterol and sitosterol concentrations also increased after plant sterol consumption, in parallel with 7ß-OH concentrations and again only after the second meal. Apparently, plant sterols and oxyphytosterols follow the same second meal effect as described for dietary cholesterol. However, the question remains whether the increase in oxyphytosterols in the postprandial phase is due to absorption or endogenous formation.

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization.

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain.

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.

Cerebral accumulation of dietary derivable plant sterols does not interfere with memory and anxiety related behavior in Abcg5-/- mice.

Plant sterols such as sitosterol and campesterol are frequently applied as functional food in the prevention of atherosclerosis. Recently, it became clear that plasma derived plant sterols accumulate in murine brains. We questioned whether plant sterols in the brain are associated with alterations in brain cholesterol homeostasis and subsequently with brain functions. ATP binding cassette (Abc)g5-/- mice, a phytosterolemia model, were compared to Abcg5+/+ mice for serum and brain plant sterol accumulation and behavioral and cognitive performance. Serum and brain plant sterol concentrations were respectively 35-70-fold and 5-12-fold increased in Abcg5-/- mice (P<0.001). Plant sterol accumulation resulted in decreased levels of desmosterol (P<0.01) and 24(S)-hydroxycholesterol (P<0.01) in the hippocampus, the brain region important for learning and memory functions, and increased lanosterol levels (P<0.01) in the cortex. However, Abcg5-/- and Abcg5+/+ displayed no differences in memory functions or in anxiety and mood related behavior. The swimming speed of the Abcg5-/- mice was slightly higher compared to Abcg5+/+ mice (P<0.001). In conclusion, plant sterols in the brains of Abcg5-/- mice did have consequences for brain cholesterol metabolism, but did not lead to an overt phenotype of memory or anxiety related behavior. Thus, our data provide no contra-indication for nutritional intake of plant sterol enriched nutrition.

Alterations in brain cholesterol metabolism in the APPSLxPS1mut mouse, a model for Alzheimer's disease.

Disturbances in cerebral cholesterol metabolism have been implicated in the pathogenesis of Alzheimer's disease (AD). Here, we provide evidence that alterations in brain cholesterol homeostasis also can be a consequence of disease progression. We found that APPSLxPS1mut mice, at the age of 9 months when AD-like pathology starts to develop, display increased levels of the cholesterol precursor desmosterol and of the cholesterol metabolite 27-hydroxy(OH)cholesterol in their cerebellum in comparison with wild-type controls. At the age of 21 months, when APPSLxPS1mut brain contains abundant amyloid deposits, desmosterol levels had further increased (> 200% in comparison with wild-type mice) in all brain regions examined. 24(S)-OHcholesterol levels were increased in hippocampus and cerebellum of the APPSLxPS1mut mice, while 27-OHcholesterol levels were increased in cerebellum exclusively. Brain cholesterol levels remained unaffected. In line with the fact that desmosterol and 24(S)-OHcholesterol are Liver X Receptor (LXR) activators, the LXR-target genes Abca1 and Apoc1 were upregulated predominantly in hippocampus of APPSLxPS1mut mice at both ages evaluated. The reduced expression of the enzyme that converts desmosterol into cholesterol, the Selective AD indicator 1 gene (Seladin-1/Dhcr24), in both cortex and cerebellum may underlie the increased desmosterol levels in 21 month-old APPSLxPS1mut mice.