Development and validation of a new UPLC-MS/MS method for quantification of ganoderic acid-A loaded nanolipidic carrier in rat plasma and application to pharmacokinetic studies.

A systematic methodology was used to quantify ganoderic acid-A (GA-A) loaded nano-lipid carriers (NLC) in rat plasma using UPLC-MS/MS. Separation of the analyte was achieved using ACQUITY UPLC BEH C18 column (1.7 µm) and mobile phase as water containing 0.1% Acetonitrile (40: 60% v/v) at a flow rate of 0.4 mL·min-1. The analyte was detected using MRM mode to track precursor-to-product ion transitions of 515.37 â†’ 285.31 m/z (time scan of 2 min) for GA-A, and 175.11 â†’ 115.08 m/z (time scan of 4 min) for ascorbic acid as an internal standard (IS), respectively. The developed method was validated for linearity, accuracy, within and between day precisions, limit of quantification and recovery of the analyte. The results indicated intra and inter-day consistency and precision values were found to be within the acceptance limit for the plasma samples. The method applicability for determination of pharmacokinetic parameters of GA-A was assessed after oral administration of free GA-A solution and GA-A-loaded NLC, which indicated significant difference (p < 0.05) in the rate and extent of absorption parameters of GA-A from the NLC formulation vis-à-vis the plain solution. Overall, the studies construed successful development and application of UPLC-MS/MS method for estimation of GA-A in the lipidic formulation.

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.

Plasma and brain pharmacokinetics of ganoderic acid A in rats determined by a developed UFLC-MS/MS method.

Ganoderic acid A (GAA), an active triterpenoid of the traditional Chinese herbal medicine Lingzhi, has been reported to exhibit antinociceptive, antioxidative, and anti-cancer activities. The present study aims to establish a sensitive and rapid UPLC-MS/MS method for studying the plasma and brain pharmacokinetics of GAA in rats. The analytes were separated on a C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3mL/min. The eluate was monitored by a mass detector using an MRM (m/z, 515.3-285.1) model in negative electrospray ionization. The calibration curve showed good linearity (r2>0.99), with limits of detection and quantification of 0.25 and 2.00 nmol/L, respectively. The intra- and inter-day precision and accuracy were less than 9.99% and ranged from 97.45% to 114.62%, respectively. The extraction recovery from plasma was between 92.89% and 98.87%. GAA was found to be stable in treated samples at room temperature (22°C) for 12h and in plasma at -20°C for 7d. The developed method was successfully applied to a pharmacokinetic study of GAA in rats. GAA could be rapidly absorbed into the circulation (Tmax, 0.15h) and eliminated relatively slowly (t1/2, 2.46h) after orally dosing, and could also be detected in the brain lateral ventricle (Tmax, 0.25h and t1/2, 1.40h) after intravenously dosing. The absolute oral bioavailability and brain permeability of GAA were estimated to be 8.68% and 2.96%, respectively.

Online solid-phase extraction-liquid chromatography-mass spectrometry to determine free sterols in human serum.

An automated method for analyzing free non-cholesterol sterols in human serum using online solid phase extraction-liquid chromatography-mass spectrometry is proposed herein. The method allows the determination of three phytosterols (sitosterol, stigmasterol and campesterol) and two cholesterol precursors (desmosterol and lanosterol). The analysis of sterols in human serum is critical in the study of cholesterol-related disorders, such as inherited familial hypercholesterolemias. Special effort was made to isolate the analytes from the serum lipoproteins, their natural conveyance through the bloodstream. The sample treatment consisted of a Bligh-Dyer extraction followed by dilution of the extract. This treatment allowed the sample to be injected into the online system and ensured the correct detection of the analytes, while avoiding the matrix effects commonly related to serum samples. The analytical performance showed linear ranges that covered two orders of magnitude, with correlation coefficients above 0.99. Limits of detection and quantification ranged from 0.2 ng/mL to 13 ng/mL and from 1.0 ng/mL to 43 ng/mL, respectively. Recovery when spiking serum with a half or a tenth of the average concentration reported in human serum ranged from 99% to 111% and from 102% to 120%, respectively. Intra-day precision and inter-day precision were below 20%.

Quantitative determination of euphol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C18 short column eluted with a mobile phase of methanol­water­formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 →109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27­9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between ­7.04 and 4.11%, and the precision was <10.83%. This LC-MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%.

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

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

Aging induces tissue-specific changes in cholesterol metabolism in rat brain and liver.

Disturbance of cholesterol homeostasis in the brain is coupled to age-related brain dysfunction. In the present work, we studied the relationship between aging and cholesterol metabolism in two brain regions, the cortex and hippocampus, as well as in the sera and liver of 6-, 12-, 18- and 24-month-old male Wistar rats. Using gas chromatography-mass spectrometry, we undertook a comparative analysis of the concentrations of cholesterol, its precursors and metabolites, as well as dietary-derived phytosterols. During aging, the concentrations of the three cholesterol precursors examined (lanosterol, lathosterol and desmosterol) were unchanged in the cortex, except for desmosterol which decreased (44 %) in 18-month-old rats. In the hippocampus, aging was associated with a significant reduction in lanosterol and lathosterol concentrations at 24 months (28 and 25 %, respectively), as well as by a significant decrease of desmosterol concentration at 18 and 24 months (36 and 51 %, respectively). In contrast, in the liver we detected age-induced increases in lanosterol and lathosterol concentrations, and no change in desmosterol concentration. The amounts of these sterols were lower than in the brain regions. In the cortex and hippocampus, desmosterol was the predominant cholesterol precursor. In the liver, lathosterol was the most abundant precursor. This ratio remained stable during aging. The most striking effect of aging observed in our study was a significant decrease in desmosterol concentration in the hippocampus which could reflect age-related reduced synaptic plasticity, thus representing one of the detrimental effects of advanced age.

Simultaneous determination of cimicifugoside H-2, cimicifugoside H-1, 23-epi-26-deoxyactein, cimigenol xyloside and 25-O-acetylcimigenoside in beagle dog plasma by LC-MS/MS.

A selective and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of five constituents (cimicifugoside H-2, cimicifugoside H-1, 23-epi-26-deoxyactein, cimigenol xyloside and 25-O-acetylcimigenoside) of Cimicifuga foetida L. in beagle dog plasma. The quantitation was performed on a LC-MS/MS with negative electrospray ionization in selected reaction monitoring (SRM) mode. A gradient mobile phase composed of methanol and water was used at a flow rate of 0.4 ml/min. All the analytes and internal standard (20 (S)-ginsenoside Rg3) were isolated from plasma samples by a liquid-liquid extraction method. The average extraction recoveries were 73-74% for cimicifugoside H-2, 89-94% for cimicifugoside H-1, 73-80% for 23-epi-26-deoxyactein, 89-91% for cimigenol xyloside, 87-96% for 25-O-acetylcimigenoside, respectively. The method showed good linearity and no endogenous material interfered with all the five compounds and I.S. peaks. The lower limit of quantification (LLOQ) of all analytes was 0.5 ng/ml. The intra- and inter-day precision of analysis was less than 15% for each analyte at concentrations of 2.0, 50, 500 ng/ml, and the accuracy ranged from 85.8% to 107%. This method was successfully applied to reveal the pharmacokinetic properties of cimicifugoside H-2, cimicifugoside H-1, 23-epi-26-deoxyactein, cimigenol xyloside and 25-O-acetylcimigenoside after oral administration.

Differences in brain cholesterol metabolism and insulin in two subgroups of patients with different CSF biomarkers but similar white matter lesions suggest different pathogenic mechanisms.

Investigate possible associations of white matter hyperintensities (WMHs) with the metabolism of cholesterol and insulin in two subgroups of patients with memory complaints and different CSF Aß42 and CSF tau levels. 59 patients from the memory clinic at Karolinska Hospital were included. Degree of WMHs was rated using the ARWMC scale and the following biomarkers were measured in CSF and plasma: insulin, cholesterol, lanosterol, lathosterol, and oxidized cholesterol metabolites. The WMHs in CSF control-like group correlated with increased brain cholesterol synthesis and reduced efflux of oxysterols and insulin in CSF. In the CSF AD-like group, the WMHs correlated with increased peripheral cholesterol metabolism. Despite having similar appearance on FLAIR images, the pathogenic mechanisms of WMHS are likely to be different in the two groups investigated.

Effects of a 2-y dietary weight-loss intervention on cholesterol metabolism in moderately obese men.

BACKGROUND: Long-term dietary weight loss results in complex metabolic changes. However, its effect on cholesterol metabolism in obese subjects is still unclear. OBJECTIVE: We assessed the effects of 2 y of weight loss achieved with various diet regimens on phytosterols (markers of intestinal cholesterol absorption), lanosterol (marker of de novo cholesterol synthesis), and changes in apolipoprotein concentrations. DESIGN: We conducted the 2-y Dietary Intervention Randomized Controlled Trial (DIRECT-a study of low-fat, Mediterranean, and low-carbohydrate diets). We assessed circulating phytosterol and lanosterol concentrations and their ratios to cholesterol and apolipoproteins A-I and B-100 in 90 DIRECT participants at 0, 6, and 24 mo. RESULTS: We observed a significant upregulation of the markers of cholesterol absorption (campesterol: +16.8%, P < 0.001) and a downregulation of the markers of cholesterol synthesis (lanosterol: -16.5%, P = 0.008) during the active weight-loss phase (first 6 mo, weight loss of 5%, 6%, and 10% in the 3 diet groups, respectively), followed by a rebound (campesterol: -6.2%, P = 0.045; lanosterol: +43.7%, P < 0.001) during the next 18 mo (weight gain of 1%, 1%, and 2% in the 3 diet groups, respectively). HDL cholesterol continuously increased during the study (17.0%, P < 0.001), whereas LDL cholesterol remained constant. At the end of the 24-mo follow-up period, campesterol (P < 0.001) and lanosterol (P = 0.016) amounts were significantly higher than baseline values. The concentration of apolipoprotein B-100 correlated with cholesterol metabolism (ρ = 0.299 and P = 0.020 for lanosterol; ρ = -0.105 and NS for campesterol), and the homeostasis model assessment of insulin resistance correlated with lanosterol (ρ = 0.09, P = 0.001). CONCLUSIONS: Long-term weight loss is related to a characteristic response suggestive of altered cholesterol and apolipoprotein metabolism. Various diets have a similar effect on these effects. DIRECT is registered at clinicaltrials.gov as NCT00160108.

Cosegregation of aortic root atherosclerosis and intermediate lipid phenotypes on chromosomes 2 and 8 in an intercross of C57BL/6 and BALBc/ByJ low-density lipoprotein receptor-/- mice.

OBJECTIVE: We sought to identify novel atherosclerosis-modifying loci and their potential functional links in a genome-wide approach using cosegregation analysis of atherosclerosis and related intermediate phenotypes in mice. METHODS AND RESULTS: We carried out an F2 intercross between atherosclerosis-susceptible C57BL/6 mice and atherosclerosis-resistant BALB/cByJ mice on the low-density lipoprotein receptor(-/-) background to examine the genetic basis for their differences in atherosclerosis susceptibility. Atherosclerotic lesion size and a comprehensive panel of 61 atherosclerosis-related phenotypes, including plasma levels of lipids, cytokines, and chemokines were measured in 376 F2 mice. Quantitative trait locus mapping revealed a novel significant locus (logarithm of odds, 6.18) for atherosclerosis on proximal mouse chromosome (Chr) 2 (Ath39), which was associated with major variations in lesion size (14%). Plasma very-low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, lanosterol, and phytosterol levels cosegregated with atherosclerosis at this locus. Moreover, these lipid traits showed significant correlations with lesion size, suggesting that they share the same underlying genetic factor. We also describe a second male-specific locus on Chr 8 (Ath40) where atherosclerosis and lipids cosegregated. CONCLUSIONS: Our study revealed new loci for atherosclerosis susceptibility on mouse Chr 2 and 8, which might exert their effects on lesion size via plasma lipid levels.

Impact of atorvastatin and omega-3 ethyl esters 90 on plasma plant sterol concentrations and cholesterol synthesis in type 2 diabetes: a randomised placebo controlled factorial trial.

OBJECTIVE: To determine the effects of statin treatment and omega-3 polyunsaturated fatty acid supplementation on plasma plant sterol concentrations and cholesterol synthesis in patients with type 2 diabetes. METHODS: Plant sterol concentrations and lanosterol (a marker of cholesterol synthesis) were measured using a high sensitivity assay to assess the effect of double-blind daily treatment for 4 months with atorvastatin 20mg or placebo and, in a 2 × 2 factorial design, omega-3 ethyl esters 90 2g or placebo. RESULTS: 658 patients were included in a per protocol analysis. The 4 treatment groups had similar mean [SD] age (63.5 years [11.7]), HbA(1c) (6.9% [1.1]) and diabetes duration (median 4 years [inter-quartile range 2, 8]). Atorvastatin treatment alone reduced low density lipoprotein (LDL) cholesterol by 1.4 mmol/l (44%, p<0.001), triglycerides by 0.3 mmol/l (20%, p<0.0001) and lanosterol by 0.36 µmol/l (72%, p<0.001). There was no significant placebo adjusted change in median [95% confidence intervals] total plant sterol concentrations (-0.77 µmol/l [inter-quartile range -2.13, 0.59]), although they were increased significantly with omega-3-acid EE90 treatment (3.23 µmol/l [1.28, 5.17]). There was a 27% smaller reduction in LDL cholesterol with atorvastatin treatment in low cholesterol synthesisers with high absorption, defined by changes at or above the median lanosterol and campesterol levels, respectively, compared with the obverse group (difference 0.42 mmol/l [0.21, 0.62]). CONCLUSION: Treatment with atorvastatin in type 2 diabetes did not change median total plasma plant sterol concentrations, but LDL cholesterol was reduced most efficaciously in high cholesterol synthesisers with low intestinal cholesterol absorption. CLINICAL TRIAL REGISTRATION INFORMATION: Current controlled trials number ISRCTN: 76737502 (http://isrctn.org).

Alterations of cholesterol precursor levels in Alzheimer's disease.

Cerebral and extracerebral cholesterol metabolism are altered in Alzheimer's disease (AD) as indicated by reduced plasma levels of the cholesterol elimination products 24S-hydroxycholesterol, which is of cerebral origin, and of 27-hydroxycholesterol, which is formed extracerebrally. However, it has to be evaluated, if changes of cholesterol metabolism in the whole body or in the CNS are exclusively due to the altered elimination of cholesterol or are also due to altered de novo synthesis in AD. We investigated CSF and plasma levels of cholesterol and of its precursors lanosterol, lathosterol and desmosterol in AD patients and non-demented controls. We found CSF levels of cholesterol (p=0.011), absolute levels of all investigated cholesterol precursors (each p<0.001) and ratios of cholesterol precursors/cholesterol (each <0.01) to be lower in AD patients as compared to controls. In plasma, the absolute levels of lanosterol (p=0.026) and lathosterol (p<0.001) and the ratio of lathosterol/cholesterol (p=0.002) but none of the other investigated parameters were reduced in AD patients (p>0.1). Furthermore, ratios of desmosterol/lathosterol in CSF (p=0.023) and plasma (p=0.009) were higher in AD patients as compared to controls. Our data support the hypothesis that cholesterol metabolism is altered in AD and further suggest that especially cholesterol de novo synthesis within the CNS of AD patients might be reduced. These findings raise doubt on a beneficial effect of cholesterol lowering treatment in manifest AD.

Plasma levels of 24S-hydroxycholesterol reflect brain volumes in patients without objective cognitive impairment but not in those with Alzheimer's disease.

OBJECTIVES: Cholesterol has been linked to Alzheimer's disease (AD) and plasma 24S-hydroxycholesterol (24OHC) has been suggested as a surrogate marker for brain cholesterol metabolism. This study investigates the relation of 24OHC as well as markers of extracerebral cholesterol homeostasis (lanosterol, lathosterol, cholesterol, LDL-C, HDL-C and 27-hydroxycholesterol) with brain volumes in memory clinic patients. METHODS: 96 patients (33 with subjective cognitive impairment--SCI; 36 with mild cognitive impairment--MCI; 27 with AD) referred to the Memory Clinic at Karolinska University Hospital, Sweden. Plasma assessments were done by isotope dilution-mass spectrometry. MRI measurements were done using custom-made software BMAP (imaging laboratory, Karolinska Institutet), running on HERMES platform. RESULTS: Ratios of 24-hydroxycholesterol, 27-hydroxycholesterol, lanosterol and lathosterol to cholesterol (R_24OHC, R_27OHC, R_lanosterol and R_lathosterol) were significantly lower in patients with AD. In the whole population, after controlling for age, sex, APOE genotype and statins, R_24OHC was positively related to gray matter (GM) fraction. However, when groups were considered separately, the relation to GM volume, GM and parenchymal fractions was significant in the SCI group only (p<0.05). There was a significant positive association between cholesterol and white matter (WM) volume, WM and parenchymal fractions in patients with AD. CONCLUSIONS: Plasma R_24OHC was lower in patients with AD, but R_24OHC was significantly related to brain volumes in the control group only. One reason may be the previously demonstrated abnormal expression of cholesterol 24S-hydroxylase in astrocytes in AD, which may limit the usefulness of this plasma marker in this specific disease. The findings on cholesterol agree with previous reports of decreasing plasma cholesterol levels in AD patients, suggesting a CNS-mediated effect on extracerebral cholesterol homeostasis.

Elevated cholesterol precursors other than cholestanol can also be a hallmark for CTX.

Cerebrotendinous xanthomatosis (CTX) is an inborn error of bile acid synthesis in which hepatic conversion of cholesterol to cholic and chenodeoxycholic acids is impaired. Patients have abnormal bile alcohols in urine, normal to increased plasma cholesterol concentrations and increased concentrations of plasma cholestanol. Little is known about cholesterol precursors in CTX, however. We studied cholesterol and phytosterol profiles in two siblings with CTX during follow-up. While cholesterol concentrations were low in both patients, plasma cholestanol was 6-fold higher compared to control values. In addition, both siblings had a more than 100-fold increase in 7-dehydrocholesterol (7DHC) and 8-dehydrocholesterol (8DHC). Lathosterol, lanosterol and sitosterol were increased in both patients while concentrations of desmosterol and campesterol were normal. In addition, plasma lathosterol/cholesterol ratios were significantly elevated. After treatment with chenodeoxycholate, both patients showed a marked decrease in cholestanol, 7DHC, 8DHC, lathosterol, lanosterol and sitosterol. In addition, the lathosterol/cholesterol ratio normalized, indicating that overall cholesterol synthesis was sufficiently suppressed. This study shows that elevated cholesterol precursors, other than cholestanol, can be a hallmark for CTX.

Inhibition of sterol 4alpha-methyl oxidase is the principal mechanism by which garlic decreases cholesterol synthesis.

Clinical and experimental evidence indicates that garlic ingestion lowers blood cholesterol levels, and treatment of cells in culture with garlic and garlic-derived compounds inhibits cholesterol synthesis. To identify the principal site of inhibition in the cholesterolgenic pathway and the active components of garlic, cultured hepatoma cells were treated with aqueous garlic extract or its chemical derivatives, and radiolabeled cholesterol and intermediates were identified and quantified. Garlic extract reduced cholesterol synthesis by up to 75% without evidence of cellular toxicity. Levels of squalene and 2,3-oxidosqualene were not altered by garlic, indicating that the site of inhibition was downstream of lanosterol synthesis, and identical results were obtained with 14C-acetate and 14C-mevalonate, confirming that 3-hydroxy-3-methylglutaryl-CoA reductase activity was not affected in these short-term studies. Several methylsterols that accumulated in the presence of garlic were identified by coupled gas chromatography-mass spectrometry as 4,4'-dimethylzymosterol and a possible metabolite of 4-methylzymosterol; both are substrates for sterol 4alpha-methyl oxidase, pointing to this enzyme as the principal site of inhibition in the cholesterolgenic pathway by garlic. Of 9 garlic-derived compounds tested for their ability to inhibit cholesterol synthesis, only diallyl disulfide, diallyl trisulfide, and allyl mercaptan proved inhibitory, each yielding a pattern of sterol accumulation identical with that obtained with garlic extract. These results indicate that compounds containing an allyl-disulfide or allyl-sulfhydryl group are most likely responsible for the inhibition of cholesterol synthesis by garlic and that this inhibition is likely mediated at sterol 4alpha-methyl oxidase.

Isotopomer spectral analysis of intermediates of cholesterol synthesis in patients with cerebrotendinous xanthomatosis.

Four patients with cerebrotendinous xanthomatosis (CTX) and 2 healthy controls received a constant proximal intraduodenal infusion of 1- 13 C-acetate as a stable-isotope-labeled marker of sterol synthesis. One patient was treated with pravastatin (20 mg twice daily) and another patient with chenodeoxycholic acid (250 mg tid). Every hour, venous blood and duodenal samples were obtained. Stable-isotope enrichment of neutral and polar sterols in serum and bile was assessed by gas chromatography/mass spectrometry. Isotopomer spectral analysis was performed on cholesterol, lathosterol, Delta-8-cholesterol, methylsterol, and lanosterol. Stable-isotope labeling of cholestanol, bile acids, and bile alcohols was analyzed by assessing the change over time of the ratio of M + 3 to M + 0. Eleven hours after marker infusion, we found up to 50% newly synthesized lathosterol in serum and up to 80% in bile, with similar results for other cholesterol precursors. In cholesterol, stable-isotope labeling could be demonstrated in all study subjects with a more prominent labeling in bile than in serum. No stable-isotope labeling was detected in cholestanol. Only minor stable-isotope incorporation was detectable in polar sterols in some subjects. Therapy with pravastatin did not have any effect on fractional or absolute synthesis rates or on the concentrations of cholestanol or cholesterol precursors compared to untreated patients with CTX. In contrast, therapy with chenodeoxycholic acid markedly lowered the concentrations of cholestanol and cholesterol precursors, led to a disappearance of bile alcohols, and reduced absolute synthesis rates of lathosterol. Isotopomer spectral analysis proved to be a powerful method to assess the endogenous synthesis of cholesterol precursors in patients with CTX. Higher fractional synthesis in bile than in serum may be due to the size of the pools in bile vs serum. Cholestanol exhibits no marker uptake and is therefore probably synthesized from preformed cholesterol. Biliary cholesterol secretion in patients with CTX is decreased compared to healthy controls.

Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase.

Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14alpha-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77). These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes.

[Serum markers in relation to cognitive functioning in an aging population: results of the Maastricht Aging Study (MAAS)]. / Serummarkers in relatie tot cognitieve functies bij ouderen. Resultaten van de Maastricht Aging Study (MAAS).

Little is known of the biochemical processes of cognitive decline during 'healthy' aging. Biological markers in body fluids, such as blood, could provide insight in those processes. In the present studies serum concentrations of different markers have been correlated to cognitive functioning of cognitively healthy aging individuals over a period of six years (mean age 57 years, SD 11, n = 93). Markers were related to mechanisms known to be involved in Alzheimer's disease, including inflammation, cholesterol homeostasis and homocysteine homeostasis. Domains of cognitive function addressed were cognitive speed (Letter-Digit Coding test), attention and information processing (Stroop test), and memory (Word Learning test: Total Words and Delayed Recall). Baseline concentrations of haptoglobine, homocysteine, lathosterol and lanosterol were negatively correlated with cognitive functioning on the Stroop test over the whole follow-up period of six years. Concentrations of all markers, i.e. haptoglobine, C-reactive protein, homocysteine, lathosterol and lanosterol, were also negatively correlated with functioning on the Word Learning test (Delayed Recall and for some markers also with the Total Words) over the whole six-years follow-up period. In conclusion, concentrations of serum markers related to inflammation, homocysteine and cholesterol homeostasis are not only associated with Alzheimer's disease, but also with cognitive functioning in the cognitively healthy aging population.

Changes in cholesterol and its precursors during the first days after major trauma.

BACKGROUND: The causes of hypocholesterolemia in the critically ill, including major trauma patients, have not yet been fully elucidated. OBJECTIVE: We tested the hypothesis that hypocholesterolemia is caused by decreased production of cholesterol precursors. DESIGN: Serum concentrations of squalene, lanosterol, and lathosterol were measured on admission, and then at 24 and 48 hours after injury using gas chromatography coupled with mass spectrometry. Serum concentrations of total low-density and high-density lipoprotein cholesterol were measured on admission and every day in the first week after injury. RESULTS: 83 consecutive patients with multiple trauma were examined. Significant drops in concentrations of lanosterol and lathosterol were found in the patients in comparison with the control group. The most profound drop was in lathosterol. CONCLUSION: Decreased synthesis of cholesterol precursors is the major cause of hypocholesterolemia in patients with multiple trauma. Lathosterol concentration is proposed as a marker of cholesterol synthesis.