OBJECTIVESTo study the potential endogenous marker of CYP3A activity, 4β-hydroxycholesterol, and its relation to sex and the CYP3A5 geno/haplotypes and compare with CYP3A4/5 catalyzed 3-hydroxylation ...of quinine in the three major races.
METHODSThe plasma concentration of 4β-hydroxycholesterol was measured in healthy Tanzanians (n=138), Swedes (n=161) and Koreans (n=149) by gas chromatography–mass spectrometry. The metabolic ratio of quinine/3-hydroxyquinine in plasma 16-h post dose was determined by high performance liquid chromatography, previously reported in Tanzanians and Swedes, and now also in Koreans. The participants were genotyped for relevant alleles of CYP3A5.
RESULTSThe mean plasma concentrations of 4β-hydroxycholesterol in Koreans, Swedes and Tanzanians were 29.3, 26.8 and 21.9 ng/ml, respectively (P<0.01 between all three populations). Within all three populations there were significant differences in 4β-hydroxycholesterol levels between the CYP3A5 genotypes. Women had higher concentrations than men, but the difference was only significant in Tanzanians (P<0.001) and Koreans (P<0.00001). The quinine/3-hydroxyquinine metabolic ratio was significantly different in all three populations with the highest CYP3A activity in Koreans and the lowest in Tanzanians. Korean women had a lower metabolic ratio than men (P<0.00001). Significant correlations between 4β-hydroxycholesterol and quinine 3-hydroxylation were found in Tanzanians and Koreans.
CONCLUSIONClear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4β-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4β-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).
To study the potential endogenous marker of CYP3A activity, 4beta-hydroxycholesterol, and its relation to sex and the CYP3A5 geno/haplotypes and compare with CYP3A4/5 catalyzed 3-hydroxylation of ...quinine in the three major races.
The plasma concentration of 4beta-hydroxycholesterol was measured in healthy Tanzanians (n=138), Swedes (n=161) and Koreans (n=149) by gas chromatography-mass spectrometry. The metabolic ratio of quinine/3-hydroxyquinine in plasma 16-h post dose was determined by high performance liquid chromatography, previously reported in Tanzanians and Swedes, and now also in Koreans. The participants were genotyped for relevant alleles of CYP3A5.
The mean plasma concentrations of 4beta-hydroxycholesterol in Koreans, Swedes and Tanzanians were 29.3, 26.8 and 21.9 ng/ml, respectively (P<0.01 between all three populations). Within all three populations there were significant differences in 4beta-hydroxycholesterol levels between the CYP3A5 genotypes. Women had higher concentrations than men, but the difference was only significant in Tanzanians (P<0.001) and Koreans (P<0.00001). The quinine/3-hydroxyquinine metabolic ratio was significantly different in all three populations with the highest CYP3A activity in Koreans and the lowest in Tanzanians. Korean women had a lower metabolic ratio than men (P<0.00001). Significant correlations between 4beta-hydroxycholesterol and quinine 3-hydroxylation were found in Tanzanians and Koreans.
Clear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4beta-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4beta-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).
The Pliensbachian and Toarcian (Early Jurassic) ages are characterised by several, relatively short-lived carbon cycle perturbations, climate change and faunal turnover. The cause(s) of biotic and ...abiotic disturbances remain unclear but most probably involved increased magmatic activity in the Karoo–Ferrar large igneous province. The Toarcian oceanic anoxic event (T-OAE) might represent the most extreme of these events, and as such, is becoming increasingly well documented worldwide. So far, other critical time intervals of the Pliensbachian–Toarcian have received considerably less attention. Here, the effects of the Middle Toarcian Variabilis event on the neritic–epeiric realm are explored making use of three well-exposed and extended stratigraphic sections in the Central High Atlas, Morocco. The carbon and oxygen isotopic compositions of 112 bulk micrite samples were analysed and placed against 39 data points from carefully screened brachiopod valves in order to differentiate between palaeo-environmental and diagenetic patterns. Additionally, the phosphorus concentrations of 109 micrite samples were determined to evaluate the P-cycling. In all studied sections, an upper middle Toarcian major change from carbonate- to clastics-dominated sedimentation is recorded, pointing to a first-order carbonate production crisis. Our results reveal that these major sedimentological patterns coincide with an increase of oxygen-isotope ratios as well as a decrease of phosphorous accumulation rates. This suggests that the late middle Toarcian carbonate ramp crisis was related to a transient cooling event, potentially triggered by pulsed massive SO4 exhalation events in the context of the Karoo large igneous province. Short-term cooling was likely amplified by the drawdown of atmospheric CO2 levels related to the coeval decline of neritic carbonate precipitation and the warm water mass circulation disruption between the Tethys and the continental shelf. The data shown here provide the first evidence for coupled changes in carbon cycling, continental weathering and neritic systems in the aftermath of the T-OAE.
•A carbonate crisis is highlighted during the middle Toarcian in W Tethys.•Oxygen isotopes of brachiopod valves are used to reconstruct seawater temperature.•The carbonate crisis is coeval to a mass extinction and a transient cooling event.•Phosphorus analyses document enhanced nutrient levels during the Late Toarcian.
Farnesoid X receptor knockout (Fxrsuperscript -/-) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxrsuperscript -/- mice ...differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxrsuperscript -/- mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxrsuperscript -/- mice, in particular hydroxylations of cholic acid in the 1{szligbeta}, 2{szligbeta}, 4{szligbeta}, 6alpha, 6{szligbeta}, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxrsuperscript -/- mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxrsuperscript -/- mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.
Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of ...additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3β-hydroxy-Δ
5 structure were converted by cholesterol oxidase to 3-oxo-Δ
4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.
One of the major oxysterols in the human circulation is 4 beta-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4 ...beta-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7 alpha-, 27-, and 24-hydroxycholesterol to be approximately 0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4 beta-hydroxycholesterol. The apparent half-life of deuterium-labeled 4 beta-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4 beta-Hydroxycholesterol was converted into acidic products at a much slower rate than 7 alpha-hydroxycholesterol in primary human hepatocytes, and 4 beta-hydroxycholesterol was 7 alpha-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 beta-hydroxycholesterol. These results suggest that the high plasma concentration of 4 beta-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7 alpha-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4 beta-hydroxycholesterol as a ligand for the nuclear receptor LXR.
Neutral steroids have traditionally been analyzed by gas chromatography/mass spectrometry (GC/MS) after necessary derivatization reactions. However, GC/MS is unsuitable for the analysis of many ...conjugated steroids and those with unsuspected functional groups. Here we describe an alternative analytical method specifically designed for the analysis of oxosteroids and those with a 3β-hydroxy-Δ5 or 5α-hydrogen-3β-hydroxy structure. Steroids were derivatized with Girard P (GP) hydrazine to give GP hydrazones, which are charged species and readily analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The resulting M+ ions were then subjected to high-energy collision-induced dissociation on a tandem time-of-flight instrument. The product ion spectra give structurally informative fragment ion patterns. The sensitivity of the analytical method is such that steroid structures can be determined from low-picogram (low-femtomole) amounts of sample. The utility of the method has been demonstrated by the analysis of oxysterols extracted from rat brain.
In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass ...spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.
The conversion of cholesterol to bile acids is a key pathway for elimination of cholesterol from the body, thereby reducing the risk of arteriosclerosis. Moderate consumption of ethanol has been ...shown to have preventive effects on cardiovascular disease and decrease the risk of gallstone formation. In the present study primary human hepatocytes were used to investigate if ethanol affected bile acid synthesis. Hepatocytes were prepared from donor liver (
n
=
11) and treated with ethanol, 7.7 or 50
mM, for 24
h. mRNA levels for enzymes in bile acid synthesis pathways were studied and bile acid synthesis was analyzed. Treatment with 7.7
mM ethanol increased cholic acid synthesis by 20% and treatment with 50
mM ethanol up-regulated cholic acid formation by 60%. The synthesis of cholic acid increased more than that of chenodeoxycholic acid, indicating that the classical pathway for bile acid synthesis was up-regulated. Increased bile acid levels in the cells treated with ethanol were seen after approximately 20
h. mRNA expression of CYP7A1, CYP27A1, and CYP8B1 in the hepatocytes was not affected by alcohol exposure.
![Fxr[superscript -/-] mice a...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0022-2275/small "Fxr[superscript -/-] mice adapt to biliary obstruction by enhanced phase I detoxification and renal elimination of bile acids")
PDF
