Methyl Ether-Derivatized Sterols and Coprostanol Produced via Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH).

Sterols are widely distributed in nature from lipids in organisms to sediments. As a conventional method, extraction and derivatization with TMS have been applied for sterol analysis, requiring a long preparation time for gas chromatography-mass spectrometry analysis. In this study, for sterol analysis, thermochemolysis using tetramethylammonium hydroxide (TMAH) was applied. This method performs hydrolysis and methylation simultaneously; thus, free and ether-bonding sterols can be analyzed as sterol methyl ethers in a relatively short time period. A sediment sample from a tideland (the Yatsu tideland, Japan) was analyzed using the TMAH method, and we detected more than 10 sterols, which include cholest-5-en-3ß-ol (cholesterol), 24-ethylcholest-5-en-3ß-ol (sitosterol), 24-methylcholesta-5,22E-3ß-ol (brassicasterol), 24-ethylcholesta-5,24(28)Z-dien-3ß-ol (isofucosterol), 4α,23,24-trimethyl-5α(H)-cholest-22E-en-3ß- ol (dinosterol), and 5ß(H)-cholestan-3ß-ol (coprostanol). The detection of the various sterols can be attributed to multiple natural and artificial sources around the Yatsu tideland. In this paper, the mass spectra of these sterols are provided together with an interpretation of their fragmentation patterns. Additionally, the fecal pollution in the Yatsu tideland is discussed in the context of the detection of coprostanol.

Sterols of the Toxic Marine Dinoflagellate, Pyrodinium bahamense.

Pyrodinium bahamense is a dinoflagellate of concern in subtropical and tropical coastal environments. To date, there is only a single published study on its fatty acids, but no published data on its sterol composition. Sterols, which are membrane-reinforcing lipids in eukaryotes, display a great diversity of structures in dinoflagellates, with some serving as chemotaxonomic markers. We have examined the sterol compositions of two isolates of P. bahamense from Indian River Lagoon and Tampa Bay, Florida, and have found both to produce three sterols: cholesterol, dinosterol, and 4α-methylgorgostanol. All three sterols are found in closely related, armored taxa.

[Determination of three anthroic acids in petri dish cultured Antrodia camphorate by quantitative analysis of multi-component with single marker].

The present study is establish the quantitative analysis of multi-component with single marker for determining three anthroic acids, (25S)-antcin K, (25R)-antcin K and (25S)-antcin C in the petri dish cultured Antrodia camphorata. The relative correction factors of (25S)-antcin K and (25R)-antcin K were established by high performance liquid chromatography with (25S)-antcin C as the internal reference. Relative correction factors were used to calculate the contents of (25S)-antcin K and (25R)-antcin K which were difficult to gain in abundance. At the same time, the contents of these three compounds were determined by external standard method. Two methods were compared to evaluate the accuracy and rationality of the multi-components with single marker method in the determination of the petri dish cultured A. camphorate. It was found that the quantitative method of multi-component with single marker and external standard method showed no significant difference. In summary, taking (25S)-antcin C as the internal reference, the method of multi-component with single marker can be applied to the quantitative analysis of (25S)-antcin K and (25R)-antcin K in the petri dish cultured A. camphorata.

Charge-tagging liquid chromatography-mass spectrometry methodology targeting oxysterol diastereoisomers.

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography - mass spectrometry with multistage fragmentation.

No Significant Increase in the Δ4- and Δ7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation.

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants.

Fast separation and quantification of steroid hormones Δ4- and Δ7-dafachronic acid in Caenorhabditis elegans.

Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 µm core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.

Absolute quantification of a steroid hormone that regulates development in Caenorhabditis elegans.

Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-ß, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.

High performance liquid chromatography used for quality control of Achyranthis Radix.

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-inokosterone (2) and 25S-inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm × 4.6 mm, 4 µm) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

Preservation potential of ancient plankton DNA in Pleistocene marine sediments.

Recent studies have shown that ancient plankton DNA can be recovered from Holocene lacustrine and marine sediments, including from species that do not leave diagnostic microscopic fossils in the sediment record. Therefore, the analysis of this so-called fossil plankton DNA is a promising approach for refining paleoecological and paleoenvironmental information. However, further studies are needed to reveal whether DNA of past plankton is preserved beyond the Holocene. Here, we identified past eukaryotic plankton members based on 18S rRNA gene profiling in eastern Mediterranean Holocene and Pleistocene sapropels S1 (~9 ka), S3 (~80 ka), S4 (~105 ka), and S5 (~125 ka). The majority of preserved ~400- to 500-bp-long 18S rDNA fragments of microalgae that were studied in detail (i.e. from haptophyte algae and dinoflagellates) were found in the youngest sapropel S1, whereas their specific lipid biomarkers (long-chain alkenones and dinosterol) were also abundant in sediments deposited between 80 and 124 ka BP. The late-Pleistocene sediments mainly contained eukaryotic DNA of marine fungi and from terrestrial plants, which could have been introduced via the river Nile at the time of deposition and preserved in pollen grains. A parallel analysis of Branched and Isoprenoid Tetraethers (i.e. BIT index) showed that most of the organic matter in the eastern Mediterranean sediment record was of marine (e.g. pelagic) origin. Therefore, the predominance of terrestrial plant DNA over plankton DNA in older sapropels suggests a preferential degradation of marine plankton DNA.

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.

Development and validation of a method for the purity determination of (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS) in pharmaceutical products containing recombinant human albumin.

A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200 ng per unit) in pharmaceutical formulations containing RECOMBUMIN (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin. After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250 nm. The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3-0.8% (depending on the impurity) corresponding to 1.9-5.1 ng. The limit of quantification was found to be 0.8-2.5% (depending on the impurity) corresponding to 5.2-16 ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100-129% for the six known impurities. The tested range for FF-MAS was from 320 to 960 ng corresponding to 50-150% of the nominal concentration (640 ng, injection volume is 100 microl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r(2)) was 0.999 for FF-MAS (50-150% level) and 0.977-0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities. The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product.

Quantitation of lanosterol and its major metabolite FF-MAS in an inhibition assay of CYP51 by azoles with atmospheric pressure photoionization based LC-MS/MS.

Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14alpha-demethylase (CYP51) is an enzyme inhibited by azoles. Only few data have been reported showing their inhibitory potency since an assay in an in vitro system is not available so far. In the present work an inhibition assay using human recombinant CYP51, coexpressed with human P450 oxido-reductase by the baculovirus/insect cell expression system, and LC-MS/MS as analytical method is described. Atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were used with a triple quadrupole mass spectrometer to compare quantitation of lanosterol (substrate) and 4,4-dimethyl-5alpha-cholesta-8,14,24-triene-3beta-ol (FF-MAS) (product of CYP51) with d(6)-2,2,3,4,4,6-cholesterol (d(6)-cholesterol) as internal standard. Optimization of analytical parameters resulted in a LC-APPI-MS/MS method with a LOQ of 10 pg on column for FF-MAS. The sensitivity of the method (LOD 0.5 ng/ml) makes it possible to analyze supernatants of inhibition experiments after precipitation of proteins by isopropanol without any sample enrichment. The coefficient of variation of the analytical method was <20% (n = 5) for FF-MAS, lanosterol and d(6)-cholesterol. The external calibration curve was linear from 1 to 10,000 ng/ml with R(2) >/= 0.999 and an accuracy of 94-115%. Compared with APCI, APPI provides a ten- to 500-fold increase in sensitivity for the analytes in this study. IC(50) values of epoxiconazole and miconazole-two widely used azole fungicides used in agriculture and in human medicine, respectively-were 1.95 microM and 0.057 microM.

Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS).

BACKGROUND: The objective of the study was to investigate the effect of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) when added to the culture media on the incidence of chromosomal abnormalities and pre-embryo development in human pre-embryos. METHODS: 243 women undergoing IVF/ICSI treatment donated 353 oocytes in a multicentre, prospective, randomized, double blind, four-arm, controlled trial performed at Danish and Swedish public and private IVF centers. Metaphase II oocytes were randomly assigned to: FF-MAS 5 microM, FF-MAS 20 microM, ethanol 0.2% (vehicle control) or water for injection (inert control). The exposure regimen of FF-MAS to the human oocytes was 4 h prior to fertilization by ICSI and 20 h exposure post ICSI. The primary endpoint was the incidence of numerical chromosomal abnormalities. Secondary endpoints were cleavage rate and pre-embryo quality. RESULT: On the pre-embryo level, no significant differences in chromosomal abnormality rate were observed among the four groups. However, the percentage of uniformly normal pre-embryos was significantly lower in the pooled FF-MAS group (5 microM: 12% and 20 microM: 17%) than in the pooled control group (inert control 32% and vehicle control 42%). A high level of mosaicism (41-60%) was found in all groups. At the blastomere level, the percentage of blastomeres categorized as normal was significantly lower in the FF-MAS 5 microM group (41%) and the FF-MAS 20 microM (29%) group versus the inert (52%) and the vehicle (61%) groups. Significantly reduced cleavage and good quality pre-embryo rates were found in both FF-MAS groups. CONCLUSION: FF-MAS increased the rate of aneuploidy and had detrimental effects on cleavage and pre-embryo development, when exposed both before and after fertilization.

[Study on the fingerprints of Achyranthes bidentata Bl. by HPLC/UV/MS].

OBJECTIVE: To establish the fingerprints of Achyranthes bidentata Bl. by HPLC/UV/MS. METHOD: Separation was performed on Aglient Zorbax SB-C18 column. Gradient elute was performed by the mobile phase consisting of methanol and 0.1% formic acid-water with the flow rate of 1.0 ml/min. RESULT: Perfect fingerprints were obtained which can be used for the evaluation of Achyranthes bidentata Bl. Four common peaks were confirmed in fingerprints. Three compounds were elucidated as 5-hydroxymethyl furaldehyde, ecdysterone, inokosterone by HPLC/MS. CONCLUSION: The method can be applied to the quality control of Achyranthes bidentata Bl.

Gas chromatography/mass spectrometry demonstration of steryl glycosides in eucalypt wood, Kraft pulp and process liquids.

The occurrence of steryl glycosides (SG) and acyl steryl glycosides (ASG) in eucalypt (Eucalyptus globulus) wood has been investigated. These compounds were analyzed as their trimethylsilyl ethers by gas chromatography/mass spectrometry (GC/MS) using a 15 m length high-temperature capillary column with a thin film, and identified on basis of their mass spectra and relative retention times comparing with those of authentic standards. Significant amounts of SG were identified in eucalypt wood whilst only traces of ASG could be detected. Eucalypt SG and ASG occur in the pyranoside form, which is readily distinguishable from the furanoside configuration by the mass spectra of their trimethylsilyl derivatives. The sterol part of the SG and ASG consisted of sitosterol, being sitosteryl 3beta-D-glucopyranoside and sitosteryl (6'-O-palmitoyl)-3beta-D-glucopyranoside, the major SG and ASG found in E. globulus wood. The presence of SG and ASG was also investigated after kraft cooking by analyzing unbleached pulp and process water samples. The GC/MS results also revealed the presence of sitosteryl 3beta-D-glucopyranoside in these samples. By contrast, no ASG could be detected. Therefore, we have shown that SG survive the kraft cooking and can be found at least partly intact after pulping, being a possible cause for pitch deposits together with free and esterified sterols.

Content of meiosis activating sterols in equine follicular fluids: correlation to follicular size and dominance.

Meiosis activating sterols (MAS) are pre-cholesterol sterols that can be isolated from follicular fluid (FF-MAS) or testes (T-MAS). Meiosis activating sterols trigger the resumption of meiosis in cultured meiotically competent oocytes. In the present work MAS, cholesterol and progesterone were assayed by HPLC in follicular fluids collected from pony mares at fixed days after the last ovulation. Follicles were divided into two groups according to whether they were aspirated before or after Day 17 after the last ovulation. The latter group was further divided according to whether the follicle diameter was < or = 22 mm or > 27 mm. Both FF-MAS and T-MAS were detected in almost all samples. Overall, the total amount of MAS in the follicular fluids increased with the size of the follicles but was accompanied by a decrease in the amount of free cholesterol. The amounts of MAS and progesterone in > 27 mm follicles aspirated after Day 17 were significantly higher as compared to the other groups. A transversal cohort analysis showed that the largest follicle at the time of aspiration had the highest level of MAS after day 17 of the cycle, which was not always true for follicle samples aspirated before Day 17 of the cycle. The study demonstrates that the content of MAS in equine follicular fluids increased during follicular maturation concomitant with a decrease in the concentration of free cholesterol. Moreover, MAS concentration is higher in dominant follicles than in subordinate follicles. The MAS may therefore play an as yet unknown physiological role during pre-ovulatory maturation.

[Isolation and identification of two novel polyhydroxylated sterol from Spongia obligue].

Two novel sterols have been isolated from Spongia obligue collected from South China sea and their structure were identified as 24(28)-methylene-1,3,4,11-tetrohydroxycholest-5-en-18-oic acid and 24(28)-methylene-cholestene-5-en-1,3,8,11-tetraol respectively on the basic of the spectral data of MS, IR, 1HNMR and 13CNMR(DEPT).

Quantitation of meiosis activating sterols in human follicular fluid using HPLC and photodiode array detection.

A chromatographic assay for 4,4-dimethyl-5alpha-cholesta-8,14, 24-triene-3beta-ol (FF-MAS), and its reduced species, 4, 4-dimethyl-5alpha-cholesta-8,24-triene-3beta-ol (T-MAS), has been established for analysis of human follicular fluid (huFF). The assay also quantifies lanosterol, free cholesterol and progesterone. It was established using a pool of more than 100 individual follicular fluids from women undergoing in vitro fertilization treatment. Both FF-MAS and T-MAS were found in huFF, and can be quantified with HPLC equipped with photodiode array (PDA) detection. The examination wavelength for each analyte was chosen at the absorption maximum between 200 and 300 nm. Spike-recovery experiments revealed mean recoveries of 91 +/- 7.3% for lanosterol, 103 +/- 5.1% for FF-MAS, 104 +/- 5.5% for T-MAS, 103 +/- 4.5% for free cholesterol and 85 +/- 5.1% for progesterone. The lower recovery value for progesterone was due to a sub-optimal extraction procedure for this particular analyte, as indicated by re-extraction. The minimum amounts of FF-MAS required for quantification were 4 ng/mL and 23 ng/mL for T-MAS and lanosterol. FF-MAS was assayed to approximately 1.6 microM. T-MAS and lanosterol was assayed to about half of this value. No esterification of either MAS or lanosterol could be detected in huFF. Less than 10% of cholesterol was underivatized cholesterol, as more than 10 times the amount of free cholesterol could be assayed after extended saponification. This method can be used for evaluating the accumulation of MAS in huFF and its correlation to oocyte quality and fertilization parameters in in vitro fertilization programmes.

Identification of 19-nor-5,7,9(10)-cholestatrien-3 beta-ol in patients with Smith-Lemli-Opitz syndrome.

We have identified the third unknown sterol in the plasma and tissues of Smith-Lemli-Opitz homozygotes as 19-nor-5,7,9(10)-cholestatrien-3 beta-ol. The structure was established from capillary gas-liquid chromatography retention index and characteristic fragmentation pattern by mass spectrometry that were identical to a synthetic reference standard. Evidence is presented that 19-nor-5,7,9(10)-cholestatrien-3 beta-ol is not an artifact formed during the chemical isolation of the relatively unstable 7-dehydrocholesterol. It is possible that 19-nor-5,7,9(10)-cholestatrien-3 beta-ol may contribute to the clinical abnormalities in patients with Smith-Lemli-Opitz syndrome.

Use of an 18O2 inhalation technique and mass isotopomer distribution analysis to study oxygenation of cholesterol in rat. Evidence for in vivo formation of 7-oxo-, 7 beta-hydroxy-, 24-hydroxy-, and 25-hydroxycholesterol.

Cholesterol oxidation products (oxysterols) have been detected in many different tissues, often at concentrations 10(3) to 10(4) times lower than cholesterol. This constitutes a considerable risk of quantitation errors, since even a minor oxidation of cholesterol during sample processing would yield a substantial increase of oxysterol levels. It has therefore been suggested that some of the oxysterols do not occur in vivo and their detection in tissues merely are artifacts produced in vitro. In the present work, an 18O2 inhalation technique was developed in order to clarify which oxysterols are produced in vivo. Rats were exposed for 3 h to an atmosphere with a composition similar to normal air, except that it contained 18O2 instead of 16O2. Control rats were kept in 16O2-containing atmosphere throughout the experiment. The 18O enrichment of oxysterols in plasma and liver was determined by gas/liquid chromatography-mass spectrometry and mass isotopomer distribution analysis. In vivo formation of oxysterols, indicated by enrichment in 18O, was established for cholest-5-ene-3 beta, 7 alpha-diol, cholest-5-ene-3 beta, 7 beta-diol, 7-oxocholesterol, cholest-5-ene-3 beta,24-diol, cholest-5-ene-3 beta,25-diol, and cholest-5-ene-3 beta,27-diol. Additionally, it seems likely that cholest-5-ene-3 beta, 4 beta-diol is formed in vivo. The 18O labeling pattern suggests that there is incomplete equilibration between the liver and plasma pools of cholest-5-ene-3 beta,27-diol. No evidence for the in vivo formation of 5,6-oxygenated oxysterols was obtained.