Analysis of Tissue Steroids By Liquid-Gel Chromatography and Computerized Gas Chromatography-Mass Spectrometry

Axelson, Magnus; Schumacher, Gitta; Sjövall, Jan

doi:10.1093/chromsci/12.10.535

Cited by 38 publications

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“…One milliliter of each extract was placed into a glass vial, dried under a stream of N 2 (N‐evap, Organomation, Berlin, MA), and 100 μl of Trisil reagent (Supelco Inc.) was used to convert sterols to their TMS ether derivatives. The derivatized sample was dried under a stream of N 2 and the extract redissolved in hexane (4 × 0.5 ml) and passed through a small column of the lipophilic gel, Lipidex 5000 to further purify the sample (Axelson, Schumacher, and Sjovall 1974). The sterol TMS ethers were then analyzed by GLC as above but using the oven temperature program, 225 °C for 2 min, an increase of 2 °C/min to 290 °C, and then held isothermal at the final temperature for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Pneumocystis carinii Sterol 14α‐Demethylase Activity in Saccharomyces cerevisiae erg11 Knockout Mutant: Sterol Biochemistry

Nkinin

Stringer

Keely

et al. 2011

J Eukaryotic Microbiology

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Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.

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Section: Methodsmentioning

confidence: 99%

Pneumocystis carinii Sterol 14α‐Demethylase Activity in Saccharomyces cerevisiae erg11 Knockout Mutant: Sterol Biochemistry

Nkinin

Stringer

Keely

et al. 2011

J Eukaryotic Microbiology

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Quantitative Massenspektrometrie in Biochemie und Medizin

Lehmann

Schulten

1978

Angew. Chem.

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Der vorliegende Aufsatz gibt einen uberblick iiber den Stand der quantitativen Analyse mit dem Massenspektrometer in Biochemie und Medizin. Die Grundlagen dafur, die gekoppelte Anwendung chromatographischer und massenspektrometrischer Methoden, die Entwicklung empfindlicher, genauer und iiberdies spezifischer massenspektrometrischer Nachweismethoden sowie das Prinzip der Verdiinnung mit stabilen Isotopen werden an Beispielen erlautert. Die wichtigsten Anwendungsgebiete sind die Verfolgung der Pharmakokinetik von Arzneimitteln und aktiven Metaboliten, die Erforschung von Stoffwechselwegen, die Unterstiitzung und Spezifizierung medizinischer Diagnosen und schlieI3lich die Qualitatskontrolle einfacherer quantitativer Verfahren der klinischen Chemie.

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Quantitative Mass Spectrometry in Biochemistry and Medicine

Lehmann

Schulten

1978

Angew. Chem. Int. Ed. Engl.

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The current state of quantitative analysis with the mass spectrometer in biochemistry and medicine is reviewed. The basic principles of mass spectrometry, the latter's combination with chromatography, the development of sensitive, exact, and in particular specific mass-spectrometric methods of detection, and the principle of dilution with stable isotopes are illustrated by examples. The most important fields of application are: the pharmacokinetics of drugs and active metabolites, investigation of metabolic pathways, supporting of medical diagnoses and enhancement of their specificity, and finally checking the quality of simpler quantitative processes in clinical chemistry.

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Analysis of Tissue Steroids By Liquid-Gel Chromatography and Computerized Gas Chromatography-Mass Spectrometry

Cited by 38 publications

References 0 publications

Pneumocystis carinii Sterol 14α‐Demethylase Activity in Saccharomyces cerevisiae erg11 Knockout Mutant: Sterol Biochemistry

Pneumocystis carinii Sterol 14α‐Demethylase Activity in Saccharomyces cerevisiae erg11 Knockout Mutant: Sterol Biochemistry

Quantitative Massenspektrometrie in Biochemie und Medizin

Quantitative Mass Spectrometry in Biochemistry and Medicine

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