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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M800313-JLR200 on July 30, 2008

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Journal of Lipid Research, Vol. 49, 2620-2626, December 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Stable carbon isotope discrimination by human 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Svena M. Lüdke1,*, Ulrich Flenker*, Wilhelm Schänzer* and Dietmar Schomburg{dagger}

* Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany
{dagger} Institute for Biochemistry and Biotechnology, Technical University Braunschweig, Langer Kamp 19B, 38106 Braunschweig, Germany

Published, JLR Papers in Press, July 30, 2008.

1 The {delta}13C value is defined as:

Formula 14(14)

The standard is carbon dioxide from PeeDee Belemnite isotopic standard limestone (29).

1 To whom correspondence should be addressed. e-mail: s.luedke{at}biochem.dshs-koeln.de

The aim of this study was to investigate the possible existence and magnitude of stable carbon isotope discrimination by human 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). The catalytic portion of HMGR was expressed and purified. The reaction product mevalonate was lactonized and extracted from the reaction mixture by a solid-phase extraction protocol. Stable carbon isotope ratios of mevalonolactone (MVL) were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. An average fractionation factor 12k/13k of 1.0031 ± 0.0004 for all carbon atoms contained in MVL was estimated by the method of internal competition. The value was calculated by nonlinear curve fitting, where the ratio 13C/12C of MVL was plotted versus the fraction of reaction.

Supplementary key words mevalonate pathway • steroids • kinetic isotope effect • isotope ratio mass spectrometry • curve fitting • doping control

Abbreviations: GC-C-IRMS, gas chromatography-combustion-isotope ratio mass spectrometry; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; KIE, kinetic isotope effect; MVL, mevalonolactone; T, testosterone


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