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The Liebermann–Burchard Reaction: Sulfonation, Desaturation, and Rearrangment of Cholesterol in Acid

  • Original Article
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Lipids

Abstract

In the Liebermann–Burchard (LB) colorimetric assay, treatment of cholesterol with sulfuric acid, acetic anhydride, and acetic acid elicits a blue color. We studied the reactivity of cholesterol under LB conditions and provide definitive NMR characterization for approximately 20 products, whose structure and distribution suggest the following mechanistic picture. The major reaction pathways do not involve cholestadienes, i-steroids, or cholesterol dimers, as proposed previously. Instead, cholesterol and its acetate and sulfate derivatives undergo sulfonation at a variety of positions, often with skeletal rearrangements. Elimination of an SO3H group as H2SO3 generates a new double bond. Repetition of this desaturation process leads to polyenes and ultimately to aromatic steroids. Linearly conjugated polyene cations can appear blue but form too slowly to account for the LB color response, whose chemical origin remains unidentified. Nevertheless, the classical polyene cation model is not excluded for Salkowski conditions (sulfuric acid), which immediately generate considerable amounts of cholesta-3,5-diene. Some rearrangements of cholesterol in H2SO4 resemble the diagenesis pathways of sterols and may furnish useful lipid biomarkers for characterizing geological systems.

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Abbreviations

ES:

Electrospray

FAB:

Fast-atom bombardment

HPLC:

High-performance liquid chromatography

LB:

Liebermann–Burchard

NBA:

3-Nitrobenzyl alcohol

TLC:

Thin-layer chromatography

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Acknowledgements

This work was supported in part by startup funding from Texas Southern University for Q.X. We thank Alemka Kisic for supplying purified cholesterol. Quantum-mechanical calculations were carried out in part on the Rice Terascale Cluster funded by the NSF (EIA-0216467), Intel, and Hewlett-Packard and on the Rice Cray XD1 Research Cluster funded by the NSF (CNS-0421109) in partnership with AMD and Cray.

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Author notes

  1. Jihai Pang

    Present address: Pharmaceutical Development Center, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Texas Southern University, 3100 Cleburne Street, Houston, TX, 77004, USA

    Quanbo Xiong

  2. Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA

    Quanbo Xiong, William K. Wilson & Jihai Pang

Authors
  1. Quanbo Xiong
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  2. William K. Wilson
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  3. Jihai Pang
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Correspondence to Quanbo Xiong.

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Xiong, Q., Wilson, W.K. & Pang, J. The Liebermann–Burchard Reaction: Sulfonation, Desaturation, and Rearrangment of Cholesterol in Acid. Lipids 42, 87–96 (2007). https://doi.org/10.1007/s11745-006-3013-5

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