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Journal of Lipid Research, Vol. 43, 2188-2195, December 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Ammonium hydroxide hydrolysis

Alba Silipo, Rosa Lanzetta, Angela Amoresano, Michelangelo Parrilli and Antonio Molinaro¹

Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli Federico II, Via Cintia 4, I-80126, Napoli, Italy

¹ To whom correspondence should be addressed. e-mail: molinaro{at}unina.it

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS.

The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.

Supplementary key words ammonium hydroxide • endotoxin • gram-negative bacteria

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