Alanine is a major end product of metabolism by Giardia lamblia: a proton nuclear magnetic resonance study

Abstract

1H-NMR spectroscopy was used to monitor the major metabolic end products released by Giardia lamblia when maintained anaerobically in culture in Diamond's TYI-S-33 medium. Spectra were acquired for the cell-free medium and the resonances of metabolites utilised and produced during cell growth identified by the addition of pure compounds and by difference spectroscopy. The major metabolites produced by the parasite were alanine, ethanol and acetate, with increases in concentrations in the media after 4 days' growth (end of log phase) of 18, 15 and 4 mM, respectively. The production of both alanine and ethanol approximated to cell growth, with ethanol formation lagging behind alanine during log growth but predominating after the parasites entered stationary phase. Acetate was formed at a more constant rate during growth. Glucose utilisation was sufficient to account for only 50% of the total carbon appearing in alanine, ethanol and acetate. The aminotransferase inhibitors l-cycloserine and carboxymethoxylamine inhibited growth and selectively inhibited the production of alanine. Analysis of the amino acid composition of the medium by HPLC showed that the only amino acid produced, apart from alanine, was proline, which increased in concentration in the medium by 4 mM after 4 days. There was also a 7 mM increase in ammonia over the same period. The only amino acids that were utilised were arginine and the components of an unresolved peak comprising serine, asparagine and glutamine. The observed changes in amino acid composition of the medium were not sufficient to account for the nitrogen in the alanine produced, nor to account for the difference in total carbon between the glucose used and the major metabolites formed. It appears that when cultured in a complex medium, G. lamblia uses other carbon sources, possibly protein, in addition to glucose.