Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry

We have established a protocol to study the kinetics of incorporation of 15N into glutamine (Gln), glutamic acid (Glu), alanine (Ala) and proline (Pro) in Aedes aegypti females. Mosquitoes were fed 3% sucrose solutions containing either 80 mM 15NH4Cl or 80 mM glutamine labeled with 15N in either the amide nitrogen or in both amide and amine nitrogens. In some experiments, specific inhibitors of glutamine synthetase or glutamate synthase were added to the feeding solutions. At different times post feeding, which varied between 0 and 96 h, the mosquitoes were immersed in liquid nitrogen and then processed. These samples plus deuterium labeled internal standards were derivatized as dimethylformamidine isobutyl esters or isobutyl esters. The quantification of 15N-labeled and unlabeled amino acids was performed by using mass spectrometry techniques. The results indicated that the rate of incorporation of 15N into amino acids was rapid and that the label first appeared in the amide side chain of Gln and then in the amino group of Gln, Glu, Ala and Pro. The addition of inhibitors of key enzymes related to the ammonia metabolism confirmed that mosquitoes efficiently metabolize ammonia through a metabolic route that mainly involves glutamine synthetase (GS) and glutamate synthase (GltS). Moreover, a complete deduced amino acid sequence for GltS of Ae. aegypti was determined. The sequence analysis revealed that mosquito glutamate synthase belongs to the category of NADH-dependent GltS.