In this poster, a tandem mass spectrometry method is developed to monitor ammonia metabolism in the mosquito Aedes aegypti, a species of mosquito which transmits yellow fever and dengue. This has been done by feeding mosquitoes with ¹⁵NH₄Cl, and then identifying and quantifying several labeled and unlabeled amino acids involved in ammonia metabolism by mass spectrometry in the multi-reaction monitoring mode. The method developed can be extended to quantify isotope labeled amino acids in other biological systems. Time course results from this study will facilitate the understanding ammonia metabolism in mosquitoes, and ultimately provide valuable information for mosquito control, which is important due to millions of deaths caused annually by the mosquito transmitted diseases worldwide.

The identification and quantification of amino acids are being used for diagnosis and monitoring of inherited diseases related to amino acid metabolism in human infants. Tandem mass spectrometry (MS) is routinely used for these studies. However, a notable deficiency of this method is its inability to distinguish between glutamine (Gln) and glutamic acid (Glu), two important amino acids involved in ammonia metabolism. In this study, the current derivatization method was modified and the related fragmentation mechanism was investigated, a method was developed to distinguish both labeled and unlabeled glutamine and glutamic acid and to quantify amino acids. Moreover, glutamine with ¹⁵N labeling at different positions (either the side chain or the backbone, or both) can be distinguished and quantified without any separation.

Results showed that the velocity of incorporation of ¹⁵N into Gln, alanine (Ala), Glu and proline (Pro) of whole body was rapid. Just 1 minute after feeding mosquitoes, labeled Gln, Ala, Glu, and Pro were detected. The data indicate that mosquitoes remove toxic ammonia at a high rate and showed that ammonia first was incorporated into the amide side chain of Gln and then into the amino group of Gln, Ala, Glu and Pro. The maximum value of labeled Gln was observed at 1 hour post feeding whereas for the rest of amino acids the peak was observed at 24 hours after feeding. At 96 hours posts feeding the labeled amino acids were significantly reduced. The addition of inhibitors of key enzymes in the ammonia metabolism pathway to the ¹⁵NH₄Cl solution showed that mosquitoes efficiently metabolize ammonia through a specific metabolic route that mainly involves glutamine synthetase and glutamate synthase.

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