Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes

Abstract

In Aedes aegypti females, the ammonia released during blood meal digestion is partially metabolized to facilitate the disposal of excess nitrogen. In this study, we used low- and high-resolution liquid chromatography-mass spectrometry (LC/MS) techniques to investigate the role of glucose during ammonia detoxification. Mosquitoes were fed a blood meal supplemented with [1,2-¹³C₂]glucose, and downstream metabolites were measured for 24 h. Quantification of [¹³C] amino acids in the entire mosquito body was conducted without sample derivatization using selected reaction monitoring of mass transitions that are indicative of the structural position of [¹³C] atom incorporation. Identification of unlabeled and [¹³C] isotopologs of 43 compounds, including amino acids, amino acid derivatives, and organic acids, was performed by high-resolution LC/MS techniques. Blood-fed mosquitoes synthesized [¹³C] metabolites in mainly 2 carbon positions from [1,2-¹³C₂]glucose. [¹³C₂]Ala and [¹³C₂]Pro were the most abundant and rapidly labeled amino acids synthesized. Additional [¹³C] amino acids, [¹³C] amino acid derivatives, and [¹³C] organic acids in 1 or 2 carbon positions were also identified. Two kinetic routes were proposed based on the incorporation of a [¹³C] atom at position 1 in specific amino acids. Our findings provide evidence that glucose is used for ammonia detoxification and [¹³C] uric acid synthesis through multiple metabolic pathways, uncovering a metabolic link at the carbon atomic level in ammonia metabolism of A. aegypti-Horvath, T. D., Dagan, S., Lorenzi, P. L., Hawke, D. H., Scaraffia, P. Y. Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes.

Keywords: mass spectrometry; metabolomics; nitrogen waste.

Figure 1.

A representative total ion current chromatogram for the cumulative instrument response for all unlabeled and [¹³C] isotopologs measured for glutamic acid, alanine, proline, and glutamine present in mosquito whole-body extracts. Each peak is labeled with the name and structure (with numbered carbon atoms) for each corresponding amino acid, and Table 1 lists the number of transitions monitored in the acquisition method, the retention time of interest, and the dMRM (a specific time-shared MRM mode utilized by Agilent triple-quadrupole mass spectrometers) acquisition window specified for each amino acid and its corresponding isotopologs. Mosquitoes were fed on a BM, BM supplemented with glucose, or BM supplemented with [1,2-¹³C₂] glucose. Data are representative of 3 independent experiments.

Figure 2.

Time-course dependence (0–24 h) of selected SRM transitions for [¹³C]alanine (A–C) and [¹³C]glutamic acid (D–F) in mosquito whole-body extracts. Mosquitoes were fed a blood meal supplemented with [1,2-¹³C₂]glucose. The amino acids were analyzed in mosquito whole-body extracts by LR-LC/MS-MS. SRM transitions were from the [M + 1]⁺ to [M + 5]⁺ precursor isotopologs to various product isotopologs. Data are presented as means ± sem of 3 independent samples. N = 15 mosquitoes for each time point.

Figure 3.

Time dependence (0–24 h) of selected SRM transitions for [¹³C]glutamine (A–C) and [¹³C]proline (D–F) in mosquito whole-body extracts. Mosquitoes were fed on a blood meal supplemented with [1,2-¹³C₂]glucose. The amino acids were analyzed in mosquito whole-body extracts by LR-LC/MS-MS. SRM transitions were from the [M + 1]⁺ to [M + 5]⁺ precursor isotopologs to various product isotopologs. Data are presented as means ± sem of 3 independent samples. N = 15 mosquitoes for each time point.

Figure 4.

Unlabeled and [¹³C] isotopologs of amino acids and amino acid derivatives present in mosquito whole-body extracts. Measurements were made using an Orbitrap HRAM-LC/MS instrument. Plots are of the normalized mass isotopomer distributions against the degree of [¹³C] labeling for metabolites that exhibited enrichment of [¹³C] labeling greater than the isotopically labeled background. Each column corresponds to a single metabolite isotopolog series, and each individual data plot corresponds to an individual time point during the time course studied. Three groups of mosquitoes were fed on 3 different diets: BM, BM supplemented with glucose, and BM supplemented with [1,2-¹³C₂]glucose, as indicated in Materials and Methods.

Figure 5.

Unlabeled and [¹³C] isotopologs of 2-HG, glutamic acid, Lac, Pyr, and uric acid present in mosquito whole-body extracts. Measurements were made using an Orbitrap HRAM-LC/MS instrument. Plots are of the normalized mass isotopomer distributions against the degree of [¹³C] labeling for metabolites that exhibited enrichment of [¹³C] labeling greater than the isotopically labeled background. Each column corresponds to a single metabolite isotopolog series, and each individual data plot corresponds to an individual time point during the time course studied. Three groups of mosquitoes were fed on 3 different diets: BM, BM supplemented with glucose, and BM supplemented with [1,2-¹³C₂]glucose, as indicated in Materials and Methods.

Figure 6.

A proposed scheme of metabolic interactions at the [¹³C] atomic levels induced by glucose on ammonia metabolism. The [1,2-¹³C₂]glucose can be converted into [1,2-¹³C₂]glucose-6-phosphate, which can be metabolized through glycolysis ([¹³C] atoms are designated in red) or entered into the PPP ([¹³C] atoms are designated in blue). Not all possible reactions between unlabeled and [¹³C] metabolites are represented in the scheme. CS, citrate synthetase; GDH, glutamate dehydrogenase; GltS, glutamate synthase; GS, glutamine synthetase; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; P5CDH, pyrroline-5-carboxylate dehydrogenase; P5CR, pyrroline-5-carboxylate reductase; P5CS, pyrroline-5-carboxylate synthase; PDH, pyruvate dehydrogenase.