LC-MS-Based Metabolic Fingerprinting of Aqueous Humor

Abstract

Aqueous humor (AH) is a transparent fluid which fills the anterior and posterior chambers of the eye. It supplies nutrients and removes metabolic waste from avascular tissues in the eye. Proper homeostasis of AH is required to maintain adequate intraocular pressure as well as optical and refractive properties of the eye. Application of metabolomics to study human AH may improve knowledge about the molecular mechanisms of eye diseases. Until now, global analysis of metabolites in AH has been mainly performed using NMR. Among the analytical platforms used in metabolomics, LC-MS allows for the highest metabolome coverage. The aim of this study was to develop a method for extraction and analysis of AH metabolites by LC-QTOF-MS. Different protocols for AH preparation were tested. The best results were obtained when one volume of AH was mixed with one volume of methanol : ethanol (1 : 1). In the final method, 2 µL of extracted sample was analyzed by LC-QTOF-MS. The method allowed for reproducible measurement of over 1000 metabolic features. Almost 250 metabolites were identified in AH and assigned to 47 metabolic pathways. This method is suitable to study the potential role of amino acids, lipids, oxidative stress, or microbial metabolites in development of ocular diseases.

Figure 1

Total compound chromatograms obtained for AH samples extracted with different volumes of methanol : ethanol. Aqueous humor samples were prepared by addition of one AH volume to one (red), two (green), or three (blue) volumes of methanol : ethanol and analyzed with 20 min gradient.

Figure 2

Metabolic pathway analysis. Metabolic pathway analysis was performed with MetaboAnalyst 3.0. Calculated p value was established based on the pathway enrichment analysis while pathway impact value based on the pathway topology analysis. Twenty of the most significant pathways are marked with the numbers: 1. phenylalanine metabolism, 2. taurine and hypotaurine metabolism, 3. arginine and proline metabolism, 4. pantothenate and CoA biosynthesis, 5. pyruvate metabolism, 6. biotin metabolism, 7. glyoxylate and dicarboxylate metabolism, 8. tryptophan metabolism, 9. alanine, aspartate, and glutamate metabolism, 10. aminoacyl-tRNA biosynthesis, 11. valine, leucine, and isoleucine metabolism, 12. beta-alanine metabolism, 13. ascorbate and aldarate metabolism, 14. sphingolipid metabolism, 15. glycolysis and gluconeogenesis, 16. nitrogen metabolism, 17. phenylalanine, tyrosine, and tryptophan biosynthesis, 18. glycine, serine, and threonine metabolism, 19. pyrimidine metabolism, and 20. arginine and ornithine metabolism.