Dynamic Assessment of Functional Lipidomic Analysis in Human Urine

Abstract

The development of enabling mass spectrometry platforms for the quantification of diverse lipid species in human urine is of paramount importance for understanding metabolic homeostasis in normal and pathophysiological conditions. Urine represents a non-invasive biofluid that can capture distinct differences in an individual's physiological status. However, currently there is a lack of quantitative workflows to engage in high throughput lipidomic analysis. This study describes the development of a MS/MS(ALL) shotgun lipidomic workflow and a micro liquid chromatography-high resolution tandem mass spectrometry (LC-MS/MS) workflow for urine structural and mediator lipid analysis, respectively. This workflow was deployed to understand biofluid sample handling and collection, extraction efficiency, and natural human variation over time. Utilization of 0.5 mL of urine for structural lipidomic analysis resulted in reproducible quantification of more than 600 lipid molecular species from over 20 lipid classes. Analysis of 1 mL of urine routinely quantified in excess of 55 mediator lipid metabolites comprised of octadecanoids, eicosanoids, and docosanoids generated by lipoxygenase, cyclooxygenase, and cytochrome P450 activities. In summary, the high-throughput functional lipidomics workflow described in this study demonstrates an impressive robustness and reproducibility that can be utilized for population health and precision medicine applications.

Cold-Activated Lipid Dynamics in Adipose Tissue Highlights a Role for Cardiolipin in Thermogenic Metabolism

“…Before extraction, a designated cocktail of internal standards was added to 25 mL plasma. 4 mL chloroform:methanol (1:1, v/v) was added to each sample, and the lipid extraction was performed as previously described (Kiebish et al, 2010; Rockwell et al, 2016). Lipid extraction was automated using a customized sequence on a Hamilton Robobtics STARlet system (Hamilton, Reno, NV, USA) to meet the high-throughput requirements.…”

Multi-OMIC analysis of brain and serum from chronically-stressed mice reveals network disruptions in purine metabolism, fatty acid beta-oxidation, and antioxidant activity that are reversed by antidepressant treatment

“…Standards were chosen so that they represented each lipid class and were at designated concentrations chosen to provide the most accurate quantitation and dynamic range for each lipid species. 4 mL chloroform:methanol (1:1, v/v) was added to each sample and the lipid extraction was performed as described 13,14 . Lipid extraction was automated using a customized sequence on a Hamilton Robotics STARlet system (Hamilton, Reno, NV) to meet the high-throughput requirements.…”

Monoacylglycerol Analysis Using MS/MSALL Quadruple Time of Flight Mass Spectrometry

“…High-resolution analysis of fragment ions using the MS/MS ALL strategy was used for accurate quantification as previously reported [ 16 , 18 ]. To evaluate the linear response of MAG and its dynamic quantification range based on the MAG-derived fragment ions, we prepared a serial dilution of MAG 17:1 in human plasma and plotted the calibration curves ranging from 2.6 fmol/µL to 40 pmol/µL, with an R 2 regression value of 0.9931 ( Figure 8 ).…”

“…Herein, we report a high throughput and highly sensitive MS/MS ALL workflow for profiling MAG molecular species via the direct infusion of lipid extracts on a quadrupole time of flight mass spectrometer [ 13 , 16 ]. MS/MS ALL is a direct infusion DIA technique, specifically designed for global lipidomics [ 13 , 16 ]. In this report, all precursor ions in the Q1 quadrupole at 1 Da m / z window were selected.…”