Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry

The heart contracts incessantly and requires a constant
supply
of energy, utilizing numerous metabolic substrates, such as fatty
acids, carbohydrates, lipids, and amino acids, to supply its high
energy demands. Therefore, a comprehensive analysis of various metabolites
is urgently needed for understanding cardiac metabolism; however,
complete metabolome analyses remain challenging due to the broad range
of metabolite polarities, which makes extraction and detection difficult.
Herein, we implemented parallel metabolite extractions and high-resolution
mass spectrometry (MS)-based methods to obtain a comprehensive analysis
of the human heart metabolome. To capture the diverse range of metabolite
polarities, we first performed six parallel liquid–liquid extractions
(three monophasic, two biphasic, and one triphasic) of healthy human
donor heart tissue. Next, we utilized two complementary MS platforms
for metabolite detection: direct-infusion ultrahigh-resolution Fourier-transform
ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography
quadrupole time-of-flight tandem MS (LC-Q-TOF-MS/MS). Using DI-FTICR
MS, 9644 metabolic features were detected where 7156 were assigned
a molecular formula and 1107 were annotated by accurate mass assignment.
Using LC-Q-TOF-MS/MS, 21,428 metabolic features were detected where
285 metabolites were identified based on fragmentation matching against
publicly available libraries. Collectively, 1340 heart metabolites
were identified in this study, which span a wide range of polarities
including polar (benzenoids, carbohydrates, and nucleosides) as well
as nonpolar (phosphatidylcholines, acylcarnitines, and fatty acids)
compounds. The results from this study will provide critical knowledge
regarding the selection of appropriate extraction and MS detection
methods for the analysis of the diverse classes of human heart metabolites.