Metabolic Profiling of the EmDia Cohort by a Scalable DIA-LC-MS Workflow

The sodium glucose cotransporter-2 (SGLT2) inhibitor empagliflozin improves glycemic control in type 2 diabetes mellitus (T2DM) and has been suggested to additionally reduce CVD comorbidities. Clinical studies, such as the EmDia trial, investigate the short-term effects of empagliflozin on left ventricular diastolic function. Over the course of the trial, the metabolic effects of empagliflozin has been monitored by a limited set of clinical assays. To expand on this data, we here established a LC-MS workflow for comprehensive metabolic profiling of EmDIA. The workflow established enables profiling of >170 metabolites in plasma covering a broad range of compound classes such as carboxylic acids, amino acids, sugars, nucleotides, steroids and drugs at a rate of >100 samples per day. The method is based on optimized metabolite separation by pentafluorophenyl chromatography and high-confidence metabolite annotation based on a well curated in-house spectral library of more than 480 reference standards. Applied to EmDIA, our methodology is characterized by high predictive power of several clinical parameters, especially fasting blood glucose (R2 = 0.97) and estimated glomerular filtration rate (R2 = 0.63) as determined by elastic net-regularized linear and logistic regression. Our data further shows that administration of empagliflozin in addition to standard T2DM medication results in reduced plasma levels of urate, which has been previously linked to improved cardiovascular disease outcome, and reduced plasma levels of deoxyhexoses such as 1,5-anhydroglucitol, a short-term biomarker for glycemic control.