Method Details

Plasma Collection and Metabolite Extraction
All blood samples used in this study were collected after an overnight fast. Peripheral blood samples from patients were collected within 24 h upon hospital admission. For patients admitted in the evening, blood samples were taken on the next morning (0500-0600) before breakfast (0700). For patients admitted during daytime, blood samples were collected only on the next morning (0500-0600) before breakfast (0700) (i.e., after one night in the hospital). Throughout the hospitalization period, patients were provided two standard meals per day, scheduled at 0700 and 1700, respectively. Blood was collected in BD Vacutainer (BD 367525). Plasma was separated by centrifugation at 2000 rpm for 10 min. Lipids and metabolites were extracted according to a modified version of the Bligh and Dyer’s protocol (Lu et al., 2019). Plasma (100 μL) for metabolomics and lipidomics analyses was inactivated via the addition of 750 μL of ice-cold chloroform: methanol (1:2) (v/v). Samples were vortexed for 15 s and then incubated for 1 h at 1500 rpm at 4°C. At the end of incubation, 250 μL of ice-cold chloroform and 350 μL of ice-cold MilliQ water were added. Samples were vortexed for 15 s and put on ice for 1 min. This step was repeated once. Samples were then centrifuged at 12 000 rpm for 5 min 4°C to induce phase separation. The lower organic phase was first extracted to a new tube. Then, another 450 μL of ice-cold chloroform was added to the remaining aqueous/methanol phase. Samples were vortexed briefly for 15 s and put on ice for 1 min, and centrifuged at 12 000 rpm for 5 min 4°C. The lower organic phase was extracted and pooled together with the first round organic extract. Double rounds of extraction ensured a better recovery and reduce variations across samples. The remaining aqueous/methanol phase was then centrifuged at 12 000 rpm for 5 min 4°C, and clean supernatant containing polar metabolites were extracted and transferred to new tube. The organic phase was dried in the SpeedVac under OH mode, while aqueous phase was dried under H2O mode. The dried metabolite extracts were shipped on dry ice to the designated laboratory for lipidomics and metabolomics analysis.

Exosome Isolation and Lipid Extraction
Exosomes were isolated from 100 μL of plasma using Invitrogen total exosome isolation kit (Thermofisher Scientific) according to the manufacturer’s protocol. The isolated exosome pellet was resuspended in 100 μL of ice-cold PBS and dispersed completely by repeatedly pipetting up and down. Following this, 750 μL of ice-cold chloroform: methanol (1:2) (v/v) was added to inactivate the samples. Lipid were then extracted in identical steps as described above for plasma samples, and organic extracts pooled from two rounds of extractions were used for lipidomics analysis. The remaining aqueous/methanol phase containing the extracted pellet was dried in the Speedvac under H2O mode. Proteins were extracted from the dried pellet using RIPA lysis buffer with protease inhibitor cocktail (Sigma-Aldrich), and total protein content was determined using Pierce BCA protein assay kit (Thermofisher Scientific) according to the manufacturer’s instructions.

Targeted Lipidomics
Prior to analysis, plasma lipid extracts were resuspended in 100 μL of chloroform: methanol 1:1 (v/v) spiked with appropriate concentrations of internal standards. All lipidomic analyses were carried out on an Exion UPLC coupled with a SCIEX QTRAP 6500 PLUS system as described previously, using an extensive, targeted library tailored for human serum lipidome that confers sufficient lipid coverage to render global lipid pathway analysis (Lam et al., 2014; Lu et al., 2019). All quantification experiments were conducted using internal standard calibration. In brief, polar lipids were separated on a Phenomenex Luna Silica 3 μm column (i.d. 150 × 2.0 mm) under the following chromatographic conditions: mobile phase A (chloroform:methanol:ammonium hydroxide, 89.5:10:0.5) and mobile phase B (chloroform:methanol: ammonium hydroxide: water, 55:39:0.5:5.5) at a flow rate of 270 μL/min and column oven temperature at 25°C. The gradient started with 5% of B and was held for 3 min, which was then increased to 40% of B over 9 min, and was held at 40% for 4 min before further increasing to 70% B over 5 min. The gradient was maintained at 70% B for 15 min before returning to 5% B over 3 min, and was finally equilibrated for 6 min. Individual polar lipid species were quantified by referencing to spiked internal standards of the same lipid class including PC-14:0/14:0, d31-PC16:0/18:1, PE14:0/14:0, d31-PE-16:0/18:1, d31-PS-16:0/18:1, PA-17:0/17:0, PG-14:0/14:0, d31-PG-16:0/18:1, C14:0-BMP, d31-PI-16:0/18:1, SM-d18:1/12:0, LPC-17:0, LPE-17:1, LPI-17:1, LPA-17:0, LPS-17:1, S1P-d17:1, Cer-d18:1/d7-15:0, GluCer d18:1/8:0, GalCer d18:1/8:0 obtained from Avanti Polar Lipids (AL, USA) and PI-8:0/8:0 from Echelon Biosciences, Inc. (UT, USA). d3-GM3 d18:1/18:0 and d3-LacCer d18:1/16:0 were from Matreya LLC (PA, USA). Glycerol lipids including diacylglycerols (DAGs) and triacylglycerols (TAGs) were quantified using a modified version of reverse phase LC/MRM. Separation of neutral lipids were achieved on a Phenomenex Kinetex-C18 2.6 μm column (i.d. 4.6x100 mm) using an isocratic mobile phase containing chloroform:methanol:0.1 M ammonium acetate 100:100:4 (v/v/v) at a flow rate of 300 μL for 10 min. Levels of short-, medium-, and long-chain TAGs were calculated by referencing to spiked internal standards of TAG(14:0)3-d5, TAG(16:0)3-d5 and TAG(18:0)3-d5 obtained from CDN isotopes (Quebec, Canada), respectively. DAGs were quantified using d5-DAG16:0/16:0 and d5-DAG18:1/18:1 as internal standards from Avanti Polar Lipids (Shui et al., 2010). Free cholesterols and cholesteryl esters were analyzed as described previously with d6-cholesterol and d6-CE18:0 cholesteryl ester (CE) (CDN isotopes) as internal standards (Shui et al., 2011). Lipid levels were expressed in nanomoles per L (nmol/L) for plasma, and in nanomoles lipids per g of total protein (nmol/g) for exosomes.

Untargeted Metabolomics
Prior to analysis, aqueous extracts were resuspended in 100 μL of 2% acetonitrile in water. Chromatographic separation was performed on a reversed-phase ACQUITY UPLC HSS T3 1.8 μm column (i.d. 3.0 × 100 mm) (Waters) using an UPLC system (Agilent 1290 Infinity II; Agilent Technologies) as described previously (Tian et al., 2020). MS detection was performed using high-resolution time-of-flight (TOF) mass spectrometry (5600 Triple TOF Plus, Sciex) equipped with an ESI source (Yuan et al., 2012). Data were acquired in TOF full scan method with positive and negative ion modes, respectively. Information-dependent acquisition methods were used for MS/MS analyses of metabolome. The collision energy was set at 35 ± 15 eV. Metabolite identification was compared with standard references, HMDB (https://hmdb.ca/), METLIN (https://metlin.scripps.edu), and literature searches. A total of 45 isotopically-labeled internal standards (IS), purchased from Cambridge Isotope Laboratories, were spiked into the samples for metabolite quantitation, including L-Phenylalanine-d8, L-Tryptophan-d8, L-Isoleucine-d10, L-Asparagine-13C4, L-Methionine-d3, L-Valine-d8, L-Proline-d7, L-Alanine-d7, DL-Serine-d3, DL-Glutamic acid-d5, L-Aspartic acid-d3, L-Arginine-d7, L-Glutamine-d5, L-Lysine-d9, L-Histidine-d5, Taurine-d2, Betaine-d11, Urea-(13C,15N2), L-lactate-13C3, Trimethylamine N-oxide-d9, Choline-d13, Malic acid-d3, Citric acid-d4, Succinic acid-d4, Fumaric acid-d4, Hypoxanthine-d3, Xanthine-15N2, Thymidine (13C10,15N2), Inosine-15N4, Cytidine-13C5, Uridine-d2, Methylsuccinic acid-d6, Benzoic acid-d5, Creatine-d3, Creatinine-d3, Glutaric acid-d4, Glycine-d2, Kynurenic acid-d5, L-Citrulline-d4, L-Threonine-(13C4,15N), L-Tyrosine-d7, P-cresol sulfate-d7, Sarcosine-d3, Trans-4-hydroxy-L-proline-d3, Uric acid-(13C; 15N3). Metabolite levels were normalized according to the following rules (1) ISs were applied to correct peak areas of their corresponding metabolites; (2) When (1) was not feasible due to unavailability of commercial standards, peak areas were corrected with IS of metabolites of the same class, comparable peak intensities, and/or proximity in retention times; (3) Results from Step (2) were evaluated based on relative standard deviation (RSD) values of each metabolite before and after IS correction. Corrected peak areas were adopted if their corresponding RSDs were smaller than that of original areas in quality control samples. For simplicity, all metabolite levels were labeled as “intensity.”