Efficient methodology for the extraction and analysis of lipids from porcine pulmonary artery by supercritical fluid chromatography coupled to mass spectrometry

Abstract

Pulmonary artery grafts are needed as cardiovascular bioprosthetics. For successful tissue recellularization after transplantation, lipids have to be removed from the donor artery. Developing a selective process to remove lipids without damaging the extracellular matrix greatly depends on knowing the amount and type of lipid compounds in the specific tissue. Here we present an efficient methodology for the study of lipids present in porcine pulmonary arteries. The performance of six extraction methods to recover lipids from artery was evaluated. For this purpose, a supercritical fluid chromatography method coupled to quadrupole time-of-flight mass spectrometry detection (UHPSFC/QTOF-MS) was adapted. The method enabled separation of lipids of a wide range of polarity according to lipid class in less than 7 minutes. One dichloromethane-based extraction method was shown to be the most efficient one for the recovery of lipids from pulmonary artery. However, one MTBE-based extraction method was able to show the highest fatty acid extraction yields (to the expense of longer extraction times). Lipids were relative quantified according to class, and the major species within each class were identified. Triacylglycerols and glycerophospholipids were the most abundant classes, followed by sphingomyelins, monoacylglycerols and fatty acyls. The matrix effect exerted no interference on the analytical method, except for some few combinations of extraction method and lipid class. These results are of relevance for lipidomic studies from solid tissue, in particular for studies on pulmonary and cardiovascular diseases. Finally, our work sets the basis for the further development of a selective processes to remove lipids from pulmonary artery without damaging the tissue prior to transplantation.

Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes

“…extraction of lipid residues from treated tissues. Lipids were recovered by a dichloromethane/ methanol/water-based extraction method adapted for porcine pulmonary arteries 34 . Briefly, freeze-dried tissue pieces (see Supplementary Fig.…”

“…Briefly, freeze-dried tissue pieces (see Supplementary Fig. S1) (in randomly assembled batches of 20 samples) were disrupted in a Qiagen TissueLyser (Qiagen GmbH, Hilden, Germany) for 10 min (1 min per cycle) at 25 Hz, followed by extraction of lipids as previously described in detail 34 . Extracts were dried under a stream of nitrogen gas, weighted for gravimetric analysis and stored at −80 °C until analysis.…”

“…Lipid fractions (n = 4 per condition) were dissolved in 50 µL of CHCl 3 :MeOH (1:1, v/v) and analyzed on an Acquity Ultra Performance Convergence Chromatography (UPC 2 , Waters, MA, USA) system equipped with an Acquity UPC 2 Torus DIOL column (130 Å, 1.7 µm, 3 mm ×100 mm, Waters, MA, USA) and fitted with a Van Guard Torus DIOL (130 Å, 1.7 µm, 2.1 mm ×5 mm, Waters, MA, USA) pre-column. Chromatography was performed as previously described in detail 34 , with a few modifications. The cosolvent (methanol containing 10 mM of ammonium formate) gradient was: 0 min, 2%; 2 min, 2%; 4 min 13%; 7 min, 27%; 7.5 min, 35%; 8.5 min, 35%; 9 min 2%; 11 min 2%.…”

“…Mass spectrometric detection was performed using a Xevo-2G quadrupole time-of-flight mass spectrometer (QTOF-MS; Waters, MA, USA). Make-up solvent (10 mM ammonium formate in methanol) was supplied at 0.25 mL/min and back-pressure regulated using two T-pieces, placed between the chromatographic system and the mass spectrometer (split ratio about 1:100) 34,35 . The capillary voltage was set at 3.0 kV and 2.5 kV for positive and negative electrospray ionization mode, respectively.…”

“…Lipids were identified by exact mass using LipidMaps ® Lipidomics gateway (San Diego, CA) and published data 34,37 retention times and by fragments.…”