C19 0 coa

LCA-CoA concentration was measured according to Blachnio-Zabielska et al. [41 (link)]. Briefly, malonyl-CoA as well as LCA-CoA were extracted according to Minkler et al. [42 (link)]. Prior to the extraction procedure, a known amount of C15:0-CoA, 16:0(d4) CoA, C17-CoA, C19:0-CoA, C21:0-CoA, C23:0:-CoA and 24:0(d4) CoA (Avanti Polar Lipids, Alabaster, AL, USA) was added as an internal standard. The molecules (C14:0-CoA, C16:0-CoA, C16:1-CoA, C18:2-CoA, C18:1-CoA, C18:0-CoA, C20:0-CoA, C22:0-CoA, C24:1-CoA and C24:0-CoA, Avanti Polar Lipids, Alabaster, AL, USA) were separated on a reverse-phase Agilent ZORBAX Extend-C18 Column, 2.1 × 150 mm, using a binary gradient with ammonium hydroxide (NH₄OH) in water and NH₄OH in ACN. The LCA-CoA concentration was quantified using multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer (Sciex QTRAP 6500+, AB Sciex Germany GmbH, Darmstadt, Germany) in positive electrospray ionization (ESI) mode against the concentration standard curves prepared for each compound.

Malonyl-CoA and LCA-CoA were extracted according to Minkler et al. [29 (link)] and their concentrations were measured according to Blachnio-Zabielska et al. [30 (link)]. Before extraction, the internal standard: C15:0-CoA, 16:0(d4)CoA, C17-CoA, C19:0-CoA, C21:0-CoA, C23:0-CoA and 24:0-(d4)-CoA (Avanti Polar Lipids, Alabaster, AL, USA) was added to all samples. The molecules were separated on a reversed-phase Agilent ZORBAX Extend-C18 column, 2.1 × 150 mm, using a binary gradient with ammonium hydroxide (NH₄OH) in water and NH₄OH in ACN. The concentration of LCA-CoA was quantified using multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer (Sciex QTRAP 6500+, AB Sciex Germany GmbH, Darmstadt, Germany) in positive electrospray ionization (ESI) mode against the concentration standard curves prepared for each compound (C14:0-CoA, C16:0-CoA, C16:1-CoA, C18:2-CoA, C18:1-CoA, C18:0-CoA, C20:0-CoA, C22:0-CoA, C24:1-CoA and C24:0-CoA, Avanti Polar Lipids, Alabaster, AL, USA).