Quantifying Adduct Uptake in HUVECs

HUVECs were plated on 6-well plates at a density of 30,000 cells/cm² and treated the next day with 200 ng/mL of isotopically labeled MG adducts (¹⁵N₅-R,S-CEdG or ¹⁵N₅-R,S-CEG). At each indicated timepoint, conditioned media was collected, centrifuged to remove cellular debris, and processed via cation exchange solid-phase extraction using Oasis MCX SPE cartridges (Waters, Milford, MA, USA) [9 (link),23 (link),24 (link)]. Additionally, cell pellets were collected and lysed using 10% w/v trichloroacetic acid, and cellular debris was separated via centrifugation for 15 min at 4 °C at 12,000× g. Both the cell pellets and conditioned media were spiked with isotopically labeled internal standards prior to their quantification via LC-MS as previously described [23 (link),24 (link)]. To quantify adduct uptake, cells treated with ¹⁵N_5-R,S-CEdG used ¹⁵N₅-S-CEG at a final concentration of 5 ng/mL as an internal standard. Conversely, cells treated with ¹⁵N_5-R,S-CEG used ¹⁵N₅-S-CEdG at a final concentration of 5 ng/mL as an internal standard.