Thermo 8404

PON1 lactonase activity was kinetically measured using 1mM dihydrocoumarin (DHC) as a substrate at and recorded at 270 nm using a SpectraMax 190 Absorbance Plate Reader (Molecular Devices LLC, San Jose, USA) as follows: 10 µL of serum diluted 1:5 in assay buffer (50 mM Tris HCL at pH 8.0 and 1 mM CaCl₂) and added per well in triplicate to a 96-well UV flat bottom plate (Thermo 8404, Thermo Fisher, Waltham, USA) The substrate, DHC (8.0 M), was mixed 1:1 in dimethyl sulfoxide (DMSO), vortexed and 6.25 µL of this was added to 25 mL of room temperature lactonase assay buffer and used within 5 min. A total of 200 µL of this mixture was added per well using a multichannel pipette and placed in the temperature controlled plate reader at 37 °C. Samples were read at 270 nm for 2 min 20 s, every 10 s.
Activities were reported as units per liter, where 1 U is defined as 1 mmol of substrate hydrolyzed per minute. Enzyme kinetics was calculated using SOFTmax PRO software version 5.4.6 (Molecular Devices LLC, San Jose, USA).

PON1 arylesterase activity was kinetically measured by following the hydrolysis of phenyl acetate at 270 nm using SpectraMax 190 Absorbance Plate Reader (Molecular Devices LLC, San Jose, USA) as follows: The reagent was prepared using 0.9 mM CaCl₂ and 20 mM Tris with vigorous stirring at pH 8.0 w/HCL. A total of 5 µL of serum diluted in 1:5 in assay buffer was pipetted per well in triplicate to a 96-well UV flat bottom plate (Thermo 8404, Thermo Fisher, Waltham, USA). The substrate, phenyl acetate 3.4 µL, was mixed into 25 mL of arylesterase assay buffer and mixed vigorously. Following this, 200 µL of this mixture was added per well using a multichannel pipette and placed in a temperature controlled plate reader SpectraMax 190 Absorbance Plate Reader (Molecular Devices LLC, San Jose, USA) set at 25 °C. The samples were read kinetically at 270 nm for 1.5 min, every 13 s.