(+)-Catechin hydrate, L-Ascorbic Acid, Iron (III) Chloride hexahydrate, 2,4,6- Tris(2 pyridyl)-s-triazine, (±)6-Hydroxy-2,5,7,8 tetramethylchroman-2-Carboxylic Acid, sodium acetate·3H20, ethanol, methanol, and hydrochloric acid were all purchased from Sigma Aldrich Co (St. Louis, MO). All clinical serum samples (S1 File : S1 and S2 Tables
For the experimental assays, aqueous antioxidant solutions were prepared as controls in a 1:4 ratio of water and 40 μM antioxidant in 10% ethanol using a microscale approach of the established protocol of Benzie and Strain. For example, 60 μL water and 180 μL of 40 μM catechin or L-Ascorbic Acid in 10% ethanol were combined. Serum samples were prepared in a similar fashion with a 1:4 ratio of serum (in lieu of water) and 10% ethanol. Briefly, these samples contain 60 μL serum (normal triglyceride values: 57–144 mg/dL, n = 11 and severe hypertriglyceridemia: 827–1096 mg/dL, n = 13) and 180 μL of 10% ethanol. Serum samples were also combined with antioxidant and prepared at the same ratio of 1:4 for the serum and antioxidant in 10% ethanol. These samples include 60 μL serum and 180 μL of 40 μM catechin or L-Ascorbic Acid in 10% ethanol. A solution of 180 μL of 10% ethanol and 60 μL water was prepared as the reagent blank. Trolox, a standard vitamin E analog used in FRAP assays, was prepared for calibration at increasing concentrations from 50 μM to 2.5 mM. In short, 60 μL of trolox and 180 μL of 80% methanol were combined as established previously. All the above solutions were incubated at 37 ºC for 1 hour following preparation. A FRAP reaction reagent was prepared with 10 mL of 20 mM FeCl3*6H2O, 10 mL of 10 mM TPTZ, and 50 mL sodium acetate buffer (pH = 3.6). After the one-hour incubation at 37ºC, the FRAP reagent (1800 μL) was added to all solutions for a 5-minute incubation at 37ºC. We measured the absorbance of all solutions in triplicate at 593 nm at various times following the assay on Biotek’s EPOCH microplate spectrophotometer. The final measurement was taken at 180 minutes when the increase in antioxidant activity became stable for catechin and ascorbic acid. A LINEST calibration (b = 0) was performed for each assay, and activities of all controls and samples are described in Trolox equivalents.
In conjunction with experimental assays, computational modeling calculations were performed with Gaussian 16 software. All geometry optimizations were carried out at the m06 [24 (link)] density functional level of theory employing the triple ζ basis set 6–311++G(d,p) [25 (link), 26 (link)] augmented with diffuse [27 (link)] and polarization [28 ] functions. Vibrational frequencies were computed at the same level of theory to confirm that the optimized geometries are minima and to obtain enthalpy and free energy values. All geometries were also optimized, and frequencies were calculated with solvent effects for water and benzene employing the self-consistent reaction field polarizable conductor model SCRF-CPCM [29 (link), 30 (link)].
Stabilization energies are calculated using free energy difference, ΔG, of the products compared to the reactant in hydrogen atom transfer (HAT) reaction represented inEq 1 Eq 2 Fig 1
For the experimental assays, aqueous antioxidant solutions were prepared as controls in a 1:4 ratio of water and 40 μM antioxidant in 10% ethanol using a microscale approach of the established protocol of Benzie and Strain. For example, 60 μL water and 180 μL of 40 μM catechin or L-Ascorbic Acid in 10% ethanol were combined. Serum samples were prepared in a similar fashion with a 1:4 ratio of serum (in lieu of water) and 10% ethanol. Briefly, these samples contain 60 μL serum (normal triglyceride values: 57–144 mg/dL, n = 11 and severe hypertriglyceridemia: 827–1096 mg/dL, n = 13) and 180 μL of 10% ethanol. Serum samples were also combined with antioxidant and prepared at the same ratio of 1:4 for the serum and antioxidant in 10% ethanol. These samples include 60 μL serum and 180 μL of 40 μM catechin or L-Ascorbic Acid in 10% ethanol. A solution of 180 μL of 10% ethanol and 60 μL water was prepared as the reagent blank. Trolox, a standard vitamin E analog used in FRAP assays, was prepared for calibration at increasing concentrations from 50 μM to 2.5 mM. In short, 60 μL of trolox and 180 μL of 80% methanol were combined as established previously. All the above solutions were incubated at 37 ºC for 1 hour following preparation. A FRAP reaction reagent was prepared with 10 mL of 20 mM FeCl3*6H2O, 10 mL of 10 mM TPTZ, and 50 mL sodium acetate buffer (pH = 3.6). After the one-hour incubation at 37ºC, the FRAP reagent (1800 μL) was added to all solutions for a 5-minute incubation at 37ºC. We measured the absorbance of all solutions in triplicate at 593 nm at various times following the assay on Biotek’s EPOCH microplate spectrophotometer. The final measurement was taken at 180 minutes when the increase in antioxidant activity became stable for catechin and ascorbic acid. A LINEST calibration (b = 0) was performed for each assay, and activities of all controls and samples are described in Trolox equivalents.
In conjunction with experimental assays, computational modeling calculations were performed with Gaussian 16 software. All geometry optimizations were carried out at the m06 [24 (link)] density functional level of theory employing the triple ζ basis set 6–311++G(d,p) [25 (link), 26 (link)] augmented with diffuse [27 (link)] and polarization [28 ] functions. Vibrational frequencies were computed at the same level of theory to confirm that the optimized geometries are minima and to obtain enthalpy and free energy values. All geometries were also optimized, and frequencies were calculated with solvent effects for water and benzene employing the self-consistent reaction field polarizable conductor model SCRF-CPCM [29 (link), 30 (link)].
Stabilization energies are calculated using free energy difference, ΔG, of the products compared to the reactant in hydrogen atom transfer (HAT) reaction represented in
