*2.4. Study Design*

The study design was a placebo-controlled, three-way crossover trial with a 1-week washout period between study visits. Subjects were randomly assigned to consume 360 mL skim milk (C), or 225 mg (L) or 450 mg (H) GAE ASP in skim milk. Although ASP were not completely soluble in skim milk, residual ASP in the glass were rinsed with water and then completely consumed. Each subject was admitted to the MRU in the morning after a 12-h overnight fast. Following a check of vital signs, an intravenous catheter was inserted into an antecubital vein of one forearm and a baseline blood sample was obtained. After subjects drank the test beverage in ~5 min, blood samples were collected at 15, 30, and 45 min and 1, 2, 3, 5, and 10 h. Lunch and dinner meals designed to contain low flavonoids and meet the recommended dietary allowances for protein and energy [28] were prepared under the supervision of the study dietitian. The same meals were served during each visit. These meals were provided at 4 and 9 h after the ASP consumption. The consumption of water, salt, sugar, and ginger ale was not limited but food and other beverages were not allowed during the intervention.

#### *2.5. Sample Collection and Storage*

Blood was collected into EDTA vacutainers and processed within 10 min. Whole blood was centrifuged at 1000× *g* at 4 ◦C for 15 min using a Sorvall RT6000 (Du Pont Co. Newtown, CT, USA). Plasma aliquots of 1.5 mL were flushed with N2, stored at 4 ◦C, and used for the Cu2+-induced LDL oxidation assay within 3 days. An aliquot of plasma was treated with an equivalent volume of 10% trichloroacetic acid, vortexed, and then centrifuged at 14,000× *g* for 10 min at 4 ◦C; then the resulting supernatant was snap-frozen for determination of reduced glutathione (GSH) and oxidized GSH (GSSG). Aliquots of plasma were snap frozen and stored at −80 ◦C until analysis of flavonoids, malondialdehyde (MDA), glutathione peroxidase (GPx), and F2α-isoprostanes.

#### *2.6. Analysis of Plasma Flavonoids*

Flavonoids in plasma were determined using the HPLC method of Chen et al. [24]. Briefly, plasma was first mixed with vitamin C-EDTA solution, internal standard (2',3',4'-trihydroxyacetophenone), and β-glucuronidase/sulfatase. After incubation at 37 ◦C for 45 min, flavonoids were extracted with acetonitrile. After centrifugation, supernatant was transferred, dried under purified N2, and reconstituted in aqueous HPLC mobile phase for HPLC analysis. Flavonoids were determined by an HPLC system equipped with a Zorbax ODS C18 column (4.6 × 150 mm, 3.5 μm) and the Coularray 5600 A detector (ESA, Inc. Chelmsford, MA, USA). The quantification of plasma catechin, quercetin, naringenin, kaempferol, and isorhamnetin was calculated according to calibration curves constructed with authentic standards, with linear relationships of *R*<sup>2</sup> > 0.999. The limit of detection on column for flavonoids was 0.5 pmol. The coefficient of variation (CV) values of intra- and inter-day assays were 3.0% and 9.0%, respectively. The recovery rate for the internal standard was 97.0 ± 0.1%.

#### *2.7. Biomarkers of Antioxidant Capacity and Lipid Peroxidation*

Blood for plasma glutathione analysis was collected using a drip approach from the catheter to avoid potential hemolysis which increases plasma GSH through contamination from red blood cell GSH. Blood from the catheter collected into an EDTA vacutainer. GSH and GSSG in the supernatant collected from acidified plasma were determined using the high-performance liquid chromatography with electrochemical detection (HPLC-ECD) method of Chen et al. [28]. The concentrations of plasma GSH and GSSG were calculated based on calibration curves of authentic GSH and GSSG. The intraand inter-day assay CV values for GSH were 2.7% and 2.9% and for GSSG the values were was 6.7% and 8.1 %, respectively.

Oxygen radical absorbance capacity (ORAC) in heparinized plasma-treated 0.5 M perchloric acid (PCA) (1:1 v/v) was determined according to the method of Ou et al. [29]. The assay provides an integrated and quantitative determination of "total antioxidant capacity" by employing the area under the curve (AUC) of the magnitude and time to the oxidation of fluorescein due to peroxyl radicals generated by the addition of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). ORAC values were calculated according to the method described by Cao et al. [30] and are expressed as μmol/L Trolox equivalents (TE).

Plasma GPx activity was determined using the spectrophotometric method of Pleban et al. [31] with a Cobas Fara II centrifugal analyzer (Roche Diagnostics, Nutley, NJ, USA). This assay measures GPx activity on the basis of the oxidation of GSH to GSSG and the reduction of H2O2 to H2O, which is coupled to the oxidation of NADPH by glutathione reductase. The intra- and inter-assay CV values were 3.4% and 3.2%, respectively.

The LDL resistance against Cu2+-induced oxidation was determined according to the method of Chen et al. [25]. Briefly, LDL particles were collected using an ultracentrifugation protocol. LDL (182 nmol/L) was oxidized by 10 μmol/L CuSO4 with (or without) in vitro addition of a final concentration of 6 μmol/L α-tocopherol in a total volume of 1.0 mL phosphate buffer. The addition of α-tocopherol was intended to amplify any potential antioxidant effect of absorbed ASP on LDL resistance against oxidation [24]. Formation of conjugated dienes was monitored by absorbance at 234 nm at 37 ◦C using a UV1601 spectrophotometer (Shimadzu Corp, Kyoto, Japan). The results of the LDL oxidation are expressed as lag time (defined as the intercept at the abscissa in the diene-time plot). The intra- and inter-assay CV was 1.8% and 7.5%, respectively.

Plasma MDA was determined by the HPLC method of Volpi and Tarugi [32], in which a thiobarbituric acid-MDA conjugate product was separated by a C18 column and fluorometrically quantified at an excitation of 515 nm and emission of 553 nm. Plasma MDA concentration was calculated based on calibration curves of authentic standard 1,1,3,3-tetraethoxypropane, with a linear relationship of *R*<sup>2</sup> > 0.995. The intra- and inter-assay CV values were 3.9% and 12.3%, respectively.

Plasma F2α-isoprostanes were measured by gas chromatograph-mass spectrometry (GC-MS) with negative chemical ionization, as described by Walter et al. [33]. Briefly, plasma lipids were isolated

with Folch extraction, followed by gentle alkaline saponification to release isoprostanes. Isoprostanes were then converted to pentafluorobenzyl esters using PFBBr and DIPEA. The resulting PFB esters of F2α-isoprostanes were isolated using an HPLC system equipped with an amino column, dried under N2, and silylated with BSTFA and DIPEA. The silylated product was dried, resuspended in undecane, and analyzed by GC-MS. The final data are expressed as ng/mL.
