Sample collection and measurement of biomarkers

Subjects were fasted overnight for more than 8 h before their blood samples being taken by a qualified phlebotomist. Plasma and serum samples were then stored at −80 °C until analysis. Anthropometric data and blood pressure were also measured before the blood sample collection. Body fat percentage was measured using a body composition scale (Tanita, Tokyo, Japan). Plasma glucose, glycated hemoglobin (HbA1c), insulin, adiponectin and C-reactive protein (CRP) were measured by a commercial diagnostic laboratory (Falco Biosystems, Hyogo, Japan).

The concentrations of caffeic acid and chlorogenic acid in plasma were determined by a highly sensitive technique using high performance liquid chromatography (HPLC; Agilent 1100LC with binary pump) (Agilent Technologies, Santa Clara, CA, USA) coupled with tandem mass spectrometer (MS/MS; Applied Biosystems Sciex API 3000, Waltham, MA, USA), as described elsewhere with minor modifications.^{6, 7, 8, 9} Briefly, 500 μl of plasma was mixed with 20 μl of 10% ascorbic acid (w/w in water) and 100 μl of 1% sulfatase H-1 (w/w in pH 5 sodium acetate buffer). After an enzymatic hydrolysis at 37 °C for 45 min, 2.5 μl of 40 μm ethyl gallate was added as an internal standard. Caffeic acid and chlorogenic acid were then extracted with 500 μl of 0.1% formic acid (v/v in ethyl acetate). The extraction process was repeated three times, and the pooled extract was evaporated under vacuum (Tomy CC-105 centrifugal concentrator, CS Bio, Menlo Park, CA, USA and Eyela Unitrap UT-2000 evaporator, Tokyo, Japan). Samples were reconstituted in 15% acetonitrile and 0.1% formic acid and then centrifuged at 15 000 g at 4 °C for 5 min. A total of 80 μl of the supernatant was transferred into HPLC-autosampler vials.

Five microliter of sample was next injected into Develosil ODS-SR C18, 5 μm, 2 × 150 mm column (Nomura Chemical) with binary gradient of 0.1% formic acid in ultrapure water (A) and 0.1% formic acid in acetonitrile (B) at a constant flow rate of 0.2 ml min⁻¹. The gradient program began at 15% solvent B, increasing to 25% by 10 min and to 50% by 14 min, before returning to 15% which was held for 10 min. The MS/MS was operated with electrospray ionization in negative mode. The ion spray potential was −4500 V, and the source temperature was set at 450 °C.

Caffeic acid, chlorogenic acid and ethyl gallate, purchased from Sigma-Aldrich (St Louis, MO, USA), were used as a standard. The peak of each compound was identified based on a comparison of its retention times and mass spectral data with the corresponding standard and published data using the ABSCiex Analyse v1.6 software^{6, 7, 8, 9} (Supplementary Figure 1A). All samples were measured in duplicates.