Figure 2.

Results of pilot study: fecal organic acid concentrations in Kawasaki disease (KD). Fecal butyrate concentrations were significantly lower in KD patients (p < 0.05, Mann–Whitney U test) while concentrations of acetate, lactate, and propionate did not differ between KD patients and healthy control children. KD: Kawasaki disease; HC: age-matched healthy children. The horizontal lines in the boxes represent the median values, and the bottoms and tops of the boxes represent the 25th and 75th percentiles, respectively. The vertical lines extend from the box to the maximum values and minimum values. Frozen fecal specimens were thawed, and a 0.1 g subsample was placed in a 2.0 mL tube with zirconia beads and suspended in 0.1 mM perchloric acid solution containing 3% phenol. Samples were heated at 80°C for 15 min, vortexed at 5 m/s for 45 s using FastPrep 24 instrument (MP Biomedicals, Irvine, CA, USA), and centrifuged at 15,350 g for 10 min. Supernatants were filtered using 0.45 μm filters. Fecal organic acids including acetate, propionate, butyrate, and lactate were measured using high-performance liquid chromatography (Prominence, Shimadzu, Kyoto, Japan) using a post-column reaction with a detector (CDD-10A, Shimadzu), two columns arranged in tandem (Shim-pack SCR-102(H), 300 × 8 mm ID, Shimadzu) and a guard column (Shim-pack SCR-102(H), 50 × 6 mm ID, Shimadzu). The mobile phase was 5 mM p-toluenesulfonic acid and the reaction solution was 5 mM p-toluenesulfonic acid containing 100 μM ethylenediaminetetraacetic acid and 20 mM Bis-Tris. The flow rate and oven temperature were 0.8 mL/min and 45°C, respectively. The detector cell temperature was kept at 48°C. Measurements were performed at Techno Suruga Lab, Shizuoka, Japan.