*3.2. Animal Experiments*

The diabetic/obese KK-*Ay* mice (4-week-old males) were obtained from CREA Japan Inc. (Tokyo, Japan). The mice were housed at 23 ± 2 ◦C and 50% humidity, with a 12 h light/12 h dark cycle and allowed free access to food and water. The control diet (A) containing 9.8% soybean oil and 0.2% MCT was prepared as shown in Table 1. MCT in the control diet was exchanged for 0.2% SO (B) in the experimental diets. Monocaprin was exchanged for soybean oil at 0.125–0.5% (C–F) in the experimental diets of each group, as shown in Table 1. After feeding the mice on a control diet for one week, six groups of seven mice were assigned so that there was no significant difference in body weight and blood glucose levels. Then, five experimental groups mice were acclimated by feeding a 0.5% monocaprin diet (C) for an additional one week. Control mice were fed a control diet (A), as shown in Table 1. After acclimation, each experimental diet and control diet (Table 1) were fed to the mice for 4 weeks. The mice were anatomized under anesthesia using isoflurane. The liver and small intestine were rapidly removed and stored at −30 ◦C. The serum was separated by centrifugation at 3000 rpm for 15 min. All procedures for animal care in this study were approved by the Ethical Committee of Experimental Animal Care of Hokkaido University (No 19-0083).

#### *3.3. HPLC Analysis of Fucoxanthin Metabolites*

The total lipid (TL) was extracted from mouse tissues and serum with chloroform:methanol (2:1, *v/v*) containing α-tocopherol (50 μg/mL) according to the Folch method [20]. The obtained TL was dissolved in *n*-hexane:acetone (7:3, *v/v*) and subjected to an HPLC system (LC-20AD and CBM-20A (Shimadzu, Kyoto, Japan), column: Mightysil Si 60 250 × 4.6 mm (Kanto Chemical Co., Inc., Tokyo, Japan), two columns were connected, column temperature: 25 ◦C, mobile phase: *n*-hexane:acetone (7:3, *v/v*), flow rate: 1.0 mL min−1, detection:

450 nm). Fucoxanthinol and amarouciaxanthin A in the serum and tissues were quantified by HPLC analysis using the standard curves prepared by authentic standards.
