Lc 2000

In the product analysis, 5 mL of substrate solution (0.5 wt%) was mixed with 10 mL of 50 mM sodium acetate buffer (pH 5.0) and the reaction was started by the addition of 25 μL of the enzyme solution (96 U/mL). To confirm the final reaction product, an additional 25 µL of enzyme solution was added to reaction mixture at 24 h, and the reaction mixture was incubated for an additional 24 h. After 0.5–48 h, 1 mL of the reaction mixture was removed, and the reaction was stopped by heating to a boiling temperature for 10 min. Each reaction mixture was applied to the HPLC system for the sugar analysis as follows. The LC-2000 (JASCO, Co. Ltd., Tokyo, Japan) system was used to connect with Shodex Asahipak NH ₂P 50-4E (4.6 × 250 mm). The mobile phase was used as the gradient concentration of the mixture with acetonitrile : 85 % H ₃PO ₄ (98.5 : 1.5) and distilled water : 85 % H ₃PO ₄ (99 : 1), and the flow rate was 1 mL/min. Products were detected using a fluorescence detector GL-7453A (GL Science, Tokyo, Japan) as post labeled method modified with 85 % H ₃PO ₄ : acetic acid : phenyl hydrazine (220 : 180 : 6) at 0.4 mL/min (flow rate). The standard sugars used were xylose (Xyl), xylobiose (Xyl ₂), xylotriose (Xyl ₃), and 1,2 ³-α-D-(4- O-methyl-glucuronyl)-1,4-β-D-xylotriose (MeGlcA ³Xyl ₃) (Megazyme, Wicklow, Ireland).

Serum vitamin A (VA) and vitamin E (VE) were measured via high-performance liquid chromatography (high-performance liquid chromatography LC-2000, JASCO Corp., Tokyo, Japan).