Quantification of hydroxyproline
Hepatic hydroxyproline content was measured as previously described (Matsumoto et al., 2013), with slight modifications. Briefly, homogenized liver samples were hydrolysed overnight in 6N HCl at 105 °C, neutralized with 6N NaOH, and filtered through a 0.2 µm PFTE filter (Advantec Toyo, Ltd., Tokyo, Japan). Aliquots of the hydroxylate were diluted in citrate buffer, oxidized with chloramine-T dissolved in citrate buffer, and incubated for 20 min at room temperature. The reaction was terminated with 20% perchloric acid. After incubation, 20 w・v-1% p-dimethylaminobenzaldehyde dissolved in ethylene glycol mono-methyl ether was added, the samples were incubated for 1 h at 60 °C, dispensed into a 96-well plate, and measured for absorbance at 570 nm. The hydroxyproline levels were determined using a standard curve. All reagents were purchased from Fujifilm Wako Pure Chem. Corp.