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.