Hydroxyproline assay of tissue collagen content.

Hydroxyproline content in the kidney was measured by the color development of Ehrlich’s reagent with furans derived from hydroxyproline oxidation essentially as previlously described (67) but with minor modifications. Kidneys (approximately one-half of a whole kidney) were dried overnight at 110°C, weighed, and then hydrolyzed in 1 ml of 6 M HCl at 110°C for 24 h. Debris was removed by centrifuging, and Amberlite MB-1 resin (8 mg, Sigma-Aldrich) was added to the supernatant to neutralize samples, which were then mixed on a tube rotator for 30 min at room temperature followed by centrifugation for 30 min at 12,000 rpm at room temperature. A 25-µl aliquot of the supernatant was mixed with 500 µl of 0.05 M chloramine-T (sodium p-toluenesulfonchloramide) in citrate-acetate buffer (0.88 M sodium acetate, 0.24 M citric acid, 0.21 M glacial acetic acid, 0.85 M sodium hydroxide, and 1 mM EDTA, pH 6.0) and incubated for 20 min at room temperature to oxidize the kidney hydrolyzate. The reaction was terminated by the addition of 500 μl of 3.15 M HClO₄ (prepared by diluting 27 ml of 70% HClO₄ to 100 ml final with water), and samples were mixed thoroughly followed by incubation for 5 min at room temperature. Next, 500 μl of Ehrlich’s solution [prepared by the addition of 15 ml of 50% p-dimethylaminobenzaldehyde in 60% (wt/vol) perchloric acid to 65 ml isopropanol] were added, and samples were mixed thoroughly and incubated for 20 min at 60°C to allow for color development. Immediately after incubation, samples were cooled in an ice bath for 5 min, and absorbance was measured at 550 nm. Samples were compared with a standard curve of hydroxyproline standards.