Quantifying Cardiac Hydroxyproline Content

Hydroxyproline content of heart tissues was measured as previously described34 (link),35 (link) with some modifications. Left-ventricle was freshly harvested from each heart and vacuum-dried. Each vacuum-dried tissue was weighed, minced and hydrolyzed in vacuum-sealed 6 M HCl (1ml) at 116°C overnight. Hydrolyzed samples were dried in a speed-vac overnight. The dried pellet was dissolved in water and re-dried in a speed-vac overnight. The dried pellet was re-dissolved in water and centrifuged 5 min. To each supernantant, 40 µl phenolphthalein was added as pH indicator, and KOH solution was added until the solution color became pale pink. Neutralized samples were centrifuged for 5 min and the supernatant was used for the hydroxyproline measurement. To each diluted (typically 20-fold) sample (2 ml), 1 ml of 0.05 M chloramine-T (0.285 g/5 ml dH₂O, 7.5 ml Methyl Cellosolve, 12.5 ml Na-citrate/Na-acetate buffer) was added and incubated 20 min. at room temperature. Chrolamine-T was then destroyed with 1 ml 3.15 M perchloric acid (7.56 ml 60% perchloric acid, 16.44 ml dH₂O). After 5 min at room temperature, 1 ml 20% p-dimethylaminobenzaldehyde solution (dissolved in Methyl Cellosolve) was added and each sample was vortexed, and incubated 20 min at 60°C. Hydroxyproline content was determined by measuring the absorbance of each sample at 557 nm. A standard curve was generated by using hydroxyproline solutions (0, 2, 4, 6, 8, 10 µg/2 ml).