L hydroxyproline

The amount of collagen in dissected corneal pieces was quantified using a hydroxyproline assay (Blain et al., 2006 (link)). Papain digests from individual samples were hydrolysed by adding equal volumes of 11.7 N HCl to supernatant at 110 °C overnight. Specimens were then lyophilized. Dried hydrolysates were reconstituted in their starting volume of distilled water and centrifuged to remove particulate material. Hydroxyproline residues were assayed against known standards (L-hydroxyproline, Sigma-Aldrich, UK) and read at 540 nm after 10 min incubation at 70 °C.

Hydroxyproline and total collagen content in the liver tissue was determined as a biochemical parameter to assess the degree of hepatic fibrosis after the treatment with NDMA. Exactly, 50‐mg wet liver tissue was hydrolysed in 6 N HCl in sealed tubes at 110°C for 16 hrs. The hydrolysed samples were evaporated to dryness in a boiling water bath to remove acid, and the residue was dissolved in 5 ml of distilled water. It was then treated with activated charcoal, vortexed well and filtered through Whatman No. 1 filter paper. Hydroxyproline content in the clear filtrate was measured as described before 32. In brief, 1 ml of sample containing 1–5 μg hydroxyproline was mixed with 1 ml of freshly prepared chloramine‐T solution and allowed to stand for 20 min. It was then mixed with 1 ml of 3.15 M perchloric acid and left for 5 min. Next, 1 ml of freshly prepared p‐dimethylaminobenzaldehyde was added, vortexed well and incubated in a water bath at 60°C for 20 min. Absorbance of the colour development was measured in a spectrophotometer at 560 nm. The concentration of hydroxyproline in the samples was determined using a known standard of L‐hydroxyproline (Sigma‐Aldrich) solution. The total collagen content in the liver tissue was calculated by multiplying the hydroxyproline content by the factor 7.46 as described previously 2.

Muscle fibrosis was measured by quantifying the hydroxyproline content according to a previously published protocol [48 (link)]. Briefly, the muscle sample was hydrolyzed in 1 ml 6 N HCl for 3 h at 115°C. After neutralization with 10 N NaOH (to the final pH of 7.5), the muscle lysate was oxidized with chlormatine-T. The hydroxyproline content was quantified by measuring the color absorbance at 558 nm. The hydroxyproline concentration was determined from a standard curve calculated from a linear dilution of L-hydroxyproline (Sigma-Aldrich, Saint Louis, MO). Due to insufficient amount of muscle tissue obtained from biopsy, the hydroxyproline content in one of the study dogs (Dog ID: E09) was not measured (Table 1).

In adaption to an essay from Reddy and Enwemeka [15 (link)] specimens were hydrolyzed in 2 mL reaction vessels with 6 M HCl (Carl Roth GmbH, Karlsruhe, Germany) for 20 h at 95 °C (Heating Block ThermoStat plus, Eppendorf, Hamburg, Germany). Due to the assay’s sensitivity regarding pH value, hydrolyzed samples were dried and dissolved again in DI water. Assay standard solutions in a range between 20 and 160 µg/mL were prepared dissolving L-hydroxy-proline (Sigma-Aldrich, Schnelldorf, Germany) in DI-water. For assay execution, 35 µL of each specimen and standard solution were transferred into a 96-well plate. 75 µL of chloramine-T reagent were added, which consisted of 1.27 g chloramine-T trihydrate (Sigma-Aldrich, Schnelldorf, Germany) in 20 mL n-propanol (Merck Millipore, Darmstadt, Germany), stocked up to 100 mL with acetate-citrate buffer per 100 mL solution. 75 µL of Ehrlich’s reagent (1.5 g 4-(dimethylamino)-benzaldehyde (Sigma-Aldrich, Schnelldorf, Germany) in 6.6 mL of n-propanol and 3.3 mL 70% perchloric acid (both Carl Roth GmbH, Karlsruhe, Germany) were added. Wells were sealed with Microseal^® ‘B’ PCR Plate Sealing Film (Bio-Rad, Watford, United Kingdom) followed by an incubation at 60 °C for 60 min. Coloration intensity measurement was performed by a photometer Infinite M200 PRO (Tecan, Crailsheim, Germany) at 570 nm.

The total protein present in the liver tissue was quantified by the method of Lowry et al.^{32 (link)}. Hydroxyproline content in the liver tissue was determined as described before^{33 (link)} using L-hydroxyproline (Sigma-Aldrich, St. Louis, MO, USA) as standard. The total collagen content in the liver tissue was calculated by multiplying the hydroxyproline content by the factor 7.46 as reported previously^{34 (link)}.