Application

This kit can be used for detection of hydroxyproline content in samples, such as animal serum (plasma), tissue, cells, culture supernatant and body fluids etc. 

Detection significance 

Hydroxyproline accounts for 13.4% in collagen, minute quantity in elastin, and not exists in other proteins. And collagen mostly exists in skin, tendon, cartilage, blood vessel, etc, so the quantity and energy of hydroxyproline can reflect collagen metabolic status of connective tissue diseases. Surgical trauma can lead to the increase of hydroxyproline excretion amount in urine. Fiber can increase through inflammation because of high fat, low protein, alcoholism and poison, malnutrition or hepatocellular degeneration and necrosis, then segments hepatic lobule and results in liver cirrhosis. When affected with hepatic fibrosis, collagenous fiber is the main increasing component in liver. And hydroxyproline is specific to collagenous fiber. So the content of liver collagen can be calculated by measuring the content of liver hydroxyproline to reflect the degree of hepatic fibrosis. The decomposition of collagen fiber primarily depends on the function of collagenase and other proteases, whose decomposition product - hydroxyproline is excreted in the urine. So measuring the content of urine hydroxyproline can reflect the degradation situation of collagen. On the one hand, measuring the content of hydroxyproline in tissue and urine can judge the degree of fibrosis; on the other hand, it can screen the drugs of prevention and treatment.

Detection principle

The oxidation product which produced by hydroxyproline under the action of oxidant react with dimethylaminobenzaldehyde and show a purplish red color. The content of hydroxyproline can be calculated by measuring the OD value at 550 nm.

Experimental instruments

Test tube

Micropipettor

Vortex mixer

60℃ and 95℃ water bath

Centrifuge

Spectrophotometer (550 nm) 

Operation steps

1. Pretreatment of sample

1) Hydrolysis of sample

(1) Serum (plasma): Take 0.5 mL serum (plasma) into a tube and add exactly 1 mL hydrolysis solution, mix fully. Cover the lid and incubate at 95℃ water bath or boiling water for 20 min. 

(2) Urine (culture supernatant): Take 1 mL urine (or 0.5 mL culture supernatant) into a tube and add exactly 1 mL hydrolysis solution, mix fully. Cover the lid and incubate at 95℃ water bath or boiling water for 20 min.

(3) Tissue: Weigh accurately 30~100 mg wet tissue into a tube and add exactly 1 mL hydrolysis solution, mix fully. Cover the lid and incubate at 95℃ water bath or boiling water for 20 min (shake the tube to mix at 10 min to hydrolyze fully).

Recommended wet weight of tissue sample: skin tissue--0.03-0.05 g, cartilaginous tissue/ liver tissue --0.08-0.1 g

2) Adjust pH to 6.0~6.8

(1) Cool the hydrolyzed sample under running water, then add 10 μL of indicator and mix fully.

(2) Add 1 mL of pH adjusted liquid A accurately and mix fully (At this time the solution is red).

(3) Carefully add pH adjusted liquid B a drop by a drop, mix fully after adding each drop, until the red is disappeared (At this time the pH is 6.0-6.8).

(4) Add double-distilled water to a final volume of 10 mL and mix fully.

(5) Take 3-4 mL diluent hydrolyzed liquid, add 20-30 mg acticarbon (the supernatant is clarified colorless after centrifugation), mix fully, centrifuge at 3500 rpm for 10 min, take 1 mL supernatant for test.

2. Operation table

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 6 months.

4. Do not use components from different batches of kit.