Urinary 17-hydroxysteroids are hormones and their metabolites secreted by the suprarenal cortex, and their excretion from urine can reflect the function of the adrenal cortex. 56% sulfuric acid and ammonium sulfate were added to promote the transfer of 17-hydroxysteroids to the organic phase, and the extracts were extracted from urine with a mixture of chloroform and n-butanol. The extract was extracted from urine by chloroform-n-butanol mixture. The extract was added with phenylhydrazine hydrochloride and sulfuric acid to generate yellow phenylhydrazine derivatives with 17-hydroxysteroids (Porter_silber reaction), and the content was obtained by colorimetric comparison with the standard solution after the same treatment. This experiment was obtained from the laboratory instruction of Mudanjiang Medical College, undergraduate 5-year testing program.
Operation method
Urinary 17-hydroxysteroid assay
Principle
Urinary 17-hydroxysteroids are hormones and their metabolites secreted by the suprarenal cortex, and their excretion from urine can reflect the function of the adrenal cortex. 56% sulfuric acid and ammonium sulfate were added to promote the transfer of 17-hydroxysteroids to the organic phase, and the extracts were extracted from urine with a mixture of chloroform and n-butanol. The extract was extracted from urine by chloroform-n-butanol mixture. The extract was added with phenylhydrazine hydrochloride and sulfuric acid to generate yellow phenylhydrazine derivatives with 17-hydroxysteroids (Porter_silber reaction), and the content was obtained by colorimetric comparison with the standard solution after the same treatment.
Materials and Instruments
Purified chloroform Purified n-butanol Ammonia sulfate anhydrous Phenylhydrazine hydrochloride Cortisol Sulfuric acid Concentrated hydrochloric acid Move I. Experimental reagents: Caveat 1. The color reaction is non-specific, glucose. Fructose. Vc. dehydro Vc. uric acid. Acetone sulfadiazine. Quinine etc. can cause interference. However, chloroform-n-butanol mixture exclusion effect is good, need to pay attention to the separation of urine to avoid droplets into the color solution. 2. centrifugation stratification of phenylhydrazine sulfate reagent in chloroform n-butanol mixture placed on top of a long time will be sunk to the bottom of the tube, so it is necessary to timely phenylhydrazine - sulfuric acid reagent suction, as long as the amount of suction to meet the colorimetric can be, in order to avoid touching the lower layer of liquid to affect the colorimetry. 3. The blank value of all the reagents reflects whether the various reagents are qualified, and this value should be noted for each determination, so the reagents should be freshly prepared. For more product details, please visit Aladdin Scientific website.
1. purified chloroform: 250 ml 2. purified n-butanol: 25 ml
3. anhydrous ammonia sulphate: 50g 4. phenylhydrazine hydrochloride: 65mg
5. Cortisol standard: 2ml 6. 56% sulfuric acid: analytically pure
7. concentrated hydrochloric acid: analytically pure
II.Experimental reagent preparation:
1. Chloroform n-butanol mixture, according to the volume ratio of 10:1 preparation.
2. 56% sulfuric acid: volume ratio
3. phenylhydrazine-sulfuric acid reagent: take 65mg of phenylhydrazine hydrochloride and add 100ml of 56% sulfuric acid plus NaCL 2g It can be kept in refrigerator for two months.
4. Standard tube: add 0.1ml of standard liquid to each tube and dry it at room temperature, cover it tightly and keep it in the dark place, take one tube to use each time.
Third, the experimental method:
l. Specimen collection: collect urine for 24 hours in a dry clean container (the first urine should be added with concentrated HCL 5ml preservative) and measure the total volume of 50ml samples for testing.
2. Extraction: take 3ml of urine in a 50ml stoppered flask, add 0.1ml of 56% sulfuric acid and anhydrous sulfuric acid according to 2.5g of light shaking to make it dissolve, add chloroform n-butanol mixture of 15ml with a stopper and vibrate for 3 minutes, pour the contents of the pointed bottom centrifugal tube, centrifuged at 1500 rpm for 10-15 minutes, suction to remove the upper layer of urine. Leave the chloroform n-butanol extract for spare.
3. Color development. Take a clean test tube with stopper and operate according to the following table: 
Shake the tubes vigorously for 2 min and leave for 30 min (or 1500 rpm centrifugation for 15 min) and suck the upper layer (mixture of phenylhydrazine sulphate) into clean tubes respectively. Place in a 56℃ water bath for 25 min. After taking out at 410nm wavelength with 0.5cm aperture colorimetric cup, with a colorimetric blank tube to zero, read the optical density value of each tube.
Calculation:
17-hydroxysteroid (mg/24h)=(A-B)/C×0.02×total urine volume
Normal range: males: 5-14mg/24 small urine
Female: 4-14mg/24h urine