Posted 22 July 2011 - 09:19 AM
Posted 26 June 2017 - 01:59 AM
Cystine colorimetric determination in urine and cell lysate (Shinohara and Padis modified method)
The reaction measures the quantity of urinary sulfhydryl groups. The cystine is reduced by the sodium sulfite to cysteine and cysteinesulfonic salt. The sulfhydryl group (SH) of cysteine, and any other urinary compound containing this group, reduces phosphotungstic acid to tungstate, a blue color that is spectrophotometrically measured at 600 nm. The reduction of phosphotungstic acid can also occur as a result of the action of other substances such as ascorbic and uric acids. In order to eliminate the interference of these substances, 2 test tubes per urine sample are prepared. Mercuric chloride is added to 1 of the tube, which binds the SH groups. This binding makes the SH group nonreactive with phosphotungstic acid and does not interfere with other compounds ability to reduce phosphotungstic acid. The SH groups are represented by the difference in the intensity of the coloring with and without mercuric chloride.
Acetate Buffer 2 M: add 2 parts of acetic acid (2 M) to 10 parts of sodium acetate (2 M), stable at 4 °C for several days.
Sodium Sulfite: add 12.6 g Na2SO3 and 0.2 g NaOH (or 5 mL NaOH 1 N) to 100 ml dH2O, resulting stable for about 30 days at 4 °C.
Mercuric Chloride: dissolve 2.7 g HgCl2 in 100 ml dH2O, stable at room temperature.
Phosphotungstic Acid: dissolve 40 g molybdenum-free sodium tungstate in about 300 ml dH2O, add 32 ml 85% orthophosphoric acid, reflux gently for 2 hours, cool to room temperature and make up to 1 liter with dH2O, mix, dissolve 32 g Li2SO4, . Water in reagent is stable indefinitely in refrigerator.
Cystine standard: dissolve 1 g/L cystine in 0.1 N HCl solution and from this mather solution obtaine through several dilution, always in 0.1 N HCL, concentrations ranging from 25 to 600 mg/L that are stable at 4 °C
Cell TNES Lysis buffer with protease inhibitors: prepare buffer to contain 0.5% (v/v) Nonidet P40, 1 mM EDTA, 150 mM sodium chloride, and 1× mammalian protease cocktail in 50 mM Tris buffer, pH 7.4. Dissolve 0.6057 g Tris base in 75 ml water and add 1 N HCl to adjust pH to 7.4. Add 0.03722 g EDTA (disodium salt, dihydrate) and 0.8766 g NaCl, mix to dissolve. Add 0.5 ml Nonidet P40. Check pH and adjust if necessary. Bring final volume up to 100 ml and refrigerate. Just before use, add a mammalian protease inhibitor cocktail (protease inhibitor cocktail – Sigma cod. P8340) to yield a 1× concentration. For cultured cells, harvest cells with 200-250 μl of lysis buffer for a 60 mm culture dish, then centrifuge to obtain supernatant usefull for test.
Test tubes: dispense in labeled tubes, 1 ml dH2O (reagent blank), 1 ml standard (standard or standard serie for calibration curve - 100, 200, 300, 400 mg/L), 1 ml urine or cell-lysate (sample blank) and 1 ml urine or cell-lysate (sample test). Dispense in each tube, 1 ml acetate buffer and 0.3 ml of sodium sulfite and mix. In tubes labeled as “reagent blank” and “sample blank” dispense 1.5 ml of dH2O and 0.2 ml of mercuric chloride solution, mix and after 2 min. add 1 ml phosphotungstic acid solution, while in standard or standard serie and in sample test dispense 1.7 ml of dH2O and 1 ml phosphotungstic acid solution and incubate for 15 min. Read the Abs of standard and sample test at 600 nm against their respective blanks.
Test microplate : dispense in wells of MTP, 50 µl dH2O (reagent blank), 50 µl standard (standard or standard serie for calibration curve - 100, 200, 300, 400 mg/L), 50 µl urine cell-lysate(sample blank) and 50 µl urine or cell-lysate (sample test). Dispense in each wells, 50 µl acetate buffer and 15 µl of sodium sulfite reagent and shaker on microplate shaker. In well contenent “reagent blank” and “sample blank” dispense 75 µl of dH2O and 10 µl of mercuric chloride solution, mix and after 2 min. add 50 µl phosphotungstic acid solution, while in wells containing standard or standard serie and in sample test dispense 85 µl of dH2O and 50 µl phosphotungstic acid solution and incubate for 15 min. Read the Abs of standard and sample test at 600 nm against their respective blanks in microplate reader. Using this method the value of cysteine in normal subjects was 80 ± 54 mg/24 hours