A new concept for urate in human serum: Colorimetric assay of total urate-equivalent chromogens in human serum

Chemicals

Sulfuric acid and sodium tungstate were from BDH Chemicals Ltd., England. Disodium hydrogen phosphate, citric acid (H₂O), sodium carbonate, sodium hydroxide and uric acid were from E. Merck, F. R. G. (reagent-grade materials).

Reagents

A solution of 12.5 ml concentrated sulfuric acid in 30 ml water is added to the solution of 50 g sodium tungstate and 20 g disodium hydrogen phosphate in 75 ml water.

After dissolving on heating, the solution is boiled gently for one hour under a reflux condenser. Then a few drops of bromine are added and the boiling is continued for further five minutes without reflux. After cooling, the solution is made up to 500 ml with water and 150 g of citric acid (H₂O) are added and dissolved. The reagent is stable at room temperature if kept protected from light.

Colorimetric study

To 100 μL of serum in each of eight test tubes, add 2.5 ml of 0.4 mol/L sodium hydroxide solution, mix and leave at room temperature. After five minutes, add to the first tube 0.5 ml of the sodium carbonate solution, 1 mol/L, followed by 1 ml of the diluted phosphotungstic acid reagent and mix. After 10 minutes, read the absorbance at 700 nm against water blank.

Repeat with the second, the third tube ... etc, after 10, 15, 20, 25, 30, 35 and 40 minutes respectively.

Ultraviolet study

Dilute 100 μL of serum with 3.9 ml of water. Transfer 0.5 ml aliquots of the diluted serum to each of two test tubes.

In the first tube, complete the total volume to 4 ml with water and record the absorbance in the U.V. light in the region between 270–300 nm against water blank.

To the second tube, while keeping the spectrophotometer ready for measurements, add 2.2 ml of water and rapidly add 1.8 ml of 1 mol/L sodium hydroxide solution (to make a final volume with 0.4 mol/L sodium hydroxide) and instantaneously record the absorbance in the region between 270–300 nm. Repeat the measurements after 30 minutes.

Determination of Total Urate-Like Chromogens (TUEC)

Add 100 μL of serum to 2.5 ml of sodium hydroxide 0.4 mol/L and leave for 30 minutes at room temperature (20–25°C), then add 0.5 ml of sodium carbonate 1 mol/L and 1 ml of the diluted phosphotugstic acid reagent and mix. After 10 minutes read the absorbance at 700 nm against water as blank.

Calibration Curve

The calibration curve was prepared by diluting the stock standard with water to have the working standards, 2.9745, 1.983, .... and 0.5949 mmol/L respectively.

100 μL of each working standard solution were added to 2.5 ml of the 0.4 mol/L sodium hydroxide solution, then very rapidly, 0.5 ml of sodium carbonate 1 mol/L, and 1 ml of the phosphotungstic acid working reagent were added and mixed. After 10 minutes, the absorbance was measured at 700 nm against water as blank.

Because of the destructive effect of sodium hydroxide solution on uric acid, the calibration curve is linear only in the small concentrations (up to 2.9745 mmol/L). Meanwhile, the measured TUEC in serum is linear up to 5.576 mmol/L (the highest value recorded).

The linear regression equation Y = 0.0185 + 0.21196 X, the correlation coefficient r = 0.9975, S_y.x = 0.0075.

Precision

The Cv_s ranged from 0.23 to 2.4% over a concentration range from 2.513 to 4.995 mmol/L.

Interference from drugs

It has been reported that L-DOPA administration produces spuriously high values for serum urate as measured by the commonly used phosphotungstate reduction methods [1]. Three of its major metabolites; dopamine, 4,4-dihydroxyphenylacetic acid, and 3-methoxyphenylacetic acid also have the same effect [2]. Moreover, we found that VELOSEF, a semisynthetic cephalosporine antibiotic, administration gives total chromogens value as high as 5.576 mmol/L compared with 2.38 mmol/L before administration. Thus before assay, a careful history must be taken to determine if one of these drugs has been ingested recently by patient.