URIC ACID (LIQUID / URICASE) PROCEDURE

Intended for the Quantitative Determination of Uric Acid in Serum and Urine

SUMMARY AND EXPLANATION

Serum uric acid is the end product of purine metabolism in the body tissues and is cleared through the kidneys by glomerular filtration (1). Increased uric acid levels may result from leukemia, polycythemia, ingestion of foods high in nucleoproteins (e.g. lever and kidney) or impaired renal function. Gout results from the deposit of uric acid in body joints.

Uric acid had been assayed by a variety of procedures including reduction of phosphotungstic acid (2), direct measurement at 293 nm before and after uricase treatment (3), ferric reduction (4) and enzymatic colorimetric methods employing uricase (5). The URICASE URIC ACID PROCEDURE is an enzymatic method using the coupling of 4 aminoantipyrine 3-5 Dichloro-2-hydrozybenzenesulfate hydrogen peroxide in the presence of peroxidase to form a colored complex which is read at 520 nm.

PRINCIPLE

Uric Acid is oxidized by Uricase to allantoin and hydrogen peroxide. DHBS + 4-aminoantipyrine + hydrogen peroxide, in the presence of peroxidase, forms a colored complex that is measured at 520 nm. The color intensity is proportional to the concentration of uric acid in the sample.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 4260 provides:

URICASE REAGENT - (Cat. No. 4251)

REACTIVE INGREDIENTS:

0.4 mM 4 - aminoantipyrine, 2 mM 3-5 Dichloro - 2 - Hydroxybenzene-sulfonate, 150 U/L Uricase, 2500 U/L peroxidase. Buffered to pH 7.5, stabilizer and 0.02% Azide statement added.

PRECAUTIONS:

Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 2 - 8ºC. Stable until expiration date on bottle if bottle remains tightly sealed.

DETERIORATION:

The reagent should be a clear liquid with an amber to slight pink. Do not use if reagent blank is greater than 0.50 absorbance at 520 nm.

URICASE URIC ACID CALIBRATOR - (Cat. No. 4252)

REACTIVE INGREDIENTS:

5.0 mg/dL Uric Acid. Preservative added.

PRECAUTIONS:

Avoid contact with skin, eyes and clothing. In case of contact, wash with water.

STORAGE AND STABILITY:

Store at 2 - 8ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

Failure to achieve proper assay values of control sera would indicate possible deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 520 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Unhemolyzed serum is recommended.

2. Lipemic samples require a sample blank.

3. 24 hour urine collection may be used.

SAMPLE STORAGE:

Uric Acid appears to be stable in serum and urine for seven days at 2-8ºC and for up to six months frozen.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Lipemic samples require a serum blank. Bilirubin and ascorbic acid can cause falsely depressed levels.Young et al, (6) have reviewed drug effects on uric acid assays.

PROCEDURE

MATERIALS PROVIDED:

URICASE REAGENT (Cat. No. 4251) and URICASE URIC ACID CALIBRATOR (Cat. No. 4252).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.025 mL and 1.0 mL pipettors

2. Incubator capable of maintaining 37ºC

3. Test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 520 nm

Filter Selection 500 - 550 nm

Reaction Type Endpoint

Incubation Temperature 37ºC

Incubation Time 5 minutes

Sample Volume 0.025 mL

Reagent Volume 1.0 mL

Total Volume 1.025 mL

Low Normal 2.5 mg/dL

High Normal 7.0 mg/dL

Calibrator Value 5.0 mg/dL

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE:

(1) Make a 1:10 dilution of urine sample and use as sample in assay.

(2) For instruments requiring a total volume >1.0 mL, increase URICASE REAGENT volume to 2.0 mL and volume of samples to 0.05 mL. Proceed as outlined.

1. Dispense 1.0 mL of Uricase Reagent into tubes labeled Reagent Blank, Control, Sample 1, etc.

2. Pre-warm tubes at 37ºC for 5 minutes.

3. Add 0.025 mL of Samples to respective tubes.

4. Incubate the tubes at 37ºC for 5 minutes.

5. Adjust instrument to zero absorbance at 520 nm using Reagent Blank.

6. Read and record absorbance values for each sample.

NOTE: For a direct read-out instrument, set read-out at concentration of Calibrator and read the Unknown concentrations directly.

PREPARATION OF A SERUM BLANK:

Add 0.025 mL of lipemic sample to 1.0 mL D-H2O. Adjust zero of instrument at 520 nm with D-H2O. Read and record absorbance of sample. Subtract this reading from the test absorbance reading. Use this corrected reading in calculation of results.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 10 minutes after color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-25 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use URICASE URIC ACID CALIBRATOR (Cat. No. 4252) which is provided in the reagent set or other suitable calibration material for this purpose. Specimens exceeding 25 mg/dL must be diluted with D-H2O and reassayed multiplying the test result by the dilution factor to obtain the final result.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for Unknowns. EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations:

Unknown (mg/dL) =

Unk. Abs.

--------------- X Cal. Conc. (mg/dL)

Cal. Abs.

EXAMPLE:

In a test series, the Calibrator had Abs. = 0.152 while the Unknown Abs. = 0.287. The Uric Acid concentration of the Unknown is:

0.287

--------- X 5.0 mg/dL = 9.4 mg/dL

0.152

The following equation is used to determine Unknown concentration when urine is the Unknown sample:

Calculate value in mg/dL using formula as stated above, then using that value, uric acid (mg)/day may be calculated.

Uric Acid = Uric Acid X 10 dL/L x Urine Volume

(mg/day) (mg/dL) (dilution correction) (Liters/day)

EXPECTED VALUES (7)

Male: 3.5 - 7.0 mg/dL

Female: 2.5 - 6.0 mg/dL

Urine: 250 - 750 mg/day

It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 25 mg/dL.

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

WITHIN RUN

MEAN / ST. DEV. / %CV

Normal 3.3 / 0.05 / 1.4

Abnormal 10.4 / 0.07 / 1.1

RUN TO RUN

Normal 3.4 / 0.12 / 2.1

Abnormal 1.03 / 0.15 / 1.6

SPECIFICITY:

A comparison of this URICASE URIC ACID PROCEDURE with another widely used commercial method showed a 99.8% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This procedure has a sensitivity of 0.05 mg/dL per 0.001 absorbance unit.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia, 1976, p. 999.

2. Carrol, J., Colburn, H., Douglas, R., and Babson, A.L., Clin Chem, 17, 158 (1971).

3. Remp, D.G., Clin Chem 6, 1 (1970).

4. Morin, L.G., and Prox, J., Am Jour Clin Path, 60 691 (1973).

5. Praetorius, E., Poulson, H., Scan. Jour Clin Invest, 5, 273 (1953).

6. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. Vol. 21, p.373D (1975).

7. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia, 1976, p. 729.