URIC ACID (TPTZ) PROCEDURE

Intended for the Quantitative Determination of Uric Acid in Serum

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., liver and kidney) or impaired renal function. Gout results from the deposit of uric acid in body joints.

Uric acid has been assayed by the reduction of phosphotungstic acid (2) and by the direct measurement at 293 nm before and after uricase treatment (3). The products of the uricase reaction have been used also as a basis for uric acid determinations (4, 5). The phosphotungstic acid method lacks specificity and the uricase procedures are cumbersome and insensitive.

The URIC ACID (TPTZ) PROCEDURE employs the ferric- reduction method proposed by Morin (6, 7). This method offers a sensitive and rapid uric acid determination in which interference is minimized.

PRINCIPLE

One mole of uric acid reduces four moles of ferric ions to the ferrous form. 2, 4, 6 - tripyridyl-s-triazine (TPTZ) reacts with the ferrous ions to form a blue colored complex which absorbs strongly at 590 nm.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 1200 provides:

FERRIC - BUFFER REAGENT - (Cat. No. 1201)

REACTIVE INGREDIENTS:

0.66 mM ferric ions in acetate buffer (pH = 4.65). Stabilizers added.

PRECAUTIONS:

Do not ingest. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be clear and slightly orange in color. If a brown precipitate forms, the reagent may have deteriorated and should not be used.

TPTZ REAGENT - (Cat. No. 1202)

REACTIVE INGREDIENTS:

12.0 mM TPTZ in methanol, stabilizer added.

PRECAUTIONS:

Methanol is toxic. Do not inhale or ingest.

STORAGE AND STABILITY:

Store at 15 - 30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be clear and pale yellow in color. If a precipitate forms, the reagent may have deteriorated and should not be used.

URIC ACID TPTZ CALIBRATOR - (Cat. No. 1203)

REACTIVE INGREDIENTS:

10mg/dL Uric Acid. Stabilizer and Preservative added.

STORAGE AND STABILITY:

Store at 15 - 30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

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

INSTRUMENTS

Use a suitable spectrophotometer or colorimeter calibrated at 590 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Separate serum from the blood clot as soon as possible.

2. Avoid bacterial contamination which can destroy uric acid.

3. Grossly hemolyzed samples should not be used.

SAMPLE STORAGE:

Serum uric acid appears stable for 7 days at 2 - 8ºC. It may also be frozen for six months (8).

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

1. Anticoagulants which complex metal ions should not be used (e.g., EDTA, citrate, oxalate).

2. Patients undergoing ascorbic acid (vitamin C) therapy may show alse elevations.

3. Young et al (9) have reviewed drug effects on serum uric acid.

PROCEDURE

MATERIALS PROVIDED:

FERRIC - BUFFER REAGENT (Cat. No. 1201), TPTZ REAGENT (Cat. No. 1202) and URIC ACID CALIBRATOR (Cat. No. 1203).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.02 mL micropipettor

2. 2.00 mL pipettor or dispenser

3. 0.20 mL micropipettor (optional)

REACTION CONDITIONS:

Wavelength 590 nm

Filter Selection 580 - 610 nm

Reaction Type Endpoint

Incubation Time and Temperature 15 min. at 25ºC / 10 min. at 37ºC

Sample Volume 0.02 mL

Reagent Volume 2.00 mL

Total Volume 2.02 mL

Low Normal 2.2 mg/dL

High Normal 7.0 mg/dL

Calibrator Value 10.0 mg/dL

PREPARATION OF WORKING REAGENT:

Add 1 part TPTZ REAGENT to 10 parts FERRIC - BUFFER REAGENT. Swirl to mix. Use within 12 hours of preparation. Do not refrigerate.

ALTERNATE METHOD:

Dispense 2.0 mL FERRIC - BUFFER REAGENT to all tubes. Add 0.2 mL TPTZ REAGENT to each tube. Mix well. Proceed with test.

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

PERFORMANCE OF TEST:

1. Dispense 2.00 mL of Working Reagent into tubes labeled: Reagent Blank, Calibrator, Control, Sample 1, etc.

2. Place 0.02 mL Specimen into the appropriately labeled tube. Use deionized water as specimen for Reagent Blank. Mix well.

3. Allow the test samples to stand at room temperature for 15 minutes (or 10 minutes at 37ºC).

4. Adjust instrument to zero absorbance at 590 nm using Reagent Blank.

5. Read and record absorbance values for Calibrator, Control and Unknowns.

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

STABILITY OF THE FINAL REACTION PRODUCT:

The test samples should be read within 30 minutes after color development.

CALIBRATION:

Use the URIC ACID (TPTZ) CALIBRATOR (Cat. No. 1203) which is provided in the reagent set. Linearity of the URIC ACID (TPTZ) PROCEDURE extends to 20 mg/dL.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner employed for the Unknowns. EAGLE DIAGNOSTICS offers CHEM - TROL NORMAL (Cat. No. 8100) and CHEM - TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve proper assay values for control sera could indicate possible reagent deterioration and Unknown values should not be reported until thoroughly investigated.

NOTE: Control sera containing azide as a preservative will give values higher than expected.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations:

Unk. Conc. (mg/dL) =

Abs. Unk

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

Abs. Cal.

EXAMPLE:

A 10 mg/dL Calibrator had an Abs. = 0.444 while the Unknown Abs. = 0.237. The uric acid concentration of the Unknown is:

0.237  

------------ X 10 mg/dL = 5.3 mg/dL

0.444

EXPECTED VALUES

MALES - 7.0 mg/dL

FEMALES - 2.20 - 6.2 mg/dL

The ranges represent the 95% confidence interval for 27 males and 26 females sampled from a clinically normal population. It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

PRECISION:

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

      MEAN / ST. DEV. / %CV

WITHIN RUN

Normal 4.3 / 0.09 / 2.1

Abnormal 11.0 / 0.13 / 1.2

RUN TO RUN

Normal 4.3 / 0.16 / 3.8

Abnormal 10.8 / 0.25 / 2.3

SPECIFICITY:

A comparison of this URIC ACID (TPTZ) PROCEDURE with another widely used commercial procedure showed a 99% correlation for 21 human serum samples in the normal and abnormal range.

SENSITIVITY:

This URIC ACID (TPTZ) PROCEDURE has a sensitivity of 0.02 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., Douglass, R., and Babson, A.L., Clin. Chem. 17, 158 (1971).

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

4. Gochman, N., and Schmitz, J.M., Clin. Chem.17, 1154 (1971).

5. Trevedi, R.C., Rebar, L., Berta, E., and Strong, L., Clin. Chem. 24, 1908 (1978).

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

7. Morin, L.G., Clin. Chem. 20, 51 (1974).

8. Dobbs, C.A., J. Biol. Chem.218, 497 (1956).

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

Form 1200

Rev. Feb. 1993