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UNIT: Renal Function(Creatinine, Urea, Nitrogen, Uric Acid) 12RENAL_stambio04.wpd

Uric Acid

Uric acid is a metabolite of nucleic acids, purines, and nucleoproteins and is the end product of protein (purine) metabolism in man. In most mammals, uric acid is oxidized in the liver toallantoin. Higher apes and man appear incapable of destroying uric acid efficiently in this

manner. As a result, uric acid accumulates in the plasma (as sodium urate) and is excreted inthe urine. Occasionally some persons are afflicted with extensive deposits of uric acid (urates)in their tissues, causing a disorder termed gout. Increased levels of uric acid have beenobserved in renal failure, chronic lead poisoning, polycythemia, some leukemias, and toxemiaof pregnancy.

Uric Acid (Stanbio Laboratory) Quantitative Enzymatic Colorimetric - 520 nm

Reaction

2 2 2 2 2Uric acid + 2H O + O Allantoin + CO + H O>Uricase

2 2H O + 4 aminophenazone + DCHB N-(4-antipyryl)-3-chloro-5-sulfonate->Peroxidase

2p-benzo-quinonemoine Dye + H O(absorbance maximum at 520 nm)

Supplies and Equipment 

1. Spectrophotometer(s) capable of measuring transmitted light at 520 nm2. Appropriate cuvets, test tubes and rack3. Centrifuge, timer, and the following pipetting devices: 0.05 mL, 5.0 mL.4. A constant temperature heat block or water bath if performing test at RT, or 37° C.

Reagents

1. Uricase – enzyme prepared from Candida utilis.2. Horseradish Peroxidase3. 4-Aminoantipyrine4. 3,5-Dichloro-2hydroxybenzenesulfonic acid5. Phosphate Buffer, pH 7.56. Uric Acid Standard Solution, 8mg/dL Cat. No. 1044 - an aqueous solution of uric

acid with solubilizer and stablizer added.

*DANGER / PRECAUTIONS:

1. Uric acid reagents are for in-vitro diagnostic use only. Normal precautions should beused as for handling laboratory reagents, and all federal, state, and local laws for handling and disposal followed.

2. Avoid contact and inhalation3. Consider all blood derivatives as potentially hazardous.

Specimens

1. Serum or plasma (heparinized or EDTA). UA is stable for 2-3 days in serum at roomtemperature, 3-7days at 2-8°C, and 6-12 months frozen. Slight hemolysis has no effectupon UA level.

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UNIT: Renal Function (Creatinine, Urea, Nitrogen, Uric Acid) (continued)

E 100 C MLAB 2401 - Clinical Chemistry Lab Manual

NOTE: Procedure can be performed on 24 hour urine collections. Measure and record urine

volume. Mix well. A 10-fold dilution (1 + 9) of the urine sample is required for the

 procedure. Once the uric acid concentration of the dilution is determined, multiply

 by the x 10 dilution factor.

2. 24 hour urine specimen in a container to which has been added 10 mL 5% sodiumhydroxide to prevent uric acid precipitation. Urine levels are stable for 3 days at roomtemperature. Refrigeration is recommended only after a 10 fold dilution has been made.

Interfering substances

1. Hemoglobin levels greater than 100 mg/dL2. Bilirubin greater than 20 mg/dL3. Ascorbic acid can result in falsely lower values.4. Grossly lipemic samples may give falsely elevated results.5. Formaldehyde must be avoided.

Steps

1. Optimize spectrophotometer at 520 nm,2. Appropriately label test tubes (13 x 100 mm) for each Blank, Standard, Control and

Patient.

3. Pipet 2.0 mL Uric Acid reagent into each tube, and bring to assay temperature. (RT,or 37 ° C)

4. Add 0.04 mL deionized water, uric acid standard, control or patient specimen to their appropriate tube, and mix by gentle inversion.

5. Incubate for 10 minutes, (if at room temperature) OR 5 minutes if at 37 ° C).6. Read and record the absorbance (A) of all the standard(s), controls and patient

samples against the reagent blank.7. Subtract the absorbance of the blank from absorbance of samples, controls and

standard to obtain change in absorbance due to uric acid.8. Determine the concentration of control and patient samples by either reading from a

prepared calibration curve, or by calculation against a standard.

Calculation (Serum)

1. Calculate control and patient results using the following formula.

(1)

Quality Control Two levels of assayed serum controls.

Linearity 

When performed as directed, procedure is linear to 20 mg/dL

Expected Values (It is recommended that each laboratory establish a normal range.)

Serum Male 3.6 - 7.7 mg/dLFemale 2.5 - 6.8 mg/dL

Urine 250-750 mg/24 hr (dependent on diet content of purines)

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UNIT: Renal Function (Creatinine, Urea, Nitrogen, Uric Acid) (continued)

MLAB 2401 - Clinical Chemistry Lab Manual CE 101

NameDate

Stanbio Uric Acid Worksheet

Uric Acid

Wavelength _____________ Linearity _____________ Spectrophotometer Used ______________________ 

Identification Absorbance Concentration (units)

Standard _____________________ 

Standard _____________________ 

Standard _____________________ 

Control 1 _____________________ 

Control 2 _____________________ 

Calculation formula(s) and examples

Uric Acid Quality Control

Your Results Controls’ range of expected results. In control?Yes / No

Level 1 ID______________ 

Level 2ID_______________ 

 Accepting Patient Results? Reason