Blood Uric Acid Determination

Uric acid is the nal product of purine catabolism in humans. Most of the uric acid in
the body dissolves in blood and travels to the kidneys. It is removed from the body in urine. A
small amount of uric acid appears in stool. If too much uric acid is ing produced, its level in the
urine will increase. High blood levels of uric acid in the body can lead to a condition known as
gout, which is characterized by the deposition of solid crystals of uric acid in joints. High levels
of uric acid in the urine can cause kidney stones. Determination of blood uric acid level is
helpful tool in the diagnosis of gout.

Principle Involved in Di erent Test Methods for Blood Uric Acid Determination
1. Phosphotungstic acid
- A bu ered phosphate solution is added to the serum followed by phosphotungstic
acid. The uric acid present is oxidized to allantoin and carbon dioxide and the resulting
reduction of the alkaline phosphotungstate to tungsten blue.

Modi ed Caraway: It involved uric acid in a protein-free ltrate reduces

phosphotungstic acid in alkaline solution to a tungsten (III) blue complex whose absorbance
can be measured spectrophotometrically.

Direct Phosphotungstate Caraway: A bu ered phosphate solution is added to the

serum following the ddition of phosphotungsticacis. The uric acid present is oxidized to
allantoin and carbon dioxide and the resulting reduction of the alkaline phosphotungstate to
tungsten blue.

2. Uricase method
- The enzymatic reaction sequence employed in the assay of Uric aciid is as follows:

Uricase

Uric acid + O2 + 2H2O ----------------------> Allantoin + CO2 + H2O2

Peroxidase

2H2O2 + 4-aminoantipyrine + DHBS -------------------------> chromogen + 4H2O

Uric acid is converted by uricase into allantoin and hydrogen peroxides. The hydrogen
peroxide initiated the coupling of 4-aminoantipyrine to 3,5-dichloro-2-hydoxybenzene sulfonic
acid (DHBS) for the chromogen which is measured at 520nm and which is proportional to the
amount of hydrogen peroxide generated from uric acid.

Materials:
Apparatus

• Spectrophotometer and accessories

• Centrifuge

Glassware/Equipment

• Test tubes

• Blood extraction materials

• Pipette

• Test tube rack

Chemicals and Samples

• Uric acid reagents and standard

• Control

• Distilled water

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 Practical Considerations:
1. Test specimen should be serum and free from hemolysis.

2. Bacterial contamination should be avoided to preserve the loss of uric acid.

3. Uric acid in serum is stable for three days at 2-8 degree celsius and up to 6 months when
frozen.

4. Bilirubin and ascorbic acid can result in falsely depressed uric acid levels.

5. Lipemic samples may cause falsely elevated uric acid levels.

6. Collection tubes containing formaldehyde as a preservative must be avoided.

Procedures:
A. Modi ed Caraway Method
1. Observed the following precautions in performing the procedure:

a. Serum uric acid is stable about 3 days a room temperature.

b. Add uoride or thymol to increase the stability of uric acid in sample and avoid bacterial
destruction.

c. Heparinized plasma can be used as a sample instead of serum.

d. Generally, fasting specimen is not necessary; however, avoid gross lipemia (fatty
appearance of serum) and avoid eating purine-rich foods like legumes, visceral organs,
and others prior to blood extraction.

e. Plasma obtained using potassium oxalate as anticoagulant must be avoided since it

forms insoluble potassium phosphate tungstate resulting to turbidity.

2. Deproteinized of unknown and control

a. Prepare the PFF

Reagents Test tube U Test tube C

Unknown Control

Distilled water 8 cc 8 cc

Serum 1 cc -

Control - 1 cc

2/3 N sulfuric acid 0.5 cc 0.5 cc

10% sodium tungstate 0.5 cc 0.5 cc

b. Miix well. Centrifuge for 5 minutes or until the ltrate is clear.

3. Colorimetric reaction

a. Prepare the following:

Reagents Test tube U Test tube C Test tube B

Unknown Control Blank

PFF 3 cc 3 cc -

Distilled water - - 3 cc

14% sodium carbonate 1 cc 1 cc 1 cc

b. Mix and let stand for 15 minutes (lag phase)

c. Add 1 cc of phosphotungstic acid to all tubes.

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 d. Cover each test tube. Mix well by inversion. Allow to stand at room temperature for 15
minutes.

e. Record the absorbance reading (Optical Density or O.D) of the unknown and control
against the reagent blank at 710 nm.

4. Preparation of standards

a. Prepare the working standard by following the procedure:

- To 0.1 cc of the uric acid standard, add 9.9 cc of distilled water

b. Using the freshly prepared working standard, set three test tubes and dispense the
following reagents as follows:
Concentration of Test tube No. Working standard Distilled water
standard

PFF 1 0.75 cc 2.25 cc

Distilled water 2 1.50 cc 1.50 cc

14% sodium carbonate 3 3.00 cc -

c. Add 1 cc of 14% sodium carbonates to all test tubes and mix.

d. Add 1 cc phosphotungstic acid. Mix and stand for 15 minutes.

e. Record the absorbance reading (optical density or O.D) of the standard against reagent
blank within the next 30 minutes.

5. Calculate the concentration of unknown and control.

Formula:

Concentration of = Optical density of unknown/control X Concentration of

unknown control Standard
Optical density of standard (mg/dL)

Note: To convert mg% to mmol/L, multiply mg% by 0.059

Reference value:

Adult Male: 3-7 mg% (0.18-0.471 mmol/L)

Adult Female: 2-6 mg% (0.12-0.35 mmol/L)

B. Uricase Method

1. Prepare four test tubes and label accordingly: reagent blank, standard, controls,
unknown.

2. Place 1.0 mL of working reagent into all tubes.

3. Pre-warm all tubes at 36 degrees celsius for three minutes.

 4. All 0.025 (25 ul) of sample (serum) to the unknown test tube and mix.

5. Incubate all tubes at 37 degree celsius for 30 minutes.

6. After incubation, zero the spectrophotometer with the reagent blank at 520 nm and
read/record the absorbance of all tubes (wavelength range 500-550)

Calculation:

Concentration of = Optical density of unknown/control X Concentration of

unknown control Standard
Optical density of standard (mg/dL)

Reference Value: 1.5-7.0 mg/dL

C. Caraway Method (Direct Phosphotungstic acid, No PFF)

1. Prepare the three test tubes containing the following:

Pipette into cuvettes Blank Standard Sample

Test Sample - - 20 μl

Standard - 20 μl -

Distilled water 20 μl - -

Bu er reagent 200 μl 200 μl 200 μl

Phosphotungstic acid 400 μl 400 μl 400 μl

Mix and incubate at room temperature for 5 minutes. Measure the absorbance of sample and standard
against the reagent blank after 5 minutes.

Formula:

Concentration of = Optical density of unknown/control X Concentration of

unknown control
Standard
Optical density of standard (mg/dL)

To convert mg/dL to mmol/L, multiply mg/dL by 0.060

Reference values:

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 Male: 3-7 mgs/dl (0.18-0.42 mmol/L)

Female: 2-6 mgs/dl (0.12-0.36 mmol/L)

Clinical indications:

1. Elevated levels (Hyperuricemia)

a. Gout

2. Decreased levels

a. Wilson's disease

b. Fanconi syndrome