Uric acid estimation in plasma

Learning Objectives

Describe the principle of uric acid estimation in serum and its clinical importance

Uric acid is produced by the breakdown of purines, which are essential components of nucleic acids (DNA and RNA) as well as other important biomolecules such as ATP, cyclic AMP and NADH.

Normal value

The normal value of uric acid in human plasma:In males: 3 - 7 mg/dl.In females: 2 – 6 mg/dl.

Clinical significance

Hyperuricemia > 7 mg/dlIn some cases, the level of uric acid is higher than 7 mg/dl; this

is known as hyperuricemia

May be due to:High intake of purine rich foodImpaired excretion of uric acid in the kidneyChemotherapy- related side effects(due to breakdown of tumour

cells and release of purines)Stress or excessive exerciseToxemia of pregnancy

Hyperuricemia may lead toFormation of kidney stones: Most of uric acid is removed by the kidneys and

disposed of in the urine.In hyperuricemia Uric acid crystals precipitate in the kidney and may block

filtering tubules leading to renal failure.

Uric acid crystals in a urine sample

Sodium urate/ kidney stones

Gout: A chronic type of inflammatory arthritis in which uric acid crystals accumulate in the joints causing severe inflammation. It usually affects the joint of big toe, other joints including ankles, knees and elbows may be affected

X-ray of gouty uric acid deposit in the big toe

Uric acid crystals in big toe joint

Principle of estimation Uricase

Uric acid + O2 +H2O Allantoin + H2O2

Peroxidase

H2O2 + 4AA + ADPS Quinoneimime + 4 H2O

The method used is a colorimetric enzymatic method. Uric acid is oxidized by Uricase to allantoin with the formation of Hydrogen peroxide. Hydrogen peroxide can oxidize a mixture of ADPS and 4-aminoantipyrine (4AA) to form a quinoneamine that absorbs maximally at 550 nm.ADPS , highly water soluble aniline derivative, (N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline, sodium salt, monohydrate ESPAS)

PHBA (Polyhalogenated benzoic acid ) may also be used instead

ProcedureBlood is drawn from a vein and transferred into a centrifuge tube containing

an anticoagulant.

Plasma is obtained by centrifugation of blood for 10 minutes.

In clean dry test tubes take 50 μl of distilled water (blank) , standard uric acid solution (standard) and plasma (test).

Then add 1 ml of Monoreagent (the monoreagent is composed of: uricase enzyme, peroxidase enzyme, 4-AA, ADPS and buffer to adjust pH to 7).

Mix the content of each tube and incubate for 5 min at 37 °C using a water bath.

Remove test tubes from the water bath.

Protocol for Colorimetric estimation of Uric acid

Measure the absorbance of the final color of the sample and standard against blank at 550nm.

The color is stable for 30 minutes protected from light.Calculate the concentration of uric acid in the sample

using the following equation

Thank you

Qassim UniversityCollege of Medicine

Phase 2, Yr. 1 , Musculoskeletal & Integumentary Block (CMD222)

Biochemistry Lab exercise Saturday, June 20, 2009 Student Name :_____________________, ID: ____________________ Exercise : Determine uric acid concentration (mg/dl) in the given sample. Mention your requirements here: Observations for the volumes mentioned in the protocol are provided below. The uric acid standard solution used in the procedure had a concentration of 0.5 mg/ml. A( sample) = ………0.36…………………. A(std) = ………0.20…………………. 2. Write your comment on the case:………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

Qassim UniversityCollege of Medicine

Phase 2, Yr. 1 , Musculoskeletal & Integumentary Block (CMD222)

Biochemistry Lab exercise Saturday, June 20, 2009 Student Name :_____________________, ID: ____________________ Exercise : Determine uric acid concentration (mg/dl) in the given sample. Mention your requirements here: Use values in the protocol below to determine the concentration of uric acid in the sample

2. Write your comment on the case:………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

BLANK STANDARD SAMPLEDISTILLED WATER 50 μl 25 μlSTANDARD URIC ACID SOLUTION (50 MG/L)

- 50 μl -

PLASMA SAMPLE - - 25 μlMONOREAGENT 1 ml 1 ml 1 mlABSRBANCE AT λ 550nm

0 0.20 0.24