1. Uric acid estimation in plasma

2. Learning Objectives
Describe the principle of uric acid estimation in serum and its clinical importance

3. 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.

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

5. Clinical significance
Hyperuricemia > 7 mg/dl
In 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 food
Impaired excretion of uric acid in the kidney
Chemotherapy- related side effects(due to breakdown of tumour cells and release of purines)
Stress or excessive exercise
Toxemia of pregnancy

6. Hyperuricemia may lead to
Formation 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

7. 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

8. 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

9. Procedure
Blood 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.

11. Protocol for Colorimetric estimation of Uric acid

12. 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

13. Thank you

14. Qassim University College of Medicine Phase 2, Yr
Qassim University College 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: ………………………………………………………………………………………

15. STANDARD URIC ACID SOLUTION (50 MG/L) - PLASMA SAMPLE MONOREAGENT 1 ml
Qassim University College 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
SAMPLE
DISTILLED WATER
50 μl
25 μl
STANDARD URIC ACID SOLUTION (50 MG/L) -
PLASMA SAMPLE
MONOREAGENT
1 ml
ABSRBANCE AT
λ 550nm
0.20
0.24