1) This document provides information about estimating uric acid levels including the normal ranges and clinical significance. It describes the enzymatic colorimetric method used which involves uricase cleaving uric acid and producing a quinonemine dye that is measured photometrically. 2) Elevated uric acid can cause gout and is associated with increased production or decreased excretion, while low levels can be caused by decreased enzyme activity or various acquired disorders and medications. 3) The estimation involves incubating serum, plasma or urine samples with reagents containing uricase, peroxidase and other compounds to produce a dye, then measuring absorbance and calculating concentration against a standard.

1. 2ND YEAR MBBS
BIOCHEMISTRY PRACTICAL
NO. 3
Thursday, 30TH July, 2020
Dr. Sana Samad Khan
MBBS,
Demonstrator/ Facilitator
Biochemistry Department,
PMC AJK

2. PART C: ESTIMATION OF URIC ACID BY
SPECTROPHOTOMETER
RENAL PROFILE

3. OBJECTIVES
1. What is uric acid?
2. Estimation of uric acid levels in blood.
3. Normal values of uric acid in serum, plasma
and urine.
4. Clinical interpretation of results.
5. What is gout?

4. URIC ACID
• Final breakdown product of purine degradation in
humans.
• Approximately 2/3rd of the body urate is produced
endogenously and rest if accounted by exogenous
purine diet.
• 2/3rd to 3/4th is eliminated by kidneys and remaining
via GIT.
• Daily synthesis: 400mg
• Dietary sources: 300mg
• Uric acid levels are estimated in order to diagnose
various clinical conditions such as hyperuricemia
and gout.

6. ESTIMATION OF BLOOD URIC ACID
(ENZYMATIC COLORIMETRIC METHOD)
• Principle:
Uric acid is cleaved by Uricase to form Allantoin and
hydrogen peroxide. Peroxide reacts in the presence of
peroxidase, DHBSA (3,4 Dichloro 2-hydroxy benzene
sulfonic acid) and Aminoantipyrine to form
quinonemine dye.
Uric acid + 2H20 + O2 Allantoin + CO2 + H2O2
2H202 + DHBSA + 4-Aminoantipyrine Quinone-diimine dye + 4H20
NOTE: The intensity of red colour dye is directly proportional to
concentration of uric acid and determined photometrically.
Uricase
Peroxidase

7. • REQUIREMENTS
1. Reagent 1 (R1)
• Phosphate buffer
• DHBSA (3,4 Dichloro 2-hydroxy benzene sulfonic acid)
• Uricase
• Peroxidase
• Preservative
• 4- aminoantipyrine
2. Specimen
• Serum, plasma (heparin and EDTA plasma) and urine.
3. Uric acid standard solution (356.9 μmol/L).
4. Spectrophotometer (wavelength = 546nm)

8. • Procedure:
1. Take three test tubes and label then as “Blank”,
“Standard” and “Unknown”.
2. Pour 1000 μL of R1 into all 3 of the test tubes.
3. Put 20 μL of the standard solution into test tube
labelled “Standard”, 20 μL of distilled water in
“Blank” and 20 μL of sample into “Unknown”.
4. Mix and incubate for 5 mins at 37˚C or 10 mins at
20˚C or 25˚C.
5. Measure the absorbance of the sample and the
standard against the blank within 30 minutes.

9. • Calculation:
Uric Acid = Absorbance of sample × concentration of standard
Absorbance of standard
• Normal values:
1. Serum / plasma:
• Male: 3.4 – 7 mg/dL
• Female: 2.4 - 7 mg/dL
2. Urine:
• On an average: 250 – 750 mg/24 hrs

10. • Limitations / Interferences:
1. Elevated levels of Ascorbic Acid produce false
low levels.
2. Icterus: no significance interference up to an
index of 12.
3. Hemolysis: No significant interference up to an
index hb of 50.
4. Lipemia (Intralipid): TGs may interfere.
5. Other purine derivatives can inhibit uric acid
reaction.

11. CLINICAL
INTERPRETAIONS

12. • Elevated levels of uric acid (>7.0mg/dL).
Causes:
1. Increased production :
• Primary (Lesch Nyhan Syndrome)
• Secondary (Myeloproliferative diseases, lymphoproliferative
diseased, hemolytic anemia, drugs such as diuretics,
pyrizinamide, ethanol, ethambutol, etc)
2. Decreased excretion:
• Kidney failure (Increased secretion, decreased reabsorption and
glomerular filtration)
• DKA
• Starvation
• Salicylate intoxication.
HYPERURICEMIA:

13. Clinical significance
• Gout:
A chronic type of inflammatory arthritis in
which uric acid crystals accumulate in the
joints causing severe inflammation.
Commonly affects the big toe followed by
ankle joints, knees and elbows.

14. HYPOURICEMIA
• Lower than normal levels of uric acid (<2 mg/dL)
Causes:
1. Decreased xanthine oxidase activity
2. Fanconi syndrome
3. Acquired disorders
4. Drugs
5. AIDS
6. Inflammation
7. Allopurinol (xanthine oxidase inhibitor)
8. Liver diseases

15. Thank you for your time and patience!
Please feel free to contact me in case of
any questions.
sanasamadkhan@gmail.com

16. STAY HOME
STAY SAFE