Lab report on Uric acid determination test

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER: Prof NJAGI
HANDING DATE: 25 /10/2016
SUBJECT: PRACTICAL REPPORT ON DETERMINATION OF URIC ACID
Lando E.
©

2. INTRODUCTION
Uric acid is a purine compound that circulates in plasma as sodium urate and is excreted by kidney. It is
derived from the break down of nucleic acids that are ingested or come from the destruction of tissue
cells; it is also synthesized in the body from simple compounds.
OBJECTIVES:
.To know the uric acid level in the body.
.To diagnose a case of hyperuricemia(Gout)
METHODS
-Chemical method (phosphotungestic acid method)
-Enzymatic method
PRINCIPLE[ Enzymatic Colometric(Uricase Method)]
Uric acid is oxidized by uricase to allantoine and hydrogen peroxide, which inder the influence of POD,4-
aminophenazone(4-AP) and 2-4 Dichlorophenol sulfonate (DCPS) forms a red quinoneimine compound:
Uric acid + 2H2O + O2 Allantoine + CO2 +2H2O2
urease
2H2O2 + 4-AP +DCPS POD Quinoneimine + 4H2O
POD
The intensity of the red color formed is proportional to the uric acid concentration in the sample.
CLINICAL SIGNIFICANCE
Uric acid and its salts are end products of the purine metabolism. With progressive renal insufficiency,
there is retention in blood urea, cratinine and uric acid. Elevated uric acid level may be indicative of renal
insufficiency and is commonly associated with gout , clinical diagnosis should not be made on a single
test result , it should intergrate clinical and other laboratory data.
PREPARATION
Working reagent: Dissolve the contents of one vial R 2 Eenzyme in one bottle R1 Buffer. Cp and mix
gently to dissolve contents.
SAMPLES
-Serum or plasma
-Urine( 24hr) ,diluted in 1/50 in distilled water.

3. PROCEDURE
1.Assay conditions:
Wavelength…………520nm
Cuvette……………….1cm light path
Temprature………….370
C
2.Adjust the instrument to zero with distilled water.
3.Pipette into cuvette.
Blank Standard Sample
WR(ml) 2.0 2.0 2.0
Standard(µl) 40.0
sample(µl) 40.0
Absorbance
4.Mix and incubate for 15min at 370
C or 10min at 15 – 250
C
5.Read the absorbance of the samples and standard, against the blank.
RESULTS AND CALCULATIONS
Blank Standard Sample
WR(ml) 2.0 2.0 2.0
Standard(µl) - 40.0 -
sample(µl) 40.0
Absorbance 0.077 0.421 0.173
Concentration of uric acid = Sample/standard x 8
=0.173/0.421 x 8
= 3.287Mg/dl
Conversion factor to µ mol/L
Mg/dl x 59.5= µ mol/L
3.287 Mg/dl x 59.5
=195.577 µ mol/L

4. REFERENCE VALUES
Women 2.5 – 6.8mg/dl(149 405 µ mol/L)
Men 3.6 – 7.7 mg/dl (214 – 458µ mol/L)
DISCUSION AND RESULTS
Uric acid (2,6,8 trioxypurine-C5H4N4O3) is an organic compound that is endogenously produced by
animals as a purine metabolite. It is formed by the liver and mainly excreted by the kidneys (65-75%) and
intestines (25-35%). Uric acid (UA ) is the end product of purine metabolism in humans due to the loss of
uricase activity, which led to humans having higher UA levels than other mammals.
Due to its double bonds, uric acid has excellent antioxidant capacity, and it can be responsible for 2/3 of
total plasma antioxidant capacity.
Because it is a weak acid that have a high dissociation constant, uric acid circulates in plasma (pH 7.4)
predominantly (98%) in the form of a monovalent sodium salt (urate). It shows low solubility in water (as
well as in plasma), and it would theoretically reach plasma saturation in the concentration of 6.4 mg/dL,
which may not occur because solubility increase is provided by its binding to proteins, namely albumin,
which is its main transporter. Protein-bound uric acid shows plasma solubility that is 70% higher than in
its free state . Uric acid pathogenesis is usually associated with gouty arthritis or nephrolithiasis.
In the kidney, uric acid and urate are initially filtered and additionally secreted. However,the largest part
(90%) is usually reabsorbed and returns to blood. Physiologically, uric acid plasma concentrations
increases with age; they are smaller in women of childbearing age and, in post menopause women, it
increase to similar values to those found in males.
High uricemia pathogenicity is associated with its low solubility in the extracellular environment leading
to crystal formation, low affinity (and deposition) to certain tissues and antigenicity (after crystal
phagocytosis). This mixture of quantitative and qualitative etiological hyperuricemia factors is
confounding because normouricemic individuals may show symptoms while others with hyperuricemia
may not . High plasma uric acid (UA) is a precipitating factor for gout and renal calculi as well as a strong
risk factor for Metabolic Syndrome and cardiovascular disease. The main causes for higher plasma UA
are either lower excretion, higher synthesis or both. Higher waist circumference and the BMI are
associated with higher insulin resistance and leptin production, and both reduce uric acid excretion. The
synthesis of fatty acids (tryglicerides) in the liver is associated with the de novo synthesis of purine,
accelerating UA production. The role played by diet on hyperuricemia has not yet been fully clarified, but
high intake of fructose-rich industrialized food and high alcohol intake (particularly beer) seem to
influence uricemia. It is not known whether UA would be a causalfactor or an antioxidant protective
response. Most authors do not consider the UA as a risk factor,but presenting antioxidant function. UA
contributes to > 50% of the antioxidant capacity of the blood. There is still no consensus if UA is a
protective or a risk factor, however,it seems that acute elevation is a protective factor,whereas chronic
elevation a risk for disease.

5. References
1. Praetorius, E.,An enzymatic method for the determination ofuric acid by ultraviolet
spectrophotometry. Scand. J. Clin.Lab.Invest. 1,222 (1949).
2. Liddle, L., Seegmiller, J. E., and Laster,L., The enzymaticspectrophotometric method for
determination of uric acid. J. Lab.Clin.Med. 54,903 (1959).
3. Lorentz, K.,and Berndt, W., Enzymic determination of urica cid by a colorimetric method. Anal.
Biochem. 18, 58(1967).
4. Domagk, G. F., and Schlicke, H. H., A colorimetric method using uricase and peroxidase for the
determination of uric acid.Anal. Biochem. 22,219(1968).