Download Uric acid estimation in plasma

Uric acid
estimation in
plasma
Learning Objectives
Describe the principle of uric acid estimation in
serum and its clinical importance
Uric acid
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:
3.5 - 7 mg/dl.
In females:
3.0 - 6 mg/dl.
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.
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
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)-3methoxyaniline, sodium salt, monohydrate ESPAS)
PHBA (Polyhalogenated benzoic acid ) may also be used instead
Lab exercise
Qassim University
College of Medicine
Phase 2, Yr. 1 , Musculoskeletal & Integumentary Block (CMD222)
Biochemistry Lab exercise
Saturday, June 13, 2010
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
DISTILLED WATER
STANDARD URIC ACID
SOLUTION (50 MG/L)
PLASMA SAMPLE
MONOREAGENT
ABSRBANCE AT
λ 550nm
BLANK
50 μl
-
STANDARD
50 μl
SAMPLE
25 μl
-
1 ml
0
1 ml
25 μl
1 ml
Write your comment on the case:
………………………………………………………………………………………
………………………………………………………………………………………
Describe the principle of the method
……………………………………………………………………………………
?
?
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 diet
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
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
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
Gout (hyperuricemia):
A metabolic disease characterized by increased levels of uric acid (as
urates) in the blood, hyperuricemia (above 7.0 mg/dL) accompanied
with increased excretion of uric acid in the urine.
-
This results in the deposition of sodium urate in the form of
monosodium urate needle-like crystals on the joint linings and in soft
tissues around it (especialy those of the big toe) skin, kidney and other
tissues.
 The precipitated urates are called tophi. It results in inflammatory
reactions in the joints, i.e., acute gouty arthritis that progresses to a
chronic form.
 Uric acid and urates precipitates in the kidney and urinary tract may
result in stone formation.
Urate
in the blood could accumulate either through an overproduction
and/or an under-excretion of uric acid.
Types of gout:
A) Primary: (inherited defect)
(1) Metabolic: is due to inherited autosomal or X-linked recessive metabolic
defects (congenital enzyme deficiencies) characterized by increased rate
of purine synthesis and/or decreased rate of salvaging purine from the
breakdown pathway, leading to overproduction of uric acid.
•
Lesch-Nyhan syndrome: that is a X-linked recessive complete deficiency of
hypoxanthine-guanine phosphoribosyltransferase (HGPRT) that leads to
increased rate of purine synthesis, breakdown and excretion with tendency
of self-mutilation and uric acid stones formation.
•
Von Gierke's disease (The glycogen storage disease) is another example, in
which absence of glucose-6-phosphatase shuttles glucose-6-phosphate to
HMP-shunt with excessive production of ribose, a precursor for purine
synthesis.
(2) Primary renal gout: is a rare condition and is due to an inherited defect in the
kidney leading to decreased secretion of urates by the renal tubules.
Lack of HGPRT activity in Lesch-Nyhan Syndrome causes a buildup of
PRPP, which activates the synthesis of purine nucleotides
hypoxanthine-guanine
phosphoribosyl transferase
Guanine + PRPP
Hypoxanthine + PRPP
Guanylate + PPi
Inosinate + PPi
Purine Salvage and Lesch-Nyhan syndrome





Salvage pathways collect hypoxanthine and guanine and recombine them with PRPP to
form nucleotides in the HGPRT reaction
Absence of HGPRT is cause of Lesch-Nyhan syndrome
This increase may be due to PRPP feed-forward activation of de novo pathways
the rate of purine synthesis is increased about 200X
Symptoms are gouty arthritis due to uric acid accumulation and severe neurological
malfunctions including mental retardation, aggressiveness, and self-mutilation
B) Secondary:
The levels (urates) in the body fluids are elevated as a result of:
Increased uric acid production secondary to
increased cell destruction and breakdown of
nucleoproteins (increased turnover of nucleic
acids) as malignancies, prolonged fasting,
Polycythaemia, Pneumonia, Psoriasis.
Decreased excretion of uric acid as in severe renal
failure (secondary renal gout).
Types of gout
• Primary gout:
Due to inherited metabolic defects leading to excessive
purine production and resultant hyperuricemia. e.g.
altered function of PRPP synthase
Clinical
disorder
Defective
enzyme
Nature of Defect
Manifestation
Gout
PRPP
Synthase
increased enzyme activity due to
elevated Vmax
Hyperuricemia
Gout
PRPP
Synthase
enzyme is resistant to feed-back
inhibition
Hyperuricemia
Gout
PRPP
Synthase
enzyme has increased affinity for
ribose-5-phosphate (lowered Km)
Hyperuricemia
Gout
HGPRT
partially defective enzyme
Hyperuricemia
Lesch-Nyhan
Syndrome
HGPRT
Complete deficiency
Hyperuricemia
–self mutilation
Other Disorders of purine metabolism
Clinical disorder Defective
enzyme
Nature of
Defect
Manifestation
Severe combined
Immunodeficiency
ADA
Complete
deficiency
T & B cell
immunodeficiency
Immunodeficiency
Purine
nucleoside
phosphorylase
Complete
deficiency
T & B cell
immunodeficiency.
Inosinuria,
deoxyinosinuria,
hypouricemia,
guanosinuria etc
Renal Lithiasis
adenine
Phosphoribosyltransferase
Complete
deficiency
2,8-dihydroxyadenine
renal Lithiasis
Xanthiuria
Xanthine
Oxidase
Complete
deficiency
Xanthine renal Lithiasis,
hypouricemia