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.

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

Principle of estimation Uricase

Uric acid + O2 +H2O Allantoin + H2O2

PeroxidaseH2O2 + 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

Lab exerciseQassim 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

Write your comment on the case:………………………………………………………………………………………………………………………………………………………………………………Describe the principle of the method……………………………………………………………………………………

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 ml

ABSRBANCE AT λ 550nm

0? ?

Clinical significanceHyperuricemia > 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 dietImpaired 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

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.

Guanine + PRPP Guanylate + PPi

hypoxanthine-guanine phosphoribosyl transferase

Hypoxanthine + PRPP Inosinate + PPi

Lack of HGPRT activity in Lesch-Nyhan Syndrome causes a buildup of PRPP, which activates the synthesis of purine nucleotides

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 nucleosidephosphorylase

Complete deficiency

T & B cell immunodeficiency. Inosinuria, deoxyinosinuria, hypouricemia, guanosinuria etc

Renal Lithiasis adenine Phosphoribo-syltransferase

Complete deficiency

2,8-dihydroxyadenine renal Lithiasis

Xanthiuria Xanthine Oxidase

Complete deficiency

Xanthine renal Lithiasis, hypouricemia

Thank you..