Uric Acid

Introduction

The breakdown product of purines in man is uric acid. The catabolism of purine nucleotides is accomplished as given below. Adenosine is first deaminated to form inosine. This reaction is catalyzed by adenosine deaminase in the liver and other tissues. Inosine is converted to hypoxanthine by the action of nucleoside phosphorylase. Guanosine is converted to hypoxanthine by the action of nucleoside phosphorylase. Guanosine is converted to guanine, which is then deaminated to form xanthine. This conversion is catalyzed by guanase in the liver, spleen, pancreas and kidneys. Hypoxanthine is oxidized to form xanthine by xanthine oxidase which also catalyzes the formation of uric acid from xanthine. In man, purine metabolism ends as uric acid excreted in urine. However, in mammals other than primates, uric acid is metabolized by uricase to form allantoin.

Methods of estimation of uric acid

1. Chemical method –
   • Phosphotungestate method
   • Caraway’s method
   • Brown’s method

2. Enzymatic method –

• • Uricase method

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Henry-Caraway’s method

Principle

Uric acid in the protein-free filtrate reacts with the Phosphotungstic acid reagent in the presence of sodium carbonate (alkaline solution) to form a blue-coloured complex. The intensity of the colour is measured at 650 – 700 nm (red filter).

Reagents

1. Deproteinizing reagent

• 10 g/dl, sodium tungstate: 50 ml
• 2/3 N, sulfuric acid: 50 ml
• Orthophosphoric acid: One drop
• Distilled water: 800 ml

Mix well and store at room temperature in an amber-coloured bottle.

2. 10 gm/dl (w/v): Sodium carbonate

• Dissolve 10 gms of Na2 CO3 in distilled water and makeup to 100 ml with distilled water.
• This reagent is stable at room temperature when stored in a polythene reagent bottle.

3. Stock Phosphotungstic acid reagent

• Sodium tungstate (Molybdate free): 50 gms
• Orthophosphoric acid: 40 ml
• Distilled water: 400 ml

Mix and reflux gently for two hours. Cool and make the final volume up to 500 ml. Store at 2–8oC in an amber-colored container.

4. Stock uric acid standard

• 100 mg/dl Heat about 80 ml of distilled water in a 250 ml beaker to 60^o Add 60 mg of lithium carbonate and mix well.
• Add 100 mg of uric acid and mix thoroughly.
• Add 2 ml formalin and slowly shake 1 ml (1:2) acetic acid. Mix well and make the final volume 100 ml by adding distilled water.

Store in an amber-coloured bottle at 2 – 8^oC.

Sample: Serum

Procedure

1. Dilute the stock Phosphotungstic acid, 1:10, by mixing 1.0 ml of the reagent and 9.0 ml of distilled water. Mix well
2. Dilute the stock uric acid standard (1:200) 0.1 ml of standard 100 mg/dl and 19.9 ml of distilled water. Mix well.
3. Pipette into a centrifuge tube labelled: – Deproteinizing reagent, ml 5.4,  Serum, ml 0.6. Mix thoroughly and centrifuge at 3000 RPM for 10 minutes.
4. Pipettes in the tubes are labelled as follows:

	Test	Standard	Blank
Filtrate, ml	3.0	–	–
Diluted standard ml	–	3.0	–
Distilled water	–	–	3.0
Na2CO3 reagent, ml	1.0	1.0	1.0
Diluted phosphotungstic acid, ml	1.0	1.0	1.0

Mix and keep in the dark for exactly 10 minutes. Read OD of test and standard at 660 nm (red filter) against blank.

Calculations

Serum uric acid, mg/dl  = OD of T / OD of S  x 5mg% x 10                          

Normal range

                     Male – 2 – 7 mg/dl

                     Female – 2 – 5 mg/dl

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Uricase method

Principle

The series of reactions involved in the assay system is as follows:

Uric Acid + O₂ + H₂O ———– Uricase ————– Allantoin + CO₂ + H₂O₂

DHBS + 4AAP + 2H₂O₂ ———- Peroxidase ———– Quinoneimine dye + 4H₂O

• • Uric acid is oxidised to allantoin by uricase with the production of H2 O2.
  • The peroxide reacts with 4-aminoantipyrine (4-AAP) and DHBS in the presence of peroxidase to yield a quinoneimine dye.

The absorbance of this dye at 505 nm is proportional to the uric acid concentration in the sample.

Reagent Composition

R1

Pipes Buffer (pH 7.0)     50 mmol/l

DHBS                                0.50 mmol/l

Uricase                             ≥ 0.32 kU/l

Peroxidase                       ≥1.0 kU/l

4-Aminoantipyrine        0.31 mmol/l

R2 standard                           ——-

Specimen

Use unheamolytic serum or plasma (heparin, EDTA) or urine.

Procedure

	Test	Standard	Blank
Reagent 1, ml	1.0	1.0	1.0
Sample, ml	0.025
Standard, ml	–	0.025	–
Distilled Water, ml	–	–	0.025

Mix and incubate 5 min. at 37^oC. Measure the absorbance of the sample and standard at 505/670nm.

Calculation

Serum uric acid, mg/dl  = OD of Test / OD of Standard x  standard conc.

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Clinical Significance

Hyperuricemia 

1. Decreased excretion

• Idiopathic
• Familial juvenile gouty nephropathy
• Renal insufficiency
• Syndrome X
• Drugs: Causative drugs include diuretics, low-dose salicylate, cyclosporine, pyrazinamide, ethambutol, levodopa, nicotinic acid, and methoxyflurane.
• Hypertension
• Acidosis
• Preeclampsia and eclampsia
• Hypothyroidism
• Hyperparathyroidism
• Sarcoidosis
• Lead intoxication (chronic
• Trisomy 21

Increased production

• HGPRT deficiency (Lesch-Nyhan syndrome
• Partial deficiency of HGPRT (Kelley-Seegmiller syndrome
• Purine-rich diet
• Increased nucleic acid turnover
• Tumor lysis syndrome
• Glycogen storage diseases III, V, and VII.