Determination of Uric Acid

Dr. Arvind Kumar

AIM: Determination of Uric Acid

Introduction

  1. The end product of purine breakdown in humans is uric acid.

  2. Purine catabolism begins with the degradation of nucleotides (AMP, GMP).

  3. Adenosine is first deaminated to inosine, and this reaction is catalyzed by
    adenosine deaminase (found in liver and other tissues).

  4. Inosine is converted to hypoxanthine by the enzyme nucleoside phosphorylase.

  5. Guanosine is also acted upon by nucleoside phosphorylase to form guanine.

  6. Guanine is deaminated to xanthine by the enzyme guanase, present in the liver, spleen, pancreas, and kidneys.

  7. Hypoxanthine is oxidized to xanthine by xanthine oxidase.

  8. Xanthine oxidase also converts xanthine to uric acid, completing the oxidation pathway.

  9. In humans and other primates, uric acid is the final product of purine metabolism and is excreted in the urine.

  10. In most mammals (non-primates), uric acid is further metabolized to allantoin by the enzyme uricase, which humans lack.

 

Methods name

  1. Chemical method –
    • Phosphotungstate method
    • Caraway’s method
    • Brown’s method
  1. Enzymatic method –
    • Uricase method

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

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.

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.

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.

Stock uric acid standard

  • 100 mg/dl Heat about 80 ml of distilled water in a 250 ml beaker to 60o 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 – 8oC.

Sample

Serum/plasma

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

 

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 for 5 min. at 37oC. 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.

 

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.