Summary

Uric acid is the end product of purine metabolism. Uric

acid is excreted to a large degree by the kidneys and

to a smaller degree in the intestinal tract by microbial

degradation. Increased levels are found in gout, arthritis,

impaired renal functions and starvation. Decreased levels

are found in Wilson’s disease, Fanconi’s syndrome and

yellow atrophy of the liver.

Principle

Uricase converts uric acid to allantoin and hydrogen

peroxide. The hydrogen peroxide formed further reacts

with a phenolic compound and 4 aminoantipyrine by

the catalytic action of peroxidase to form a red colored

quinoneimine dye complex. Intensity of the color formed

is directly proportional to the amount of uric acid present

in the sample.

 Uricase

Uric acid + H2O Allantoin + H2O2

 Peroxidase

H2O2 + 4 Aminoantipyrine Red + Phenolic

Compound Quinoneimine dye + H2O

Normal Reference Values

Serum/plasma (Males) : 3.4–7.0 mg/dL

(Females) : 2.5–6.0 mg/dL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 25 mL 75 mL

L1: Buffer reagent 20 mL 60 mL

L2: Enzyme reagent 5 mL 15 mL

S: Uric acid standard (8 mg/dL) 5 mL 5 mL

Storage/Stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: Pour the contents of 1 bottle of L2

(Enzyme reagent) into 1 bottle of L1 (Buffer reagent). This

working reagent is stable for at least 4 weeks when stored

at 2–8°C. Upon storage the working reagent may develop a

slight pink color however, this does not affect the performance of the reagent. Alternatively for flexibility as much

of working reagent may be made as and when desired by

mixing together 4 parts of L1 (Enzyme reagent 1) and 1

part of L2 (Enzyme reagent 2). Alternatively 0.8 mL of L1

and 0.2 mL of L2 may also be used instead of 1 mL of the

working reagent directly during the assay.

Sample Material

Serum, plasma. Uric acid is reported to be stable in the

sample for 3 to 5 days when stored at 2–8°C.

Procedure

Wavelength/filter : 520 nm (Hg 546 nm)/yellowgreen

Temperature : 37°C/RT

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank (B),

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Working reagent 1.0 1.0 1.0

Distilled water 0.02

Uric acid standard (s) - 0.02 -

Sample - - 0.02

Mix well and incubate at 37°C for 5 minutes or at RT

(25°C) for 15 minutes. Measure the absorbance of the

standard (Abs S), and test sample (Abs T) against the

blank, within 30 minutes.

Calculations

 Abs T

Uric Acid in mg/dL = ________ × 8

 Abs S

Linearity

This procedure is linear upto 20 mg/dL. If values exceed

this limit, dilute the serum with normal saline (NaCL 0.9%)

and repeat the assay. Calculate the value using the proper

dilution factor.

System Parameters

Reaction : End point Interval :

Wavelength : 520 nm Sample volume : 0.02 mL

Zero setting :  Reagent

blank

Reagent volume : 1.00 mL

Contd...

Clinical Chemistry 493

Incubation

temperature

: 37°C / RT Standard : 8 mg/dL

Incubated time :  5 min/

15 min

Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 20 mg/dL

No. of read : — Units : mg/dL

Normal Values

SI units

Adult females 2.5–6.0 mg/dL 143–357 µmol/L

Adult males 3.4–7.0 mg/dL 202–416 µmol/L

Children 2.5–5.5 mg/dL 119–327 µmol/L

Panic level > 12 mg/dL > 714 µmol/L

Clinical Relevance

Factors Affecting Serum Uric Acid levels

Increased Production, Raised Serum Levels

¾ Idiopathic mechanisms associated with primary gout

¾ Excessive dietary purines (organ meats, legumes,

anchovies, etc.)

¾ Cytolytic treatment of malignancies, especially

leukemias and lymphomas

¾ Polycythemia

¾ Myeloid metaplasia

¾ Psoriasis

¾ Sickle cell anemia.

Decreased Excretion, Raised Serum Levels

¾ Alcohol ingestion

¾ Thiazide diuretics

¾ Lactic acidosis

¾ Aspirin doses < 2 g/day

¾ Ketoacidosis especially diabetes or starvation.

¾ Renal failure due to any cause.

Increased Excretion, Lowered Serum Levels

¾ Probenecid, sulfinpyrazone, aspirin doses above 4 g/

day

¾ Corticosteroids and ACTH

¾ Coumarin anticoagulants.

¾ Estrogens.

Decreased Production, Lowered Serum Levels

¾ Allopurinol.

CALCIUM

OCPC Method

(Courtesy: Tulip Group of Companies)

For the determination of calcium in serum or plasma (for

in vitro diagnostic use only).