URIC ACID, Determining uric acid level by The Caraway method and kit systems

URIC ACID, Determining uric acid level by The Caraway method and kit systems

URIC ACID

Uric acid is the final breakdown product of purine metabolism. Purines such as adenosine and guanine result from the breakdown of nucleic acids. 

They are either ingested or come from the destruction of tissue cells, and are converted mainly in the liver into uric acid. From the liver, uric acid is transported to the kidneys where it is filtered by the glomeruli.

Nearly all the filtered uric acid is reabsorbed in the proximal tubules; and small amounts are then secreted by the distal tubules and ultimately appear in the urine.

Nearly all of the uric acid in the plasma is in the form of monosodium urate. Urates in this form are relatively insoluble. At high levels (> 6.4 mg/dl ), the plasma is saturated; urate crystals may form and precipitate in the tissues.

Assay Methods for Serum/Plasma Uric Acid

Uric acid is readily oxidised to allantoin and so can function as a reducing agent in many chemical reactions. The Caraway method is the most popular method for determining uric acid level. This method is based on the oxidation of uric acid in a protein-free solution, with the eventual reduction of phosphotungstic acid to tungsten blue. Sodium carbonate is used to provide the alkaline pH necessary for colour development. Unfortunately, this method is relatively non-specific, and so several methods that make use of the specific enzyme, uricase, have been developed. This enzyme catalyses the oxidation of uric acid to allantoin with the subsequent production of hydrogen peroxide (H2O2). The uricase method is based on the fact that uric acid has a significant UV absorbance with a peak at 293 nm, whereas allantoin does not have. Therefore, the difference in absorbance in an ultraviolet spectrophotometer before and after incubation with uricase is proportional to the concentration of uric acid.

Caraway Method

Reagents 

(i) Phosphotungstic acid reagent Dissolve 40.0 g of high grade sodium tungstate in about 300 ml of distilled water. Add 32 ml of orthophosphoric acid (H2PO4, 85% w/v). Reflux for 4 hours under a vertical condenser. Cool to room temperature, and add about 300 ml of distilled water. In this solution, dissolve 32.0 g lithium sulphate monohydrate and make volume up to 1 litre. Add 81.0 ml of 2 M sodium hydroxide solution and mix. The final pH is adjusted to 2.5; and the colour should be pale greenish-yellow. 

(ii) Sodium carbonate solution Dissolve 140 g anhydrous sodium carbonate in, and make up to 1 litre with distilled water. Mix well and store in a plastic bottle. 

(iii) Uric acid standard (stock) 50mg/dl Add 1.0 g of lithium carbonate to about 500 ml distilled water. Warm to 60°C to dissolve. Place 500 mg of pure uric acid in a 1 litre volumetric flask and add the lithium carbonate solution to it. Warm to 60°C to dissolve. Cool and add 5.0 ml formalin. 

Add 400 ml distilled water and 10 ml of 50 % acetic acid. Dilute to 1 litre with distilled water. 

(iv) Uric acid standard, working (5mg/dl) Dilute the stock standard 1:10 by diluting 10 ml to 100 ml with distilled water.

Technique

Prepare a set of 3 tubes as follows:

	test	Standard	Blank
Test Phosphotungstic acid reagent	2 ml	2 ml	2 ml
Sample 0.2 ml	0.2 ml
Working standard uric acid (5 mg/dl) -		0.2 ml
Distilled water			ml

(I)Add the reagents as shown above

(ii) Mix well and leave at room temperature for 15 minutes.

(iii) Centrifuge all tubes at 3000 rpm for 10 minutes.

(iv) Decant the supernatant directly into 3 separate correspondingly labelled tubes.

(v) Add 1.0 ml of sodium carbonate solution to each tube.

(vi) Leave at room temperature for 15 minutes.

(vii) Read the absorbance at 650-700 nm, setting the zero with the blank.

         

Kit systems Nearly all the kit systems are based on the enzyme uricase method. They are specific, and the manufacturer's leaflet clearly states in simple language, the method of assay.

Reference Ranges for Uric Acid

Children: 2-5.5 mg/dl (0.12-0.32 mmol/L)

Adult males: 3.5-7.2 mg/dl (0.21-0.42 mmol/L)

Adult females: 2.6-6.0 mg/dl (0.15-0.35mmol/L)

Interpretation

Factors affecting uric acid levels in the blood Increased production, raised serum levels (hyperuricaemia)

(i)                Idiopathic primary gout

(ii)             Excessive dietary purines (liver, kidney, legumes, etc.)

(iii)           Cytotoxic treatment of malignancies such as leukaemias and lymphomas

(iv)           Polycythaemia

(v)             Multiple myeloma

(vi) Sickle cell anaemia.

Decreased excretion, raised serum levels	Decreased level of uric acid (hypouricaemia) It is not very common but can be caused by :
(i)                Alcohol ingestion	(i) Over treatment with allopurinol
(ii) Lactic acidosis, Ketoacidosis	(ii) Ingestion of drugs like aspirin with doses above 4 g a day
(iv) Thiazid diuretics	(iii) Corticosteroids
(V) Renal dysfunction	(iv) Estrogens