TEST FOR KETONE BODIES (KETONES)

TEST FOR KETONE BODIES (KETONES)

KETONE BODIES (KETONES):-

Ketone bodies belong to a group of three related substances-acetone, acetoacetic acid (acetoacetate) and B-hydroxybutyric acid (hydroxybutyrate). They are not found in normal urine or blood. The presence of ketones in the urine is called ketonuria and in the blood it is called ketonemia. An increased level of ketones in blood and urine is referred to as ketosis.

Ketones are products of fat metabolism when fat is broken down to serve as a source of energy as in the cases of untreated or uncontrolled diabetes (lack of insulin), starvation or dehydration (vomiting and diarrhoea). When there is absence of or insufficient glucose metabolism, the result is an accumulation of ketones in blood. Ketosis may also result in an individual who is on a ketogenic diet, that is, diet high in fat and low in carbohydrate.

Ketosis is a serious physiological condition. Acetoacetic acid and B-hydroxybutyric acid cause lowering of the blood pH by transferring excess hydrogen ion to the blood. This results in acidosis such as seen in coma found in diabetic ketoacidosis. Acetone and acetoacetic acid, more specially acetone, are toxic to the brain when present in sufficiently high concentration in the blood. In the presence of high level of ketone in the blood, the body, in order to maintain the buffering system, excretes excess acid through urine. The urine pH can be as low as 4.5. The presence of ketone bodies in the urine is therefore associated with low urinary pH.

In the normal formation of the ketones, acetoacetic acid is produced first and then converted to B-hydroxybytyric acid and acetone. If urine containing the three ketone bodies is allowed to stand at room temperature after collection, the acetoacetic acid and the B-hydroxybutyric acid are converted to acetone. The acetone being volatile, may disappear from the urine specimen. For this reason, freshly voided urine is used to detect ketone bodies.

Most of the laboratory tests for ketones detect acetone and acetoacetic acid or only acetoacetic acid. There is no simple laboratory test for B-hydroxybutyric acid. The commonly used methods for ketonuria are based on the principle of Rothera's nitroprusside test. These methods are available in the form of dry reagent strips, tablet tests (Acetest) and tube method.

Acetest Tablet Test (Ames):- Acetest tablet contains sodium nitroprusside, glycine and lactose in alkaline buffer. The principle is virtually the same as the strip tests. Acetest can however be used to test whole blood, plasma or serum. The test kit is supplied with the manufacturer's instructions which should be followed. The addition of lactose enhances the colour.

Procedure:-

1. Place the tablet on a clean surface preferably a piece of white paper

2. Place one drop of urine, serum, plasma orwhole blood on the tablet.

3. Compare the colour of the tablet with the colour chart 30 seconds after the application of urine or 2 minutes after the application of plasma, or serum or 10 minutes after the application of whole blood.In the presence of acetoacetic acid and acetone the tablet will show varying shades of colour purple.

Result:-  Report as negative, small, moderate or large depending on the manufacturer's direction. Acetest detects 5 to 10 mg/dl of acetoacetic acid and 20-25 mg/dl of acetone.

Note;-1.Urine specimens containing large amounts of phethaleins, phenylketones or the preservative 8. hydroxyquinoline may give colour ranging from orange-red to mauve which may be mistaken for the ketones. This is a false positivereaction.

2. Urine specimens containing various pigments, drugs or substances causing abnormally brightly coloured urine may pose a problem in reading the ketone tests.

3. False negative or reduced reactions may be seen in improperly preserved urine due to the conversion of acetoacetic acid to acetone.

   

Causes of ketonuria

1. Diabetes indicates ketoacidosis which maylead to coma if unchecked.

2. Acute febrile states especially in infants.

3. Excessive vomiting and diarrhoea. .

4. Glycogen storage impairment.

Other test for ketone bodies

Reagent Strip Tests for Ketones
 
NitroprussideTube Test (Modified Rothera's Test)

Gerhardt'sTest for Acetoacetic Acid and Salicylates
 

Test for B-hydroxybutyric acid