Ketone bodies and it's measurement

TESTING FOR KETONES

The three ketone bodies are acetone (2%), acetoacetic acid (20%) and 3-β-hydroxybutyrate (78%). The primary substrates for ketone body formation are free fatty acids from adipose stores. When glucose is not available ketone bodies supply the majority of the brains energy. After 3 day fast, ketone bodies provide 30% to 40% of the body’s energy requirements. In uncontrolled diabetes the low insulin concentrations result in increased lipolysis and decreased reesterification, thereby increasing plasma free fatty acids. In addition, the increased glucagon: insulin ratio enhances fatty acid oxidation in

Fig. Ketone bodies Synthesis

the liver (as seen in type 1 diabetes). Increased counter-regulatory hormones also augment lipolysis and ketogenesis in adipose tissue and liver respectively. Thus increased hepatic ketone production and decreased peripheral tissue metabolism lead to acetoacetate accumulation in the blood. A major fraction is converted to β-hydroxybutyrate. In healthy people beta hydroxybutyrate and acetoacetate are present at equimolar concentrations, here acetone is minor component. In severe diabetes the ratio between beta hydroxybutyrate to acetoacetate may increase to 6: 1 owing to the presence of large concentration of NADH which favors beta hydroxybutyrate production. Measurement of ketone bodies is recommended for patients with type 1 diabetes during acute illness, stress, pregnancy or elevated blood glucose >300 mg/dL or when the patients has signs of ketoacidosis.

None of the commonly used methods for the detection and determination of ketone bodies in serum or urine reacts with all 3 ketone bodies. Gerhardt’s ferric chloride test reacts with acetoacetate only. Nitroprusside test are least 10 times more sensitive to acetoacetate than to acetone and give no reaction at all with beta hydroxybutyrate. So, most of the tests for ketosis essentially detect or measure acetoacetate only, so presence of ketosis may not be detected. Traditional tests for beta hydroxybutyrate are indirect; they require brief boiling of the urine to remove acetone and acetoacetate by evaporation (acetoacetate first break down spontaneously to acetone) followed by gentle oxidation of beta hydroxybutyrate to acetoacetate and acetone with peroxide, ferric ions or dichromate.  The acetoacetate thus formed may be detected with Gerhardt’s test or one of the procedures using Nitroprusside.

Excessive formation of ketone bodies results in increased blood concentrations (ketonemia) and increased excretion in the urine (ketonuria). This process is observed in conditions associated with decreased availability of carbohydrates (like in starvation or frequent vomiting) or decreased use of carbohydrates (like in diabetes mellitus, glycogen storage disease, von Gierke’s disease and alkalosis). The popular high-fat, low-carbohydrate diets are ketogenic and increase ketones in circulation. Diabetes mellitus and alcohol consumption are the most common cause of ketoacidosis in adults. Ingestion of isopropyl alcohol and salicylate poisoning can also produce ketoacidosis. Urine ketones are widely used for monitoring control in patients with type 1 diabetes, pregnancy with preexisting diabetes and GDM.

Although quantitative determination of individual ketone bodies is possible these methods are not used as routine tests. The semiquantitative acetest and ketostix are frequently used but are insensitive to beta hydroxybutyrate so negative Nitroprusside test result does not rule out ketoacidosis.

The test strip contains a mixture of glycine, sodium Nitroprusside, disodium phosphate, and lactose. Acetoacetate or acetone in the presence of glycine forms a lavender-purple complex with Nitroprusside.

Beta hydroxyl butyrate can be determined by using beta hydroxybutyrate dehydrogenase to convert it to acetoacetate with the production of NADH; this NADH is oxidized to NAD in the presence of NBT and enzyme diaphorase and producing reduced NBT which is purple compound whose absorbance can be measured.

Urine testing for ketone bodies are useful for type 1 diabetes mellitus. This testing is important when glucose levels are persistently higher and associated with symptoms compatible with ketoacidosis like nausea, vomiting or abdominal pain. The commercial test uses Nitroprusside-based (Rothras test) reactions to measure acetoacetate. Certain drugs may cause positive results like captopril and levodopa, while ascorbic acid intake may cause false negative results.