Ketosis occurs when there is an excess of fatty acid metabolism due to inadequate carbohydrate intake or metabolism. This produces ketone bodies - acetone, acetoacetic acid and beta-hydroxybutyric acid - which are excreted in urine as ketonuria. Ketonuria is commonly seen in uncontrolled diabetes and other conditions involving excessive fat metabolism and limited carbohydrate availability. It can be detected using tests like Rothera's test, reagent strips, or nitroprusside tablets which detect ketone bodies in urine and produce a color change.

1. Mr.Manoj kumar Mehata

2. KETOSIS
 Whenever there is a defect in carbohydrate metabolism or
absorption or an inadequate amount of carbohydrate in the diet,
the body compensates by metabolizing increasing amounts of
fatty acids.
 When this is increase is large, ketone bodies the products of
incomplete fat metabolism , begin to appear in the blood known
as ketonemia and is followed by kenonuria and kussmaul
respiration (acetone breath).these all are collectively known as
ketosis.

3. KETONE BODIES
 Ketones bodies are water soluble molecules produced by liver,
intermediate product of fat metabolism, they are acetone,
acetoacetic acid and beta hydroxybutyric acid.
 Ketone is found when there is excessive fat metabolism.
excessive fat metabolism occurs in various situation
 Impaired ability to metabolize carbohydrate
 Inadequate carbohydrate intake
 Excessive carbohydrate loss
 Increased metabolic demand.

4. KETOGENESIS
Acetyl coA + Acetyl coA
acetoacetyl coA synthase
Acetoacetyl coA
HMG coA synthase
b-hydroxy b-methyl glutaryl coA
HMG coA lyase
Acetoacetate
decarboxylaton dehydrogenase
acetone b-hydroxy butyrate

5. KETONURIA
 In ketonuria, the three ketone bodies present in the urine are
 Acetoacetic (diaacetic) acid (20%)
 Acetone (2%)
 3-hydroxybutarate(about 78%)
According to killander (1962) up to 2mg acetoacetic acid per
deciliter is normal. Ketonemia and ketonuria are commonly seen in
uncontrolled diabetes mellites, as well as several other conditions.

6. DIABETIC KETONURIA
 Ketonuria implies the presence of ketoacidosis (ketosis) and may
provide a warning of impending coma.
 Upto 50mg of acetoacetic acid per deciliter may be present
without clinical evidence of ketosis.
 Type 1 diabetic patients are prone to episodes of ketosis, often
associated with infection ,stress or other problems in
management.
 Whereas there are large amounts of ketones and glucose in urine
in diabetic ketoacidosis, ketonuria is not found with the
hyperosmolar hyperglycemic coma sometimes occuring in type
2 diabetics.

7. NON DIABETIC KETONURIA
 In infants and children, ketonuria commonly occurs in a variety
of condition, such as acute febrile diseases and toxic states
accompained by vomiting or diarrhoea.
 Inherited metabolic disease should be suspected when there is
severe persistent neonatal ketosis. Ketonuria may be present in
hyperemesis of pregnancy, in cachexia, and following
anesthesia. In these case, ketoneuria is likely related to
increased tissue (especially fat) catabolism in the face of limited
food intake.
 In pregnancy, a normal patient may have a low fasting blood
glucose level and mild ketonuria.

8. LACTIC ACIDOSIS
 Lactic acidosis may coexist with many conditions including
shock diabetes mellitus, renal failure, liver disease , infections
and in response to certain drugs, especially phenformin and
salicylate poisoning.
 Acetoacetate and 3-hydroxybutyrate may both be highly
elevated, although usually the butyrate is high and acetoacetate
low. Under these circumstances, ketonuria may not be detected
by usual nitroprusside test.

9. METHODS
 Rothera's test for acetone.
 Reagent strip test
 Nitroprusside tablet test
 Gerhard's test for diacetic acid
 Lindeman's test for diacetic acid
 Han’s method for beta-hydroxybutyric acid.
 Enzymatic method
 Flourometric technique and automatic colorimetric
method

10. ROTHERA’S TEST
Principle: Nitroprusside used in this test reacts with both acetone
and acetoacetic acid in presence of alkali to produce purple ring
at the junction.
Requirements
 Test tubes
 dropper
 Rothera’s powder mixture
 Sodium nitroprusside:1gm
 Ammonium sulphate:20gm
 Sodium carbonate:20gm

11. Procedure
 Weigh out require amount of sodium nitropruside, ammonium
sulphate and anhydrous sodium carbonate.
 Mix completely, but do not grind together. keep dry and it will
keep for at least a year.
 Place a small pinch of powder on a white surface tile or in test
tube and add 1 drop of urine.
 Acetone and acetoacetic acid give an immediate violet colour.
 Report as trace, (+) , (2+), (3+) as the intensity of purple colour.
ROTHERA’S TEST

12. REAGENT STRIP TEST
 This method is based on a
nitroprusside (sodium
nitroferricyanide) reaction for ketones.
Different formulations are available.
 Reagent strips without alkali react to
acetoacetic acid and not to acetone.
With large (3+) results, urine may be
diluted and remeasured, reporting a
‘moderate’ result and dilution factor.

13.  Chemstrip reagent strip contains sodium nitroferricyanide and
glycine, which react with acetoacetic acid and acetone in an
alkaline medium to form a violet dye. A positive result is
indicated by a colour change from beige to violet, which is
read at 60 seconds.
 The method detects about 10mg/dl of acetoacetic acid and
70mg/dl of acetone, and the sensitivity and reaction of the
reagent strip are similar to those of the tablet.
 Multistrip contains buffer and sodium nitroferricyanide,
which react with acetoacetic acid ,producing a pink maroon
color in 15 seconds.
 The reagent area detects 5-10 mg acetoacetic acid per deciliter
of urine. It does not react with acetone.
REAGENT STRIP TEST

14. Disadvantages of reagent strip test
 False positive occurs :
 After use of phthaleins(BSP or PSP dyes) or in the
presence of extremely large amounts of phenylketones
and the preservatives 8-hydroxyquinoline or 1-dopa
metabolites.
 Acetylcysteine(aerosol) produces a strong red color.
 Antihypertensive drugs methyldopa and captopril give
positive results.
 False negative results occur :
 loss of reagent reactivity
 Presence of salicylates
REAGENT STRIP TEST

15. NITROPRUSSIDE TABLET TEST
 A tablet test method may be useful if the urine has interfering color.
 These are very sensitive to humidity and will deteriorate if not stored
properly.
 The acetest tablet contains sodium nitroprusside, glycine, and a
strongly alkaline buffer.
 It can be used to assay whole blood, plasma, serum or urine.
 Acetest will detect 5-10 mg of acetoacetic acid per deciliter of urine
and 20-25mg acetone per deciliter of urine. Like the reagent strip, it
does not react with 3-hydroxybutyrate.
 It will give positive results with 1-dopa and large amount of
phenylketones and with BSP and PSP dyes, which react with the
alkali in tablets.

16. Procedure for tablet test
 Place the tablet on a clean surface, preferbly a piece of white
paper.
 Place one drop of urine, serum, plasma or whole blood on the
tablet
 For urine measurement, compare the color of the tablet with a
color chart at 30 seconds.
 For serum or plasma measurement, compare color of tablet
with color chart at 2 minutes.
 For whole blood measurements, remove clotted blood from
tablet and compare color of tablet with color chart, 10 minutes
after application of the specimen.

17.  If acetone and acetoacetic acid are present , the tablet will
show a color varying from lavender to deep purple.
 Report the result as negative, small , moderate, or large.
 If large , a dilution may be made. Report these analyses in
a form such as this undiluted “large” 1:2 dilution “large”
1:4 dilution “ moderate” etc.

18. Clinical Significance
Increased In
 Diabetes mellitus
 Propanol poisoning
 Severe starvation.
 Severe carbohydrate restriction
 Anorexia
 Fasting
 Fever
 Prolonged vomiting
 Lactic acidosis
 Salicyclate toxicity.