UA 3: SPECIAL TESTS Pt. I (KETONES, BLOOD)

Question 1

intermediate products of fat metabolism

Answer 1

Ketones

• acetone (2%)
• acetoacetic acid (20%)
• B-hydroxybutyrate (78%)

Question 2

increased accumulation of ketones in the blood leads to:

Answer 2

• electrolyte imbalance
• dehydration
• acidosis, eventual diabetic coma (if not corrected)

Question 3

tests for ketone bodies

Answer 3

1. Rothera’s test
2. Reagent strip
3. Gerhardt’s ferric chloride test
4. Hart’s test
5. Confirmatory tests

Question 4

T/F:

measurable amounts of ketones normally appear in the urine

Answer 4

FALSE

- all metabolized fat is completely broken down into CO2 & H2O

Question 5

clinical reasons for increased fat metabolism

Answer 5

• inability to metabolize carbohydrate (in DM)
• increased loss of carbohydrate from vomiting
• inadequate intake of carbohydrate associated with starvation & malabsorption

Question 6

testing for urinary ketones is most valuable in the management & monitoring of (Type I, Type II) DM

Answer 6

Type I DM

• “insulin-dependent”
• ketonuria shows a deficiency of insulin
  
  • • an early indicator of insufficient insulin dosage

Question 7

clinical significance of urine ketones

Answer 7

• diabetic acidosis
• insulin dosage monitoring
• starvation
• malabsorption/pancreatic disorders
• strenuous exercise
• vomiting
• inborn errors of amino acid metabolism

Question 8

for at-home testing by patients, ketone tests are combined with ____________ on test strips

Answer 8

Glucose

Question 9

other causes of positive ketone test results

Answer 9

• illness prevents the adequate intake or absorption of carbohydrates
• illness produces an accelerated loss of carbohydrate (i.e. in vomiting)
• frequent strenuous exercise –> overuse of available carbohydrates –> ketonuria

Question 10

this reaction is used in the reagent strip test for ketones

Answer 10

Sodium nitroprusside reaction

• “nitroferricyanide”
• acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a PURPLE color

Question 11

reporting results in the reagent strip test

Answer 11

READ AFTER 40 SECONDS
- color without reaction: TAN

A. Qualitative
Negative = tan-colored
Trace = pink
Small (+) = dark pink
Moderate (++) = darker pink
Large (+++) = purple

B. Semi-Quantitative
Negative
Trace (5mg/dL)
Small (15mg/dL)
Moderate (40mg/dL)
Large (80-160mg/dL)

Question 12

one of the confirmatory tests for urine ketones

Answer 12

Acetest tablets

• in cases of severe ketosis, tests are performed on serial dilutions
• provide more info on the extent of ketosis
• can also be used for serum & other body fluids
• hygroscopic
  
  • • if specimen is not completely absorbed within 30 minutes, use a new tablet

Question 13

components of the Acetest tablet

Answer 13

• sodium nitroprusside
• glycine
• disodium phosphate
• lactose
  
  • • gives better color differentiation

Question 14

nitroprusside in alkaline medium reacts with a ketone group to form a purple ring

Answer 14

Rothera’s test

• given by acetone & acetoacetate
• NOT BY B-HYDROXYBUTYRATE

Principle:
sodium nitroprusside decomposition

Reagents:

• Rothera’s reagent
  
  • • sodium nitroprusside
  • • ammonium sulfate
• conc. NH4OH

Question 15

Rothera’s test procedure

Answer 15

1. prepare Rothera’s reagent
   - 7.5g sodium nitroprusside + 200g ammonium sulfate
2. add 1 gram Rothera’s reagent to 5mL urine
   - mix well
3. overlay mixture with 1mL conc. NH4OH
4. observe for the appearance of a reddish-PURPLE RING
   - at the interphase of the 2 layers
   - within 1 minute 30 seconds

Question 16

Rothera’s test reporting of results

Answer 16

Negative

• no ring
• brown ring
• red/purple color fades during the first 5 seconds

+
- faint pinkish purple ring appearing slowly

++
- narrow dark purple ring

+++
- wide dark purple ring appearing very rapidly

*the width of the purple ring is roughly proportional to the concentration of ketone bodies

Question 17

identify test:

principle:
sodium nitroprusside decomposition

components:

7. 1% (w/w) sodium nitroprusside
8. 9% (w/w) buffer

Answer 17

Reagent strip

• reading time: 40 seconds
• available in the following brands:
  
  • • Multistix = sensitivity: 5-10mg/dL acetoacetate
  • • Chemstrip = 9mg/dL acetoacetate; 70mg/dL acetone

*5-10mg/dL = 0.25-0.5mmol/L

Question 18

sources of error for the reagent strip test

Answer 18

a. False Positive
- highly pigmented red urine
- phthalein dyes
- Levodopa (med for Parkinson’s disease)
- - may produce atypical color reactions
- meds containing free sulfhydryl groups
- - may produce atypical color reactions

b. False Negative
- improperly preserved specimen
- - breakdown of acetoacetic acid by bacteria
- volatilization of acetone

Question 19

this test is only given by acetoacetate

Answer 19

Gerhardt’s ferric chloride test
- sensitive to acetoacetate (diacetic acid); NOT to B-hydroxybutyrate

Principle:
on boiling, acetoacetate is converted to acetone and produces a purplish color when added with ferric chloride

Reagent:
conc. HNO3

Question 20

T/F:

acetoacetate conversion to acetone gives a positive result for Gerhardt’s test

Answer 20

FALSE

Question 21

Gerhardt’s test procedure

Answer 21

1. take 3mL urine in a test tube
2. add 1-2 drops of conc. HNO3
3. boil & cool
4. add 1mL 10% FeCl3 solution

Question 22

this test is sensitive to B-hydroxybutyric acid

Answer 22

Hart’s test

• reagent: Hart’s reagent
  
  • • acetic acid
  • • H2O2
• result: formation of RED ring

Question 23

disadvantage/s of reagent strip test for ketones

Answer 23

• does not measure B-hydroxybutyrate

- slightly sensitive to acetone when glycine is present

Question 24

tests to detect ketone bodies in fresh serum AND urine

Answer 24

1. Nitroprusside test

2. Enzymatic

Question 25

nitroprusside test

Answer 25

• acetone or acetoacetate reacts with nitroprusside to form a VIOLET color
• if the reagent contains glycerin, acetone can be detected by Acetest tablets or Ketostix