UA 3: SPECIAL TESTS Pt. I (KETONES, BLOOD) Flashcards
intermediate products of fat metabolism
Ketones
- acetone (2%)
- acetoacetic acid (20%)
- B-hydroxybutyrate (78%)
increased accumulation of ketones in the blood leads to:
- electrolyte imbalance
- dehydration
- acidosis, eventual diabetic coma (if not corrected)
tests for ketone bodies
- Rothera’s test
- Reagent strip
- Gerhardt’s ferric chloride test
- Hart’s test
- Confirmatory tests
T/F:
measurable amounts of ketones normally appear in the urine
FALSE
- all metabolized fat is completely broken down into CO2 & H2O
clinical reasons for increased fat metabolism
- inability to metabolize carbohydrate (in DM)
- increased loss of carbohydrate from vomiting
- inadequate intake of carbohydrate associated with starvation & malabsorption
testing for urinary ketones is most valuable in the management & monitoring of (Type I, Type II) DM
Type I DM
- “insulin-dependent”
- ketonuria shows a deficiency of insulin
- an early indicator of insufficient insulin dosage
clinical significance of urine ketones
- diabetic acidosis
- insulin dosage monitoring
- starvation
- malabsorption/pancreatic disorders
- strenuous exercise
- vomiting
- inborn errors of amino acid metabolism
for at-home testing by patients, ketone tests are combined with ____________ on test strips
Glucose
other causes of positive ketone test results
- 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
this reaction is used in the reagent strip test for ketones
Sodium nitroprusside reaction
- “nitroferricyanide”
- acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a PURPLE color
reporting results in the reagent strip test
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)
one of the confirmatory tests for urine ketones
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
components of the Acetest tablet
- sodium nitroprusside
- glycine
- disodium phosphate
- lactose
- gives better color differentiation
nitroprusside in alkaline medium reacts with a ketone group to form a purple ring
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
Rothera’s test procedure
- prepare Rothera’s reagent
- 7.5g sodium nitroprusside + 200g ammonium sulfate - add 1 gram Rothera’s reagent to 5mL urine
- mix well - overlay mixture with 1mL conc. NH4OH
- observe for the appearance of a reddish-PURPLE RING
- at the interphase of the 2 layers
- within 1 minute 30 seconds
Rothera’s test reporting of results
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
identify test:
principle:
sodium nitroprusside decomposition
components:
- 1% (w/w) sodium nitroprusside
- 9% (w/w) buffer
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
sources of error for the reagent strip test
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
this test is only given by acetoacetate
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
T/F:
acetoacetate conversion to acetone gives a positive result for Gerhardt’s test
FALSE
Gerhardt’s test procedure
- take 3mL urine in a test tube
- add 1-2 drops of conc. HNO3
- boil & cool
- add 1mL 10% FeCl3 solution
this test is sensitive to B-hydroxybutyric acid
Hart’s test
- reagent: Hart’s reagent
- acetic acid
- H2O2
- result: formation of RED ring
disadvantage/s of reagent strip test for ketones
- does not measure B-hydroxybutyrate
- slightly sensitive to acetone when glycine is present
tests to detect ketone bodies in fresh serum AND urine
- Nitroprusside test
2. Enzymatic
