An AST method follows the fall in NADH absorbance at 340 nm, with malate dehydrogenase as the coupling enzyme, and calculates activity from 60 to 180 seconds after the start reagent is added. Today both QC levels read about 40% below target. The reagent pack has been on board for 6 weeks (stated on-board stability 4 weeks). Raw data for the level 2 control: starting absorbance 1.72 (expected 1.60-1.90); the absorbance then falls by 0.006, 0.011, 0.016 and 0.020 in the four 30-second intervals of the read window. The cuvette temperature log shows 37.0 °C throughout. The most likely cause is:

Loss of malate dehydrogenase activity

Oxidation of NADH in the reagent

Substrate depletion by high AST activity

Reaction temperature below 37 °C

In the IFCC ALT method, serum is first incubated with a reagent containing alanine, NADH, LD and pyridoxal-5'-phosphate, and the reaction is started later by adding 2-oxoglutarate. What is the purpose of this pre-incubation?

To use up endogenous pyruvate before timing begins

To warm the reagent so that NADH can absorb at 340 nm

To let ALT convert all of the alanine to pyruvate

To inactivate LD released from hemolyzed red cells

A malnourished patient with alcohol-related liver disease has an ALT of 48 U/L by a method without pyridoxal-5'-phosphate (P5P). The same sample gives 82 U/L by the IFCC method, which adds P5P. What explains the difference?

P5P activates ALT apoenzyme lacking its coenzyme

P5P inhibits the side reaction of endogenous LD

P5P stabilizes NADH, slowing its breakdown

The P5P method measures AST as well as ALT

A healthy 40-year-old has had an isolated AST of 180-200 U/L (reference < 40 U/L) for 2 years. ALT, CK, GGT, bilirubin and liver ultrasound are normal, and the samples are not hemolyzed. After polyethylene glycol (PEG) precipitation, AST activity in the supernatant falls far more than in control sera. The most likely explanation is:

Macro-AST: AST bound to immunoglobulin

Early cirrhosis with normal ALT

Subclinical skeletal muscle disease

Enzyme kinetics and measurement

12 DOH Abu Dhabi (HAAD) lab licensing practice questions on Enzyme kinetics and measurement, part of Clinical Chemistry. The first 12 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
An AST method follows the fall in NADH absorbance at 340 nm, with malate dehydrogenase as the coupling enzyme, and calculates activity from 60 to 180 seconds after the start reagent is added. Today both QC levels read about 40% below target. The reagent pack has been on board for 6 weeks (stated on-board stability 4 weeks). Raw data for the level 2 control: starting absorbance 1.72 (expected 1.60-1.90); the absorbance then falls by 0.006, 0.011, 0.016 and 0.020 in the four 30-second intervals of the read window. The cuvette temperature log shows 37.0 °C throughout. The most likely cause is:
1. AOxidation of NADH in the reagent
2. BSubstrate depletion by high AST activity
3. CReaction temperature below 37 °C
4. DLoss of malate dehydrogenase activity
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Answer: D · Loss of malate dehydrogenase activity
In a valid kinetic assay the absorbance falls by the same amount in every interval, because the reaction is zero order and the coupling enzyme turns oxaloacetate into malate as fast as AST makes it. Here the fall grows in each interval, so the reaction is still in its lag phase: too little coupling enzyme is left, oxaloacetate builds up slowly and the measured rate underestimates AST. Malate dehydrogenase loses activity when a reagent is kept past its on-board stability. Oxidized NADH would give a low starting absorbance, and substrate depletion or a cold cuvette would give a slowing or a steady rate, not an accelerating one. Replace the pack, recalibrate and repeat QC before rerunning patients.
- A: Oxidized NADH would lower the starting absorbance, which is normal here.
- B: Substrate depletion makes the rate slow down, not speed up, and needs a very high activity.
- C: The log shows 37.0 °C; a cold cuvette would give a steady but lower rate.
Question 2 · Medium
In the IFCC ALT method, serum is first incubated with a reagent containing alanine, NADH, LD and pyridoxal-5'-phosphate, and the reaction is started later by adding 2-oxoglutarate. What is the purpose of this pre-incubation?
1. ATo warm the reagent so that NADH can absorb at 340 nm
2. BTo let ALT convert all of the alanine to pyruvate
3. CTo use up endogenous pyruvate before timing begins
4. DTo inactivate LD released from hemolyzed red cells
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Answer: C · To use up endogenous pyruvate before timing begins
Serum contains pyruvate and other keto acids that would react with LD and oxidize NADH, causing a fall in absorbance unrelated to ALT. During pre-incubation these side reactions run to completion, and pyridoxal-5'-phosphate can also bind any ALT apoenzyme. ALT cannot act until 2-oxoglutarate is added, so the rate measured afterwards reflects only ALT activity. A sample very high in ALT can still exhaust NADH, which the analyzer flags.
- A: NADH absorbs at 340 nm at any temperature; warming controls the reaction rate, not absorbance.
- B: ALT cannot act until 2-oxoglutarate is added, and using up the substrate would end the zero-order rate.
- D: LD is a reagent in this method; the step does not inactivate it.
Question 3 · Hard
A malnourished patient with alcohol-related liver disease has an ALT of 48 U/L by a method without pyridoxal-5'-phosphate (P5P). The same sample gives 82 U/L by the IFCC method, which adds P5P. What explains the difference?
1. AP5P inhibits the side reaction of endogenous LD
2. BP5P activates ALT apoenzyme lacking its coenzyme
3. CP5P stabilizes NADH, slowing its breakdown
4. DThe P5P method measures AST as well as ALT
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Answer: B · P5P activates ALT apoenzyme lacking its coenzyme
Aminotransferases need P5P, the active form of vitamin B6, as a coenzyme. In vitamin B6 deficiency, which is common with heavy alcohol use and poor nutrition, part of the circulating ALT is inactive apoenzyme. Adding P5P to the reagent restores this activity, so the IFCC method gives higher and more representative results. Methods without P5P can underestimate aminotransferase activity in such patients.
- A: P5P is a coenzyme; it does not inhibit LD side reactions.
- C: P5P has no effect on the stability of NADH.
- D: Both methods are specific for ALT; adding P5P does not make the method detect AST.
Question 4 · Hard
A healthy 40-year-old has had an isolated AST of 180-200 U/L (reference < 40 U/L) for 2 years. ALT, CK, GGT, bilirubin and liver ultrasound are normal, and the samples are not hemolyzed. After polyethylene glycol (PEG) precipitation, AST activity in the supernatant falls far more than in control sera. The most likely explanation is:
1. APyridoxal phosphate deficiency
2. BEarly cirrhosis with normal ALT
3. CSubclinical skeletal muscle disease
4. DMacro-AST: AST bound to immunoglobulin
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Answer: D · Macro-AST: AST bound to immunoglobulin
In macro-AST, the enzyme is bound to an immunoglobulin, usually IgG, forming a large complex that is cleared slowly. Activity accumulates in serum without any tissue damage, giving a persistent isolated rise. PEG precipitates immunoglobulins and the bound enzyme, so low recovery of AST in the supernatant supports the diagnosis. Recognizing this benign finding prevents unnecessary investigation, including liver biopsy.
- A: Coenzyme deficiency lowers, rather than raises, measured aminotransferase activity.
- B: Cirrhosis does not make AST precipitate with PEG and usually shows other abnormal results.
- C: Muscle disease would raise CK, which is normal here.
Question 5 · Easy
The Michaelis constant (Km) of an enzyme is the substrate concentration at which the reaction velocity is:
1. AOne quarter of Vmax
2. BTwice Vmax
3. CHalf of Vmax
4. DEqual to Vmax
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Answer: C · Half of Vmax
In the Michaelis-Menten equation, v = Vmax[S]/(Km + [S]); when [S] equals Km, v is exactly half of Vmax. A low Km means the enzyme reaches half its maximum rate at a low substrate concentration. For activity assays, substrate is set well above Km, often 10 times or more, so the rate depends on the amount of enzyme rather than substrate. Km is a property of the enzyme-substrate pair under the stated conditions.
- A: At [S] = Km the velocity is one half, not one quarter, of Vmax.
- B: Velocity can never exceed Vmax.
- D: Velocity only approaches Vmax at substrate concentrations far above Km.
Question 6 · Easy
IFCC reference measurement procedures for enzymes such as ALT, AST, CK and LD are performed at which temperature?
1. A37 °C
2. B40 °C
3. C30 °C
4. D25 °C
Answer and explanation in LearnFree account
Question 7 · Easy
The SI unit of catalytic activity, equal to the conversion of one mole of substrate per second, is the:
1. AMichaelis constant
2. BTurnover number
3. CInternational unit
4. DKatal
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Question 8 · Medium
An ALT result of 120 U/L must be reported in SI units. Given that 1 U = 16.67 nkat, the result is:
1. A2,000 µkat/L
2. B2.0 µkat/L
3. C7.2 µkat/L
4. D0.12 µkat/L
Answer and explanation in LearnFree account
Question 9 · Easy
In a kinetic enzyme assay, the zero-order phase appears on the absorbance-versus-time plot as:
1. AA slope that steepens with time
2. BA straight line with a constant slope
3. CA flat line showing no change
4. DA curve that flattens as time goes on
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Question 10 · Easy
From the double-reciprocal (Lineweaver-Burk) plot shown, the enzyme's Km is:
Figure: MedExams
1. A2 mmol/L
2. B0.02 mmol/L
3. C0.01 mmol/L
4. D0.5 mmol/L
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Question 11 · Medium
ALT is measured by following the fall in NADH absorbance at 340 nm, and the analyzer calculates activity from the read window. Refer to the reaction curves shown. What should be done with the patient's sample?
Figure: MedExams
1. AReport the activity as calculated
2. BDilute the sample and repeat the assay
3. CReplace the reagent and repeat
4. DRecalibrate and repeat the assay
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Question 12 · Medium
When measuring enzyme activity, why is the substrate added in excess?
1. ASo the reaction stops faster
2. BSo the rate depends on substrate concentration (first-order kinetics)
3. CSo the substrate equals the Km
4. DSo the reaction rate depends only on the enzyme (zero-order kinetics)
Answer and explanation in LearnFree account

Enzyme kinetics and measurement

In the IFCC ALT method, serum is first incubated with a reagent containing alanine, NADH, LD and pyridoxal-5'-phosphate, and the reaction is started later by adding 2-oxoglutarate. What is the purpose of this pre-incubation?

A malnourished patient with alcohol-related liver disease has an ALT of 48 U/L by a method without pyridoxal-5'-phosphate (P5P). The same sample gives 82 U/L by the IFCC method, which adds P5P. What explains the difference?

The Michaelis constant (Km) of an enzyme is the substrate concentration at which the reaction velocity is:

IFCC reference measurement procedures for enzymes such as ALT, AST, CK and LD are performed at which temperature?

The SI unit of catalytic activity, equal to the conversion of one mole of substrate per second, is the:

An ALT result of 120 U/L must be reported in SI units. Given that 1 U = 16.67 nkat, the result is:

In a kinetic enzyme assay, the zero-order phase appears on the absorbance-versus-time plot as:

From the double-reciprocal (Lineweaver-Burk) plot shown, the enzyme's Km is:

ALT is measured by following the fall in NADH absorbance at 340 nm, and the analyzer calculates activity from the read window. Refer to the reaction curves shown. What should be done with the patient's sample?

When measuring enzyme activity, why is the substrate added in excess?