BCHM2072/2972

2004 THEORY of PRACTICAL PAPER

MCQs

18 - 33

Enzymic methods (questions 18 to 33).

The alternative methodology for estimating ketone bodies in urine and serum is to use the enzyme β-hydroxybutyrate dehydrogenase (3-HBDH). This enzyme catalyses the reversible oxidation of β-hydroxybutyrate to acetoacetate, with the concomitant reduction of NAD+. The reaction is shown below.

H3C CHH2C COOH

OH

-hydroxybutyrate

H3C CH2C COOH

O

Acetoacetate

+ NAD+ + NADH

3-HBDH

Measuring the level of acetoacetate in biological samples: The reaction will proceed quantitatively in the direction of β-hydroxybutyrate formation at pH 7 in the presence of excess NADH (NADH:acetoacetate of at least 2:1). Under these conditions the conversion of acetoacetate to β-hydroxybutyrate is >98 %. The progress of the reaction can be monitored by measuring the amount of NADH in the reaction. NADH and not NAD+ absorbs strongly at 340 nm.

Measuring the level of β-hydroxybutyrate in biological samples: At an alkaline pH (pH 9.5) the conversion of β-hydroxybutyrate to acetoacetate is favoured. β-hydroxybutyrate, in the presence of a large excess of NAD+ is oxidized to acetoacetate. Again the reaction is monitored by absorbance at 340 nm. To ensure this reaction proceeds to completion in this direction a trapping reagent, hydrazine, is also added. Hydrazine will react with the acetoacetate forming a hydrazone derivative.

To improve the sensitivity of the β-hydroxybutyrate reaction: Your research team has developed a synthetic derivative of NAD (syn-NAD) which has much stronger absorbing properties in the reduced state to NADH. The absorption maximum for syn-NADH is 360 nm, giving an extinction coefficient of 200 mM-1cm-1, whereas NADH has an extinction coefficient of 6 mM-1cm-1. Like NAD, oxidized syn-NAD has no absorbance at 360 nm. Syn-NAD has the same affinity for 3-HBDH as natural NAD. With either cofactor both the forward and reverse reaction are completed within 30 min under the conditions described.

The measurement of acetoacetate in samples.

Acetoacetate + NADH β-hydroxybutyrate + NAD+

You begin by setting up the acetoacetate assay as in the table below.

Component Volume (l)

5 X buffer pH 7.0 200

NADH 50

Standard acetoacetate 0 - 200

3-HBDH (1 U/l) 10

water Made up to 1000 l

18. What is the most appropriate [NADH] stock

Given that the working range of the spectrophotometer is 0.1 to ~1.2, what would be the most appropriate concentration of the stock NADH solution to use in the assay described in the table above (ε = 6 mM-1cm-1)?

A. 0.2 mM

B. 0.2 μM

C. 1 mM

D. 4 mM

E. 60 mM

During the reaction, the NADH is going to be used – and the absorbance of the cuvette will DECREASE

The absorbance needs to start at an absorbance of about 1.2 (higher would be beyond the useful range of the spec, lower would not give us enough substrate to fuel the reaction

If a 1 mM NADH solution has an absorbance of 6, an abs of 1.2 would be given by an [NADH] of 0.2 mM

0.2 mM is 0.2 umol/ml – so there’s 200 nmol NADH in the cuvette to begin with.

This came from 50 ul stock… so the stock must be 4 nmol/ul = 4 mM

19. What is the maximum measurable amount of acetoacetate

Again, using the working range of the spectrophotometer of 0.1 to ~1.2, what is the maximum amount of acetoacetate the assay above can accurately measure (final volume 1 ml), using NADH? Hint: NADH must be in excess; NADH:acetoacetate of at least 2:1

A. 200 nmoles

B. 100 nmoles

C. 0.2 nmoles

D. 0.1 nmoles

E. 10 nmoles

From the last question, the [NADH] starts at 0.2 mM Which is 0.2 umol/ml

So, in the 1 ml cuvette, there’s 200 nmol NADH to start with

So we can have 100 nmol of AcAc

21. Time Course

You perform a time course over 30 min with 5 different concentrations of the acetoacetate, measuring the absorbance at 340 nm every 5 min. From this you obtain a standard curve.

Which graph (A-E) below shows the time course for standards for this reaction?

0.0

0.2

0.4

0.6

0.8

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1.4

0 10 20 30Time (min)

Ab

so

rba

nc

e 3

40

nm

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sorb

ance

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nm

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ance

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nm

A B

C D

E Start at Abs 1.2 and go down

At lowest abs (when all AcAc in the top standard has run out) is 0.6 (showing that there’s still 100 nmol of NADH available

Measuring the amount of β-hydroxybutyrate.

β-hydroxybutyrate + NAD+ acetoacetate + NADH

Below are the assay components for the β-hydroxybutyrate assay, using NAD.

Component Volume (l)

5 x glycine buffer containing hydrazine, pH 9.5200

60 mM NAD+

50

Standard -hydroxybutyrate0 - 200

3HBDH (1 U/l)10

H2O

Made up to 1000 l

22. What [β-hydroxybutyrate] gives absorbance of 0-1.2 What concentration of standard β-hydroxybutyrate would generate a standard curve with an absorbance range between 0 and ~1.2, when measured at 340 nm?

A.10 mM

B. 0.2 mM

C. 0.2 μM

D. 1.0 mM

E. 1.0 μM

Same logic as before: at the end of the reaction, all the NAD will have gone to NADH and an abs of 1.2 would be given by a 0.2 mM solution

Since one NAD reacts with one bOHBu, thin initial bOHBu in the top standard should be 0.2 mM

This 200 nmol will have come from 200 ul

So the stock bOHBU is 1 nmol/ul = 1 umol/ml = 1 mM

23. What [components] to change for syn-NAD You are planning to use the same assay components, simply replacing the 60 mM NAD+ solution with a solution of the syn-NAD derivative of a similar concentration. However, you would like to set up the new standard curve with an absorbance range from 0 to 1.0 at 360 nm. To do this, what component(s) would you need to decrease in concentration in the new assay?

A. β-hydroxybutyrate dehydrogenase

B. Syn-NAD

C. Standard β-hydroxybutyrate

D. A & B

E. A, B & C

Enzyme still necessary to take reaction to completion

Still need syn-NAD to be in excess to drive coversion to completion

A smaller amount of bOHBu is required because a tiny concentration gives a big absorbance. If we keep the bOHBu the same as it was before, the final absorbances will be too high

Use the figures below (A-E) to answer questions 24 to 30.

You have worked up the new assay, using the syn-NAD derivative and the β-hydroxybutyrate standard. The ideal standard curve should have an absorbance range of 0 to 1 when measured at 360 nm. The following time courses (A-E) above were produced during the course of your investigations.

Which was the result of:

The graphs right (A-E) have been obtained with the new assay, using syn-NAD. Series 1-5 are increasing concentrations ofβ-hydroxybutyrate.

A

0.0

0.1

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Time (min)

Ab

so

rban

ce

360 n

m

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E

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Series1

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D

0.00.10.20.30.40.50.60.70.80.91.0

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Time (min)

Ab

so

rban

ce

360 n

m

Bunching at higher bOHBURunning out of syn-NAD

Reactions not reaching completion, but otherwise OK

Perfect! Perfect! Even faster than C

bOHBu running out prematurely

24. Pipette dispensing 30% too little

Using a pipette to dispense the standard

β-hydroxybutyrate, which consistently dispenses 30% less volume?

bOHBu running out prematurely

Option E

25. Adding 2x enzyme

Adding twice as much enzyme to each of the assay tubes?

Very fast completion

Option D

26. Using old enzyme

Using an old preparation of the β-hydroxybutyrate dehydrogenase enzyme, lacking much of the original activity?

Getting there slowly but surely

Option B

27. Adding 2x syn-NAD

Adding twice the volume (100 ul instead of 50 ul) of the syn-NAD solution to each of the assay tubes?

Syn-NAD is already in excess

Won’t make syn-NADH any faster

Option C

28. Using impure standard

Using an impure preparation of the β-hydroxybutyrate standard?

Not enough bOHBU

Option E

29. Adding 2 nmoles syn-NAD

Adding 2 nmoles syn-NAD to each assay tube?

Without calculation, tempted to say “the one where the syn-NAD is running out”!

Option A

When 2 nmol syn-NAD all used up, the [syn-NADH] will be 2 nmol/ml = 2 uM

Since 1 uM syn-NADH gives abs of 0.2, a 2 uM soln would give abs of 0.4

30. Measuring at too low a wavelength

Measuring the absorbances at 340 nm instead of 360 nm?

Absorbance never gets quite as high as expected

Option E

The following information refers to questions 31 to 33.

Once you have worked up the assay using standard β-hydroxybutyrate, you then try the assay on a number of urine samples. These samples have been collected from well nourished individuals and from volunteers who have been on the Atkin’s diet for one month. The level of all ketone bodies in urine increases on this diet.

Urine is a complex biological sample which may contain compounds which interfere with this procedure. There are a number of compounds known to do this:

1) Ascorbic acid (vitamin C) at high concentrations can slowly reduce NAD+ non-enzymically.

2) Vitamin B supplements sometimes result in the excretion of urinary metabolites which strongly absorb at 340 - 360 nm.

3) The β-hydroxybutyrate dehydrogenase enzyme can be inhibited competitively by a number of other aldehyde and ketone containing compounds, which may be

present in urine. The presence of sufficient amounts of these compounds can interfere with the reaction.

To overcome the problem of potential interference you have decided to assay 10 and 20 μl of each urine sample with and without a known amount of β-hydroxybutyrate standard, termed a spike. You have measured the absorbance of the assays, containing the two volumes of urine, at various times (0, 10, 20, 30, 40 min) throughout the course of the reaction. In a duplicate set of assays you have added a known amount of β-hydroxybutyrate (*enough to produce a 0.4 change in absorbance) and incubated these spiked tubes for 40 min. The results are presented below. **The control urine contains no β-hydroxybutyrate. A “no enzyme blank” was also measured for each sample.

Assay components Absorbance 360 nm

Sample # Enzyme

(l) Volume

urine (l) 0 min 10 min 20 min 30 min 40 min

40 min + spike*

0 10 0.01 0.01 0.01 0.01 0.01 0.01 10 10 0.01 0.02 0.02 0.02 0.02 0.42 **control 10 20 0.02 0.04 0.04 0.04 0.04 0.44 0 10 0.04 0.04 0.04 0.04 0.04 0.04 10 10 0.04 0.18 0.18 0.18 0.18 0.58 A 10 20 0.09 0.37 0.37 0.37 0.37 0.77 0 10 0.08 0.08 0.08 0.08 0.08 0.08 10 10 0.08 0.29 0.45 0.45 0.45 0.85 B 10 20 0.15 0.58 0.91 0.91 0.91 1.31 0 10 2.1 2.1 2.1 2.1 2.1 2.1 10 10 2.1 >3 >3 >3 >3 >3 C 10 20 >3 >3 >3 >3 >3 >3 0 10 0.05 0.05 0.05 0.05 0.05 0.05 10 10 0.05 0.10 0.15 0.21 0.26 0.46 D 10 20 0.06 0.16 0.27 0.36 0.46 0.66 0 10 0.02 0.08 0.16 0.24 0.32 0.32 10 10 0.02 0.24 0.48 0.56 0.64 1.04 E 10 20 0.04 0.48 0.96 1.12 1.28 1.68

A: Spike works, so assay deals with bOHBu fine. Final abs is proportional to amount of urine put in. Zero enzyme gives low absorbance. So all appears good.

B: Spike works, so assay deals with bOHBu fine. Final abs is proportional to amount of urine put in. Zero enzyme gives low absorbance. So all appears good.C: Everything just maxed out. Must be something in the urine that gives a massive absorbance – even when there is no enzyme present.

D: The spike is not fully effective, so something is compromising the bOHBu assay. Final abs partially proportional to starting amount of urine. Bit slow??

E: The spike works, so the bOHBu assay is fine. Urine gives increase in abs with time even when there is no enzyme present.

31. Which has vitamin C

Which urine sample (A-E) has been collected from someone who takes large doses of vitamin C?

Something that causes a slow, but steady, rise in absorbance – even in the absence of enzyme

Option E

32. Which has vitamin B

Which urine sample (A-E) has been collected from someone on vitamin B supplements?

Something that shows a strong absorbance when urine is added, even in the absence of enzyme

Option C

33. Which interferes with enzyme

Which urine sample (A-E) contains a compound which interfers with the β-hydroxybutyrate dehydrogenase reaction?

Something that causes a slowing of the rate, with the reaction not really reaching completion even after 40 min

Option D