Determination of ketone bodies in blood serum.
Virtual experiment.

1. Preparing standards to calibrate the assay

Reagents:

A series of tubes needs to be prepared (standard solutions) with known decreasing concentrations of acetoacetate: 16, 8, 4, 1.5 and 0.5 mM.
Each standard solution will be preparaed by dilution of the previous one. The total volume of each one must be between 200 and 500 µL.

tube
number		 desired
concentration		 previous
standard		 volume to take
from the previous
standard solution (µL)		 volume of
water (µL)
to add 		 
P1 16.0 mM P0
P2 8.0 mM P1
P3 4.0 mM P2
P4 1.5 mM P3
P5 0.5 mM P4

You may then if your procedure is correct. All volumes are correct. 👍

2. Performing the assay

If you don't see the capillary on the left of tube m1, click here
toggle sounds
m1 m2 m3
A
B
C
Calibration standards:

In this experiment we intend to semiquantitatively estimate the concentration of ketone bodies in blood serum samples m1, m2 and m3.

Instructions

  1. To move the capillary, hold its lower end with the mouse pointer.
  2. Drag the capillary to one of the tubes with sample.
  3. Drag the capillary to position its lower end on the centre of a pad (coloured zone) in one of the reactive strips, in order to deposit the sample. Reaction for ketone bodies will only work in the second pad from the left.
  4. Drag the capillary to the waste container (soon a new capillary will show up on the left-hand sde)
  5. Repeat the process with another sample, applying it to a different reactive strip.
  6. Wait for the colour to develop.

In you lab notebook: write down the code asigned to your particular experiment (in this case it is and which of the samples (m1, m2 or m3) you have applied onto each reactive strip (A, B, C). By comparing the colour displayed with those of the standards, estimate the concentration of ketone bodies in each sample. Finally, interpret your results: Is there ketosis? Signs of diabetes?

Rationale of the reaction:

Acetoacetate is detected through a reaction with nitroprusiate that forms a coloured product:

sodium nitroprusside



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