Lactate dehydrogenase is an enzyme that catalyses the reaction;
Lactic acid + NAD+ Pyruvic acid + NADH + H+
Under suitable conditions the reaction will proceed to the right giving almost complete conversion of lactate to pyruvate. Since NADH absorbs light at 340nm, by monitoring the change in absorbance of the solution at 340nm, the quantity of lactate converted to pyruvate may be measured.
The data below were obtained from experiments to determine the quantity of lactate present in the blood of rats subjected to severe exercise.
A 2mL sample of blood was removed from each of four rats. Following removal of the cells and protein, an aliquot of each sample was assayed for lactate in the following manner. 1mL of the de-proteinised sample was diluted to 4mL with phosphate buffer. 250µL of the resultant solution was placed in a spectrophotometer cell of 1cm light path containing 750µL of 12mM NAD+ and 2mL lactate dehydrogenase (0.01mg. mL-1). The absorbance of the mixture at 340nm was recorded at 1min intervals until the reaction was completed and the results were as follows;
Absorbance at 340nm
Sample Initial Final
1 0 1.204
2 0 0.295
3 0 0.595
4 0 0.619
Calculate the mean concentration of lactate in the initial blood sample expressed as µg.mL-1.
(The molar absorption coefficient of NADH is 6220 and the molecular weight of lactic acid is 90.1)
Honestly i dont have a clue as to how to approach this question. If someone could point me in the right direction, it would be appreciated.
I do understand that i have to work backwards to get the answer. There is 1 dilution from 1 mL to 4 mL which means the eventual concentration result must be divided by 4 as i am starting off with 4mL in my calculations. Or have i got this way wrong?
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Topic: Concentration of lactate in the initial blood : Spectrophotometer question. (Read 3580 times)