I am using an enzymatic kit for Lipid analysis from liver tissue and I have trouble in converting the 0.018 mM of TG to mg/g of liver.
Please help me with this. The details of the experiments are given below.
Weight of liver tissue used =106 mg.
Briefly, the tissue was extracted using chloroform:methanol and filtered to make upto 6 mL volume with the same solvent.
Then, 250 μL was taken from it and added 250 μL 1X triton-100.
the solution was allowed to dry under Nitrogen gas and after complete evaporation of the solvent the residue was dissolved in 0.5 mL of distilled water (considered as the final volume for calculation by me).
50 μL of samples were used for the enzymatic assay.
The molecular weight of TG= 885.4 g/mol (as given in the kit manual)
I tried to follow the equation mass (mg)=concentration (mM)* volume (mL)* molecular weight (g/mol), which give an answer 75.5 mg/g of liver.
But I don't know if this is the right method.
Please help me with a detailed calculation if possible.
If I had made any error in this calculation please point that out.
Thank you.
I really appreciate and respect your expertise in this calculation things.
You started with 106 mg of tissue, which was used to make an extract with a volume of 6 ml.
You used 0.25 ml of this extract, which is equivalent to (0.25/6)x106 = 4.42 mg of tissue.
After further treatment, this material ended up in 0.5 ml, of which 0.05 ml was used in the assay, which is therefore equivalent to (0.05/0.5)*4.42 = 0.442 mg of tissue.
The assay gave the result of 0.018 mM of TG.
At this point, there is a missing piece of information, which is what volume the measurement of 0.018 mM was made in. Was the 50 µl sample diluted in the assay?
As an example of the rest of the calculation, I will guess that the assay volume was 1 ml and the TG concentration measured in that volume was 0.018 mM. Therefore the TG concentration in the 50 µl sample was (1000 µl/50 µl)*0.018 mM = 0.36 mM.
The number of mg of TG in that sample is (50 x 10-6 L) x (3.6 x 10-4 mol/L) x 885.4 g/mol) x (1000 mg/g) = 0.016 mg
That amount was in 0.442 mg of tissue, so in mg/g that is (0.016 mg TG)/(0.442 mg tissue)*(1000 mg/g)= 36 mg TG/g tissue. Remember that I assumed an assay volume of 1 ml, since you didn't specify the volume. Correct for the difference in the assay volume (in bold), if any.
Hello Dr. Shapiro,
Thank you so much for the detailed explanation.
The final volume of the assay solution was 102 µL (52 µL assay reagent+ 50 µL sample). I have changed the volume while doing my calculation as you mentioned.
I really appreciate your detailed response.
Can you let me know the reason why 1000 mg/g was used to find the mg of TG in the sample, in the following line?
The number of mg of TG in that sample is (50 x 10-6 L) x (3.6 x 10-4 mol/L) x 885.4 g/mol) x (1000 mg/g) = 0.016 mg?
Once again, thank you for your time and patience.
Note that this assumes that the assay was calibrated per final volume (102 µL in your case). In practice, I would usually calibrate per sample volume (52 µL), as the final volume is of little interest. Which method you use is up to you, as long as you do it consistently for both your sample and your standards. If you do not use standards, but a conversion factor supplied by the test manufacturer, read the instructions carefully on this point.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue