Materials and Reagents

EtOH
KOH
MgCl₂
Triglyceride (GPO Trinder) reagent A (Sigma-Aldrich, catalog number: 337-40A )
[Please note that this product has been replaced by free glycerol reagent (F6428) from Sigma]
Glycerol Standards (Sigma-Aldrich, catalog number: 339-11 or G7793 )
Ethanolic KOH (see Recipes)

Equipment

Microfuge tube
Glycerol
Microreader
Cuvette

Procedure

Creation of saponified, neutralized liver extract
1. Moved 100-300 mg of liver to pre-weighed microfuge tube.
2. Record tube + liver weight.
3. Add 350 μl ethanolic KOH.
4. Incubate overnight 55 °C
5. Vortex early in incubation till the tissue is completely digested.
6. By morning, tissue should be digested and no oil layer should be visible (if oil layer is present then one must digest longer, and may need more ethanolic KOH).
7. Bring volume to 1,000 μl with H₂O: EtOH (1:1).
8. Spin in microfuge 5 min, move supernatant to new tube.
9. Bring volume of supernatant to 1,200 λ with H₂O: EtOH (1:1), vortex.
10. Move 200 μl to new eppendorf tube, add 215 μl 1 M MgCl₂, vortex.
11. 10 min on ice.
12. Microfuge 5 min, move supernatant to new tube.
Assay of glycerol content
1. Reconstitute reagent A according to instructions for determination of glycerol content.
2. Pipette 1 ml of reconstituted reagent A into cuvettes.
3. Add samples, standards and blank.
  Standards: 10 μl + 20 μl H₂O
  Samples: 30 μl
  Blank: 30 μl H₂O
4. Shake.
5. Incubate 15 min & read at 540 nm.
Calculation of liver TG content
1. Note that the Sigma glycerol standards are expressed as triglyceride (triolein) equivalents.
2. Subtract blank from all samples/standards.
3. Create standard curve by linear regression.
4. Determine cuvette (triolein equiv) (CTE) glycerol concentration by comparison to standard curve.
5. Liver TG content (in mg TG / gram liver)
  = CTE (mg/dl)*(10/30)*(415/200)*0.012 (dl)/wt (gr)

Recipes

Ethanolic KOH (2 parts EtOH: 1 part 30% KOH)
1 M MgCl₂
H₂O: EtOH (1:1)

Acknowledgments

This protocol was previously used in Norris et al. (2003).

References

Norris, A. W., Chen, L., Fisher, S. J., Szanto, I., Ristow, M., Jozsi, A. C., Hirshman, M. F., Rosen, E. D., Goodyear, L. J., Gonzalez, F. J., Spiegelman, B. M. and Kahn, C. R. (2003). Muscle-specific PPARgamma-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones. J Clin Invest 112(4): 608-618.