Imaging Cytoplasmic Lipid Droplets in Enterocytes and Assessing Dietary Fat Absorption - Methods in Cell Biology - page 155

c. Rinse the test tube that contained the feces or diet with Folch, and pour
through the filter system.
d. Rinse filter with Folch mixture.
9. Bring the volume of the filtrate to approximately 10 ml.
10. Add 2 ml of water, vortex, and allow the mixture to separate.
11. Pre-weigh scintillation vials, weighing them to three decimal places.
12. Pipette off the top layer (aqueous phase), then pour the lower layer (organic
phase) into the scintillation vials.
13. Evaporate organic solvent in scintillation vials under nitrogen.
14. Place the vials in an oven (80-90 C) for 15 min.
15. Allow the vials to cool to room temperature and weigh immediately. The
difference in the weights of the empty vial and vial with lipid extract is the lipid
content of the measured amount of feces or diet used for the extraction.
9.1.3.2.3 Results
This experiment will provide two different sets of numbers: (1) stool output (g/day)
and (2) lipid content (% lipid/stool). The extracted lipid can be redissolved in chlo-
roform, separated by thin layer chromatography using hexane:ether:acetic acid
(80:20:1), and visualized using iodine vapor. This will allow for a quick characteri-
zation of the lipid species in the extracted fecal fat ( Uchida et al., 2011 ). The majority
of lipid excreted should be free fatty acids if digestion of dietary fat is not inhibited.
High levels of TAG may indicate a problem in digestion as opposed to absorption or
secretion.
9.1.3.3 Blood TAG in response to dietary fat
Assessing the rate of appearance of TAG in circulation will allow for determining the
rate at which the TAGs are being secreted out of the intestine, as well as the rate at
which the other organs process the dietary fat. Understanding the intestinal rate of
TAG secretion and the postprandial triglyceridemic response also helps determine
ideal timing for imaging and tissue collection.
9.1.3.3.1 Materials
1. 0.5 M EDTA pH 8.0
2. Goldenrod Lancet (MediPoint Inc.)
3. L-Type TG Measuring Kit (Wako)
4. Gavage needles 20 G (Instech)
5. Oil (olive/peanut/vegetable)
6. 1 ml syringe with 25 G needle
7. Vis microplate spectrophotometer
8. Tyloxapol (Sigma)
Weigh out Tyloxapol in a small beaker, and then add the appropriate amount of
PBS into the beaker (0.25 g/ml in PBS). Stir the mixture using a small stir bar.
Tyloxapol can take several hours to completely dissolve in PBS.