Imaging Cytoplasmic Lipid Droplets in Enterocytes and Assessing Dietary Fat Absorption - Methods in Cell Biology - page 154

9.1.3.2 Dietary fat absorption—assessed by percent lipid in feces
Quantitative fat absorption allows for determining how much of the ingested fat is
absorbed and not excreted in feces. One method for assessing whether fat malabsorp-
tion is present is to assess the percent of lipid present in feces. This is determined by
collecting the feces and quantifying how much lipid remains in the feces. Fecal lipid
is expected to be less than 5% in a healthy mouse without fat malabsorption.
9.1.3.2.1 Materials
1. Wire fasting racks (Ancare)
2. Mechanical blender (or mortar and pestle)
3. Folch solution (chloroform:methanol 2:1)
4. Whatman #2 filter paper
5. Funnels
6. Glass Pasteur pipette and bulbs
7. 15 ml glass test tubes (screw top preferred)
8. Scintillation vials
9. Drying oven
10. Analytical balance (0.000 g)
11. Nitrogen flow
12. Vortex
9.1.3.2.2 Methods
1. House mice individually in cages with wire rack bottoms. Allow the mice to
adapt to this cage for at least 24 h before collecting feces. This is important for
accurate quantitative measure of feces excreted, because mice eat their feces.
2. Collect feces daily for 3-5 days. Store feces in the freezer until ready to analyze.
Keep feces collected from each day in a separate container so that dried stool
output can be determined (g/day).
3. Dry collected feces in an oven at 65-80 C overnight. To double-check that drying
is complete, weigh the container with the drying fecal samples and once the
weight stops decreasing, drying is complete. Record weight and determine stool
output (g/day). As a control, also dry 1.0 g of diet of known lipid content.
4. Grind the feces and control diet into a fine powder with a mechanical blender (or
using a mortar and pestle).
5. Extract lipid from 0.2 to 0.5 g of ground feces or diet using the Folch method.
Quantitatively measure and record the amount of ground feces or diet used.
6. Add 5 ml of Folch reagent to a screw top glass test tube containing the ground
feces or diet and stir gently. If not using a screw top glass test tube cover with
aluminum foil. Incubate in a water bath or on a hot plate at 65 C for 1 h.
7. Mix the contents of the test tube every 15 min by vortexing at the lowest setting.
8. Filter the mixture, using a #2 filter paper and a funnel. Collect filtrate in a clean
glass test tube. Precise filtering method:
a. Pre-rinse the filter paper with Folch.
b. Pour feces or diet and Folch mixture through the filter system.