Abstract
The here described method can be used to estimate the uptake of orally provided cholesterol in mice. Briefly, mice are gavaged with radiolabeled cholesterol and 4 h later, organ distribution of the radiolabel is determined by liquid scintillation counting. The method has been applied successfully to determine dietary cholesterol handling of mice housed at different ambient temperatures
Keywords: Lipid metabolism, Oral fat tolerance, Cholesterol, Lipid/Cholesterol biodistribution, Radioactive tracer study, Organ uptake
Background
Cholesterol facilitates membrane fluidity and is a precursor for steroid hormones, bile acids and vitamin D. Animals are provided with cholesterol either from the diet or by de novo synthesis. Surplus of cholesterol may be harmful as its accumulation in blood vessel walls can cause atherosclerosis. It has been shown that the activation of brown adipose tissue (BAT) by cold reduces hypercholesterolemia (Berbee et al., 2015). It was, however, unclear whether BAT activity alters acute peripheral dietary cholesterol handling. To address this question, we orally administered mice with recombinant triglyceride-rich lipoprotein particles (rTRL) labeled with [4-14C]-Cholesterol and measured the organ distribution of the radiolabel 4 h after gavage. The biodistribution of radiolabeled cholesterol in vivo has been measured before (Szigeti et al., 1972; Townsend et al., 2001). However, in these studies, the radioactive cholesterol was either supplied by the diet or injected intravenously. Furthermore, other studies have assessed the fractional cholesterol absorption rate over a longer period of time (48 and 72 h) (Zilversmit and Hughes, 1974; Turley et al., 1994), As we were especially interested in the plasma clearance and the cholesterol uptake into the BAT after cold exposure, we chose to analyze the organs after a rather short period of time (4 h). Instead of applying an oil gavage, we used rTRL since the oil gavage resembles a rather unphysiologic condition (very high amount of lipids) but not a postprandial situation. Additionally, supplying the tracer with the diet is not suitable in cold-treated mice as they eat twice as much as when kept at warm ambient temperatures (30 °C).
Materials and Reagents
Equipment
Procedure
Figure 1. Schematic depiction of TRL Preparation Note: All steps are performed at room temperature in a fume hood and in an isotope laboratory.
Data analysis
Raw data from the counter was processed in Microsoft Excel. For every sample, counts per minute were measured twice and mean was calculated. Exemplary calculation for the rTRL solution is depicted below. As shown in Figure 4A, and as expected, the number of counts in each duplicate was roughly equal. Further, it was expected that counting 10 µl of rTRL solution would roughly result in twice as many counts detected in 5 µl TRL solution which is shown in Figure 4A. Additionally, calculated counts per µl were equal (Figure 4B).
Figure 4. Counts in rTRL solution. A. Five or ten microliters of rTRL solution were counted in duplicates and counts per minute were detected. B. Five or ten microliters of rTRL solution were counted in and counts per minute/µl were calculated.
Recipes
Acknowledgments
This protocol is adapted from Bruns et al. (2009) and Worthmann et al. (2017). This work was supported by grants funded by the Deutsche Forschungsgemeinschaft (SFB841; Liver inflammation: Infection, immune regulation and consequences). The authors have no conflicts of interest to declare.
References
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