Abstract
The continuous isotope feeding method of Crouse and Grundy (1978. J. Lipid Res. 19: 967-971) for measurement of dietary cholesterol absorption has been modified by using markers labeled with stable isotopes ([2,2,4,4,6-2H5]cholesterol or [25,26,26,26,27,27,27-2H4]cholesterol or [26,26,26,27,27,27-2H6] cholesterol and [5,6,22,23-2H4]sitostanol) quantified by gas-liquid chromatography-selected ion monitoring. Tracing of the isotope distribution of the authentic markers and after their intestinal passage, including the microbiological products (coprostanol and coprostanone) revealed stability of the labels. The new method was evaluated in six monkeys on two occasions by comparison with the original method using radioactively labeled cholesterol and sitosterol. The results obtained by the two different methods were in excellent agreement, and absorption ranged from 49% to 73% (mean 60%) for the stable isotope method and from 51% to 69% (mean 62%) for the radioactive method. The coefficient of variation of cholesterol absorption in animals ranged from 3.9% to 15.1% (mean 7.1%) for stable isotopes and 1.9% to 13.6% (mean 5.7%) for radioactive isotopes. In twelve subjects cholesterol absorption was measured by the new method from total fecal samples frozen immediately and compared to results obtained from small fecal aliquots (approximately 1 g) sent by ordinary mail to the laboratory. A significant correlation of cholesterol absorption between the two different sample handlings was obtained (r = 0.981, P < 0.001). In addition, measurement of cholesterol absorption twice in seven volunteers 2 weeks apart revealed identical results. Thus, the new method is extremely safe and reproducible without radioactive exposure to the subjects and labortory staff and can be used on women of child-bearing age.
Highlights
S nor the intestinal intubacholesteroland [5,6,22,23-2H4]sitostanolq)uantifiedby gas-liquid tion procedures are available for routine and repeated chromatography-selected ion monitoring
I n the first study twelve volunteers participated in cholesterol absorption measurements
Gas-liquid chromatography-mass spectrometry deuterated cholesterol was added to the corrected areas of deuterated coprostanol plus deuterated coprostanone for Combined gas-liquid chromatography (GLC)-SIM was performed on a Hewlett- calculation of cholesterolabsorption accordingto equation 1
Summary
A mixture (1:l; w/w) of [2H5]cholesterool r [2H7]cholesterol or [2H6]cholesterol and [2H4]sitostanol was dissolved in sunflower oil by gently heating and stirring. I n the first study twelve volunteers participated in cholesterol absorption measurements They received one capsule t.i.d. for 7 days. One volunteer received one capsule of 3 mg [2H,]cholesterol and 3 mg [2H4]sitostanol t.i.d. for 1 week and cholesterol absorption was determined as described above. In a second set of experiments five patients with mild hypercholesterolemia received one capsule t.i.d. of deuterated markers as described above and collected fecal samples from day 5 to 7. In a third set of experiments cholesterol absorption was measured twice in seven normolipemic male volunteers 2 weeks apart. For this purpose they received 3 mg t.i.d. of [2H6]cholesteroltogether with [2H4]sitostanol[3 mg t.i.d.1 for 7 days. The study was in accordance with the principles of the Helsinki Declaration and approved by the local ethical committee
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