ABSTRACT: Cholesterol absorption is frequently determined using the plasma dual stable-isotope ratio method (PDSIRM). However, this method involves intravenous injection of stable- isotope-labeled cholesterol with simultaneous oral administra- tion of differently labeled cholesterol, which results in high study costs and involves additional ethical considerations. The objective of the present study was to validate a simpler single- isotope method for determining cholesterol absorption against PDSIRM by using data from two previous studies. Enrichments of carbon-13 ( 13 C) and deuterium in red blood cells were ana- lyzed by using differential isotope ratio MS. The area under the curve of 13 C-enrichment in the plasma free-cholesterol pool was found to be significantly correlated with cholesterol absorption measured by using PDSIRM for study 1 (r = 0.85, P < 0.0001) and study 2 (r = 0.81, P < 0.0001). Average 13 C-enrichment corre- lated with the area under the curve of 13 C-enrichment in the plasma free cholesterol for both study 1 (r = 0.98, P < 0.0001) and study 2 (r = 1.00, P < 0.0001). Study 1 examined the effi- cacy and mechanisms of unesterified plant sterols and stanols on lipid profiles in hypercholesterolemic men and women, while study 2 investigated the effects of phytosterol vs. phy- tostanol esters on plasma lipid levels and cholesterol kinetics in hyperlipidemic men. Experimental approaches to determine cholesterol absorption were identical between the two studies. Consequently, in both studies, correlations (r = 0.88, P < 0.0001 for study 1, and r = 0.82, P < 0.0001 for study 2) were found between the average 13 C-enrichment of plasma free cholesterol and cholesterol absorption measured by PDSIRM. These results suggest that a single-isotope-labeled cholesterol tracer approach can be used as a reliable noninvasive method to replace PDSIRM for examining changes in cholesterol absorption. Paper no. L9373 in Lipids 39, 87–91 (January 2004). Dietary cholesterol and the large enterohepatic recirculation of endogenous cholesterol readily mix to form a single pool of intestinal cholesterol (1–3). Cholesterol absorption from the gastrointestinal tract is a key component of whole-body cholesterol metabolism. Measurement of cholesterol absorp- tion provides important insights into the relationships among diet and plasma cholesterol levels, cholesterol homeostasis, genetic variations, and drug effects. For this purpose, a vari- ety of different methods have been developed for estimating cholesterol absorption in humans (4,5). Presently, three meth- ods exist for measuring cholesterol absorption: the plasma dual-isotope ratio method (6–8), the fecal dual-isotope ratio method (9), and mass (sterol) balance (10). The simplest of these methods is the plasma dual-isotope ratio method origi- nally introduced by Zilversmit (6). In 1993, Lutjohann et al. (4) introduced the use of stable iso- topes into the fecal dual-isotope ratio method for measuring cholesterol absorption. In the same year, Bosner et al. (11) de- veloped a plasma dual stable-isotope-ratio method (PDSIRM) to measure cholesterol absorption, based on the plasma dual- isotope ratio method developed by Zilversmit (6). This modi- fied method has been used extensively (12–17). Although this technique can measure cholesterol absorption with good preci- sion and accuracy, it involves intravenous injection of labeled cholesterol. As such, the technique increases study costs dra- matically, involves an invasive procedure, and requires addi- tional ethical considerations. In PDSIRM, the amount of labeled cholesterol absorbed is calculated by the enrichment of orally administered and in- travenously injected cholesterol tracers. Isotope enrichment in the cholesterol pool following an oral dose of single-iso- tope-labeled cholesterol is believed to be indicative of the cholesterol absorption rate (18). However, the relationship between the isotope enrichment of plasma free cholesterol following an oral dose of single-isotope-labeled cholesterol and cholesterol absorption remains to be established as a non- invasive method to compare relative changes in cholesterol absorption. It was hypothesized that the area under the iso- tope enrichment curve, or the average isotope enrichment, in the plasma free-cholesterol pool following an oral dose of sin- gle-isotope-labeled cholesterol tracer correlates with the cho- lesterol absorption rate. The objective of the current study was to devise a method that would simplify the process of measuring cholesterol absorption in humans. MATERIALS AND METHODS Study protocols. Data from two published studies (14,15) were analyzed for the correlations between the area under the curve of a single-isotope enrichment and the cholesterol absorption rate, as measured by PDSIRM. Correlations between the mea- sured cholesterol absorption rate and the average enrichment of the single isotope were also conducted. Experimental ap- proaches to determine cholesterol absorption were identical be- tween the two studies. Study 1 involved 15 otherwise healthy Copyright © 2004 by AOCS Press 87 Lipids, Vol. 39, no. 1 (2004) *To whom correspondence should be addressed at School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, Ste-Anne-de- Bellevue, Quebec, Canada, H9X 3V9. E-mail: jonesp@macdonald.mcgill.ca Abbreviations: IRMS, isotope ratio MS; PDB, Pee Dee belemnite; PDSIRM, plasma dual stable-isotope ratio method; SMOW, Standard Mean Ocean Water. METHOD Validation of a Single-Isotope-Labeled Cholesterol Tracer Approach for Measuring Human Cholesterol Absorption Yanwen Wang, Catherine A. Vanstone, William D. Parsons, and Peter J.H. Jones* School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9