Measurement of reverse cholesterol transport pathways in humans: in vivo rates of free cholesterol efflux, esterification, and excretion

Abstract

Background: Reverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high-density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and fecal sterol excretion of plasma-derived FC.

Methods and results: A constant infusion of [2,3-(13)C(2)]-cholesterol was administered to healthy volunteers. Three-compartment SAAM II (Simulation, Analysis, and Modeling software; SAAM Institute, University of Washington, WA) fits were applied to plasma FC, red blood cell FC, and plasma cholesterol ester (13)C-enrichment profiles. Fecal sterol excretion of plasma-derived FC was quantified from fractional recovery of intravenous [2,3-(13)C(2)]-cholesterol in feces over 7 days. We examined the key assumptions of the method and evaluated the optimal clinical protocol and approach to data analysis and modeling. A total of 17 subjects from 2 study sites (n=12 from first site, age 21 to 75 years, 2 women; n=5 from second site, age 18 to 70 years, 2 women) were studied. Tissue FC efflux was 3.79±0.88 mg/kg per hour (mean ± standard deviation), or ≍8 g/d. Red blood cell-derived flux into plasma FC was 3.38±1.10 mg/kg per hour. Esterification of plasma FC was ≍28% of tissue FC efflux (1.10±0.38 mg/kg per hour). Recoveries were 7% and 12% of administered [2,3-(13)C(2)]-cholesterol in fecal bile acids and neutral sterols, respectively.

Conclusions: Three components of systemic reverse cholesterol transport can be quantified, allowing dissection of this important function of high-density lipoprotein in vivo. Effects of lipoproteins, genetic mutations, lifestyle changes, and drugs on these components can be assessed in humans. (J Am Heart Assoc. 2012;1:e001826 doi: 10.1161/JAHA.112.001826.).

Keywords: cholesterol efflux; esterification; isotope labeling, stable; reverse cholesterol transport; sterol excretion.

Figure 1.

Multicompartment model of fluxes of FC. SAAM II model of cholesterol fluxes in and out of the plasma FC pool (V1), RBC FC (V2), plasma CE (V3), and rapid-exchange (mixing) pool in equilibrium with V1 (V4). k indicates rate constants 1/hour; s, sampling sites – data input into model; Infusion, site of constant infusion of FC. Parameters: R, infusion rate (mg/kg per hour); V1, plasma FC pool size (mg/kg body weight); V2, RBC FC pool size (mg/kg body weight); V3, plasma CE pool size (mg/kg body weight); k(0,1), rate constant for transfer of tracer out of plasma FC pool (hour⁻¹); k(0,3), rate constant for transfer of tracer out of plasma CE pool (hour⁻¹); k(3,1), rate constant for transfer of tracer from plasma FC to plasma CE pool (hour⁻¹); k(1,2), rate constant for transfer of tracer from RBC FC pool to plasma FC pool (hour⁻¹); k(2,1), rate constant for transfer of tracer from plasma FC pool to RBC pool (hour⁻¹); and s₁, s₂, and s₃ sampling sites, corresponding with V₁, V₂, and V₃. R, V₂, and V₃ entered as fixed parameters into the model; others are calculated by SAAM II. Steady-state equations: Flux 1=k(0,1)×V1=flux of plasma FC out of V1 (mg/kg per hour); Flux 2=k(2,1)×V1=k(1,2)×V2=exchange flux between plasma FC and RBC FC (mg/kg per hour); Flux 3=k(0,3)×V3=k(3,1)×V1=flux of plasma FC to plasma CE pool (mg/kg per hour); Flux 1+Flux 3=TCE (mg/kg per hour).

Figure 2.

A, Example of a typical isotope enrichment profiles in plasma FC (circles), RBC FC (triangles) and CE (squares) in subject during a 32-hour isotope infusion. Solid lines represent the SAAM II–generated fits. B, Enrichment profiles in stool for a NS, coprostanol (closed), and a BA, deoxycholic acid (open). Infusion was on day zero, and enrichment was measured in daily stool collections, when available. Preinfusion enrichments were used to determine baseline isotope enrichments, which were subtracted from all subsequent measurements.