Intravenous administration of apolipoprotein (apo) A

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][4][5][6][7][8][9][10][11][12][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][4][5][6][7][8][9][10][11][12][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][4][5][6][7][8][9][10][11][12][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)Key Words: cholesterol efflux • esterification • reverse cholesterol transport • isotope labeling, stable • sterol excretion T he regulation of cellular cholesterol homeostasis is crucial for membrane function and cell survival and is maintained by multiple mechanisms, including control of uptake, synthesis, storage, and efflux. Compared to the pathways of cellular uptake and de novo synthesis of cholesterol, however, less information exists about the control of flux though pathways that remove cholesterol from cells and from the whole organism, 1,2 particularly in humans. [3][4][5] These pathways collectively have been termed reverse cholesterol transport (RCT). RCT is postulated to play a fundamental role in cholesterol homeostasis and distribution among tissues 5 and thereby in the development and reversal of atherosclerosis. 6,7 The atheroprotective effects of high-density lipoprotein cholesterol (HDL-C) in both human and animal studies often hav...

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.

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Crystallization of mixtures of the host compounds 1,5-naphthalenedisulfonate and guanidinium results in a new three-component network structure in the presence of linear fatty acid esters. The formation of this new topology is temperature-dependent and arises from the disruption of the basic guanidinium-sulfonate sheet structure through the incorporation of the solvent 2-methoxyethanol within the host network. Examination of the hightemperature crystal phase, the fatty acid ester-free 2-methoxyethanol solvate, reveals that the GS sheet topography of this network is directed by the hydrogen-bond interactions of the solvent and host. In the fatty acid ester inclusion compound, which crystallizes at lower temperatures, the host-guest interaction is much more explicit, with each 2-methoxyethanol molecule participating in three hydrogen bonds, becoming the third component of the host structure. The result of this new assembly is the creation of hydrophobically lined pores with an available cross-section area of 4.1 × 4.7 Å 2 within which the linear fatty acid ester guests are contained. Hydrophobic interactions and tight host-guest packing provide an explanation for formation of the entropically unfavorable three-component host network.