There is currently no direct, facile method to determine total-body skeletal muscle mass for the diagnosis and treatment of skeletal muscle wasting conditions such as sarcopenia, cachexia, and disuse. We tested in rats the hypothesis that the enrichment of creatinine-( methyl-d3) (D3-creatinine) in urine after a defined oral tracer dose of D3-creatine can be used to determine creatine pool size and skeletal muscle mass. We determined 1) an oral tracer dose of D3-creatine that was completely bioavailable with minimal urinary spillage and sufficient enrichment in the body creatine pool for detection of D3-creatine in muscle and D3-creatinine in urine, and 2) the time to isotopic steady state. We used cross-sectional studies to compare total creatine pool size determined by the D3-creatine dilution method to lean body mass determined by independent methods. The tracer dose of D3-creatine (<1 mg/rat) was >99% bioavailable with 0.2–1.2% urinary spillage. Isotopic steady state was achieved within 24–48 h. Creatine pool size calculated from urinary D3-creatinine enrichment at 72 h significantly increased with muscle accrual in rat growth, significantly decreased with dexamethasone-induced skeletal muscle atrophy, was correlated with lean body mass ( r = 0.9590; P < 0.0001), and corresponded to predicted total muscle mass. Total-body creatine pool size and skeletal muscle mass can thus be accurately and precisely determined by an orally delivered dose of D3-creatine followed by the measurement of D3-creatinine enrichment in a single urine sample and is promising as a noninvasive tool for the clinical determination of skeletal muscle mass.

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...

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This article is available online at http://www.jlr.org lesterol absorption in man, each with their own strengths and weaknesses (reviewed in Refs. 3,4 ). Generally, these methods are laborious and costly, as they require administration and analysis of isotopic tracers, thereby limiting their use in large-scale studies. For this purpose, more than two decades ago, plasma noncholesterol sterol concentrations were introduced as markers of cholesterol absorption and synthesis. The plant sterols campesterol and sitosterol and the cholesterol metabolite cholestanol were shown to be associated with cholesterol absorption ( 5, 6 ), whereas the cholesterol precursors lathosterol and desmosterol correlated with cholesterol synthesis ( 7,8 ). The conception of cholesterol precursors as a refl ection of the cholesterol synthesis pathway is intuitively plausible and has subsequently been corroborated by repetitive positive validation against various methods ( 7-11 ). Plant sterols derive strictly from the diet and share a high structural similarity with cholesterol. Nevertheless, their validity as markers of cholesterol absorption has been less well established, given that these absorption markers were initially validated against the cholesterol balance method in two relatively small study populations of 17 ( 5 ) and 63 ( 12 ) subjects. Subsequently, reported associations in larger study populations were not only weak but also have remained without additional prospective validation since. Notwithstanding, plasma plant sterol concentrations are currently used to describe cholesterol absorption in steadystate ( 13, 14 ) or intervention-induced changes in absorption ( 15-17 ). Finally, plasma plant sterol concentrations have also been suggested as a clinical tool to customize cholesterol-lowering treatment ( 18,19 ). In our opinion, these markers warranted thorough assessment before such suggestions can be supported. Human intestinal cholesterol absorption displays a large inter-individual variation, ranging from 20 to 80% ( 1, 2 ). Several techniques have been described to measure cho-