A new method for measuring cholesterol efflux capacity uses stable isotope-labeled, not radioactive-labeled, cholesterol

Abstract

Supplementary key words apolipoprotein • reverse cholesterol transport • high density lipoprotein • mass spectrometry • stable isotope tracers • [d 7 ]cholesterol Many epidemiologic studies have reported an inverse relationship between coronary artery disease (CAD) events and HDL-cholesterol (HDL-C) levels (1-3). It is commonly known that the inhibition of cholesteryl ester transfer protein (CETP) leads to an increase in HDL-C levels in blood. Bowman et al. (4) recently reported that the inhibition of CETP by anacetrapib resulted in a lower incidence of major coronary events. In contrast, recent intervention trials have shown that treatment with CETP inhibitors or niacin fails to reduce CAD risk despite increasing HDL-C levels. Moreover, paradoxically, a high mortality rate was reported among individuals in the general population with extremely high HDL-C (5). It has since been suggested that quality is as important as quantity when measuring HDL (6-9). HDL is known to mediate reverse cholesterol transport (RCT), act as an antioxidant, suppress inflammation, and improve endothelial function (10). In RCT, accumulated cholesterol is transported from peripheral tissues back to the liver, a process that requires HDL and apoA-1 (10-12). The first step in RCT is cholesterol efflux from macrophages; this is thought to be the most important Abstract The incidence of cardiovascular events correlates inversely with cholesterol efflux capacity (CEC) more than with HDL-cholesterol level. The measurement of CEC is used to qualify cardiovascular disease risk and is conventionally performed with radioisotope (RI)-labeled cholesterol. Here, we established a CEC measurement technique using stable isotope-labeled cholesterol as an alternative, and we compared the new method with RI and fluorescence (boron dipyrromethene difluoride-cholesterol) methods in cells and in patient serum. We incubated J774 cells labeled with [d 7 ] cholesterol ([d 7 ]C) with patient serum depleted of apoB, and [d 7 ]C extracted from the culture medium was quantified by liquid chromatography/quadrupole time-of-flight mass spectrometry. [d 7 ]C efflux increased with greater apoB-depleted serum concentration and longer incubation time. The assay coefficient of variation (CV) of five consecutive measurements of three sets of samples ranged from 7.3% to 9.5%, and the interassay CV determined by measuring three samples four times ranged from 4.1% to 8.5%, both indicating good precision. We then measured CEC levels of 41 outpatients with serum HDL-cholesterol levels between 36 and 94 mg/dl (mean: 61.7 ± 18.0 mg/dl); in the presence of cAMP, we observed a significant, positive correlation between CEC levels determined with the stable isotope and RI methods that was stronger than the correlation between measurements obtained by the fluorescence and RI methods (r = 0.73, P < 0.0001 vs. r = 0.55, P < 0.001). Therefore, our stable isotope method can be considered useful as a non-RI method and thus deserves evaluation in future clinical studies.-Shimizu, T., O. Miyaz...

A meta-analysis of HDL cholesterol efflux capacity and concentration in patients with rheumatoid arthritis

“…Macrophage cholesterol efflux is the first critical step of RCT and considered as a key atheroprotective property [ 13 ]. The general methods for measuring cholesterol efflux capacity (CEC) include using radioisotope-labeled cholesterol that is labeled with [ 3 H]-cholesterol ([ 3 H]-C) and fluorescence-labeled cholesterol to measure CEC [ 14 , 15 ]. Impaired CEC has been proven to increase the risk of CVDs, which is independent of the plasma HDL-C levels [ 16 ].…”

The Antiatherogenic Effects of Flavonoid on Cholesterol Efflux Capacity

“…Standard techniques employ cultured cells containing cholesterol labeled by radioisotopes to test HDL's efflux capacity, or ability to remove cholesterol from the serum [75], [76]. For example, J774, Raw264, or differentiated THP-1 cells are tagged with [3 H] cholesterol ([3H]C) and then treated with serum HDL, after which the radioactivity of [3H]C emitted by cells is assessed [77]. Due to the complexity of this treatment, it cannot be used in a clinical situation.…”

Detection of lipid efflux from foam cell models using a label-free infrared method

“…14 The incidence of cardiovascular disease is more inversely related to cholesterol efflux capacity, than to high density lipoprotein (HDL) levels. 15,16 Regarding foam cells, debate still remains as to the effect statins, specifically atorvastatin, have on initiating cholesterol efflux/reverse cholesterol transport (RCT). Some studies have reported an inhibitory effect of atorvastatin on foam cell models.…”