High-density lipoprotein cholesterol efflux, nitration of apolipoprotein A-I, and endothelial function in obese women

Abstract

Subjects at risk of atherosclerosis might have dysfunctional high-density lipoprotein (HDL) despite normal cholesterol content in the plasma. We considered whether the efflux of excess cellular cholesterol to HDL from obese subjects is associated with impaired arterial endothelial function, a biomarker of cardiovascular risk. A total of 54 overweight (body mass index [BMI] 25 to 29.9 kg/m(2)) or obese (BMI ≥30 kg/m(2)) women, aged 46 ± 11 years, were enrolled in a worksite wellness program. The HDL cholesterol averaged 57 ± 17 mg/dl and was inversely associated with the BMI (r = -0.419, p = 0.002). Endothelial function was assessed using brachial artery flow-mediated dilation. Cholesterol efflux from (3)H-cholesterol-labeled baby hamster kidney cells transfected with the adenosine triphosphate-binding cassette transporter 1 showed 8.2% to 22.5% cholesterol efflux within 18 hours when incubated with 1% serum and was positively correlated with brachial artery flow-mediated dilation (p <0.05), especially in the 34 subjects with BMI ≥30 kg/m(2) (r = 0.482, p = 0.004). This relation was independent of age, HDL or low-density lipoprotein cholesterol concentrations in plasma, blood pressure, or insulin resistance on stepwise multiple regression analysis (β = 0.31, R(2) = 0.21, p = 0.007). Nitration of apolipoprotein A-I tyrosine residues (using sandwich enzyme-linked immunosorbent assay) was significantly greater in women with a BMI ≥30 kg/m(2) and the lowest cholesterol efflux than in women with a BMI of 25 to 29.9 kg/m(2) and the greatest cholesterol efflux (p = 0.01). In conclusion, we have shown that decreased cholesterol efflux by way of the adenosine triphosphate-binding cassette transporter 1 is associated with increased nitration of apolipoprotein A-I in HDL and is an independent predictor of impaired endothelial function in women with a BMI of ≥30 kg/m(2). This finding suggests that the functional measures of HDL might be better markers for cardiovascular risk than the HDL cholesterol levels in this population.

Figure 1

Validation of nitrated apoA-I ELISA assay. Purified human apoA-I was incubated with increasing doses of peroxynitrite (0, 25, 50, 75, 100, 125 mol peroxynitrite/mol apoA-I) at 37°C for 1 hour. Aliquots were removed for either trypsin digestion followed by LC/MS or for nitrated apoA-I determination by the ELISA assay. Panel A: Mass spectrometry analysis of purified apoA-I incubated with peroxynitrite. The number of apoA-I peptides containing nitrotyrosine (normalized to the number of peptides recovered) by mass spectrometry increased with peroxynitrite dosage. Panel B: ELISA analysis of purified apoA-I incubated with peroxynitrite. Absorbance (405) increased with peroxynitrite dosage. Panel C: Validation of nitrated apoA-I ELISA assay by mass spectrometry. The number of apoA-I peptides containing nitrotyrosine (normalized to the number of peptides recovered) by mass spectrometry strongly correlated with absorbance (405) in the nitrated apoA-I ELISA assay.

Figure 2

Association between brachial artery flow-mediated dilation as a measure of endothelial nitric oxide bioactivity and cholesterol efflux from BHK cell line expressing the human ABCA1 transporter incubated for 18 hours with subject's whole serum at 1% concentration in 34 women with BMI≥ 30 kg/m².

Figure 3

Interaction of cholesterol efflux and HDL cholesterol on brachial artery flow-mediated dilation in subjects with BMI≥ 30 kg/m²: Those with efflux values below the median had reduced brachial artery flow-mediated dilation (P= 0.011 versus efflux> median values) regardless of whether HDL cholesterol values were above or below the median.

Figure 4

Nitrated apoA-I (by sandwich ELISA) in serum from the 10 women with BMI 25 – 29.9 kg/m² and the highest ABCAI-dependent cholesterol efflux (left) and from 10 women with BMI≥ 30 kg/m² and the lowest cholesterol efflux (right). Absorbance at 405 nm (A₄₀₅) corresponds to the amount of nitrated apoA-I (in microliters) in serum.