This study was conducted at the Clinical Center of the National Institutes of Health with employees enrolled in a worksite wellness program initiated by the National Heart, Lung, and Blood Institute. The protocol, approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (NCT00666172), was open to women according to BMI (weight in kilograms divided by height in meters2
) classification of overweight (25 – 29.9 kg/m2
) or obese (≥ 30 kg/m2
), who were without history of atherosclerotic vascular disease and were not participants in structured exercise or weight loss programs. All participants provided written consent to participate in the protocol. All subjects underwent focused cardiovascular physical examinations, and venous blood samples were drawn following overnight fast. Standard lipid profiles were measured, using enzymatic assay (Wako Chemical USA Inc, Richmond, VA). Insulin resistance was estimated from fasting glucose and insulin values using the Homeostasis Model Assessment (HOMA).7
For women of reproductive age reporting menses, testing was performed during the follicular phase (days 1–10) of the menstrual cycle.
Brachial artery flow-mediated dilation testing, as an index of endothelial nitric oxide bioactivity, was conducted by a single investigator (GZ) as follows: Imaging of the left brachial artery proximal to the antecubital fossa was performed using a high-resolution ultrasound (12.5-MHz linear-array transducer) after 10 minutes of rest. Arterial diameter was measured in millimeters from the leading edge of the intima–lumen interface of the near wall to the leading edge of the lumen–intima interface of the far wall, coincident with the R wave on the electrocardiogram (end-diastole), at ≥ 6 sites and averaged. The maximum increase in brachial artery diameter was then measured during reactive hyperemia following 5 minutes of forearm ischemia caused by inflation of a blood pressure cuff on the forearm to suprasystolic pressure (225 mm Hg). Brachial artery flow-mediated dilation (%) = (post-ischemia minus baseline diameter) divided by baseline diameter × 100.
HDL-associated proteins apoA-I and apoA-II were measured using turbidimetric immunoassay (Wako Chemicals USA, Richmond, VA). The HDL subparticle preβ-1 was measured by an enzyme-linked immunosorbent assay (Polymedco, Cortlandt Manor, NY).8,9
The subparticle HDL2b was measured electrophoretically as previously described10
with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and expressed as a percentage of the total HDL particle. The capacity of a serum specimen to accept cholesterol effluxed by ABCA1 was measured, using a stably transfected BHK cell line expressing the human ABCA1 transporter.11
BHK cells transfected with a hygromycin-resistant control plasmid were used as the control cell line. Cholesterol efflux was conducted at 37 °C in cells labeled with 3
H-cholesterol for 24 hours, washed, and incubated for 18 hours with subject's whole serum at 1% concentration. The percent of efflux specific to the ABCA1 transporter was calculated by subtracting the radioactive counts in the blank medium (α-minimal essential medium with 0.1% bovine serum albumin) from the radioactive counts in the presence of HDL and then dividing the result by the sum of the radioactive counts in the medium plus the cell fraction.
Nitrated apoA-I in serum of study participants with high and low efflux capacity was quantitated by a sandwich enzyme-linked immunoabsorbent assay.12
Briefly, sera from overweight and obese subjects with high and low cholesterol efflux were incubated in wells of a 96-well plate that was coated with anti-nitrotyrosine antibodies, enabling the capture of nitrated serum proteins, such as nitrated apoA-I. The plate wells were washed and an antibody to human apoA-I was added, specifically identifying nitrated apoA-I. Serum with higher levels of nitrated apoA-I will have increased nitrated apoA-I bound to the well, resulting in higher levels of anti-apoA-I antibody binding. HRP-conjugated secondary antibody followed by the HRP substrate OPD was used for detection. Absorbance at 405 nm was measured and corrected to absorbance of control wells in which no serum was added. The assay was validated experimentally through the range of values detected in subject samples by adding increasing concentrations of peroxynitrite to purified apoA-I and either performing ELISA on the nitrated apoA-I or digesting the nitrated apoA-I with trypsin and using LC-MS to quantify tyrosine nitration of apoA-I. The number of apoA-I tryptic peptides containing nitrotyrosine, normalized to the total number of apoA-I tryptic peptides recovered, increased with increasing peroxynitrite dosage (). Between absorbance (405) of 0.3 and 0.7 units, the fit between peroxynitrite dosage and absorbance (405) from the ELISA assay (y= −2E-05x2
+ 0.004x + 0.368) was highly significant (). Finally, the number of apoA-I tryptic peptides containing nitrotyrosine, normalized to the total number of apoA-I tryptic peptides recovered, was correlated with absorbance (405) in the ELISA assay (). Second-order polynomial equations and R2
values were calculated using Microsoft Excel.
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 (more ...)
Data are reported as mean ± SD, unless otherwise indicated. All analyses were performed using the Instat3 or Prism statistical software (GraphPad Software Inc., San Diego, CA) or SAS (SAS Institute Inc, Cary, NC). Simple linear regression and the Pearson correlation coefficient were used to quantify associations between dependent and independent variables. Two-way analysis of variance (ANOVA) with interaction was performed to determine the effects of cholesterol efflux and HDL-C on brachial artery flow-mediated dilation. Multiple regression models for explaining brachial artery flow-mediated dilation were constructed, using stepwise model-building approaches by entering cholesterol efflux, age, HDL cholesterol, low-density lipoprotein cholesterol, triglycerides, blood pressure and HOMA as covariates using the SAS statistical analysis package and STEPWISE, SQUARE, GLM, and MEANS procedures (SAS User's Guide: Statistics, Version 9 Edition: SAS Institute Inc, Cary, NC). Statistical significance of differences in apoA-I tyrosine nitration were assessed by unpaired t-test. Both high and low efflux groups passed normality testing (Prism). P< 0.05 was considered to achieve statistical significance.