Kullo, Iftikhar J.

Abstract Background: Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays. Methods: We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias. Results: Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose–response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms. Conclusions: Multiplex measurements present difficult challenges that require further analytical and statistical developments.

Context: Stress-mediated hypothalamic-pituitary-adrenal axis activation, regulated by arginine vasopressin (AVP), may have a role in the pathophysiology of metabolic syndrome (MetSyn). Objective: The objective of the study was to investigate whether plasma C-terminal provasopressin fragment (copeptin), a surrogate for circulating AVP, was associated with measures of insulin resistance and presence of MetSyn. Design, Setting, and Participants: This was a multicenter, community-based study, investigating novel biomarkers for vascular disease. Participants included 1293 African-Americans (AA) (64 ± 9 yr) and 1197 non-Hispanic whites (NHW) (59 ± 10 yr) belonging to hypertensive sibships. Main Outcome Measures: Plasma copeptin levels were measured by an immunoluminometric assay. MetSyn was defined per Adult Treatment Panel III criteria. Generalized estimating equations were used to assess whether plasma copeptin was associated with measures of insulin resistance and MetSyn. Results: The prevalence of MetSyn was 50% in AA and 49% in NHW. In each group, after adjustment for age and sex, plasma copeptin levels significantly correlated with body mass index, fasting plasma glucose and insulin, homeostasis model assessment of insulin resistance, triglycerides, and (inversely) high-density lipoprotein cholesterol (P < 0.05 for each variable). In multivariable logistic regression models that adjusted for age, sex, smoking, statin use, serum creatinine, education, physical activity, and diuretic use, plasma copeptin levels in the highest quartile were associated with an increased odds ratio of having MetSyn compared with bottom quartile: odds ratio (95% confidence interval) in AA, 2.07 (1.45–2.95); in NHW, 1.74 (1.21–2.5). Conclusions: Our findings indicate a novel cross-sectional association between plasma copeptin and measures of insulin resistance and MetSyn.

Abstract Background: Higher plasma concentrations of soluble adhesion molecules have been shown to be associated with increased risk of cardiovascular events. We investigated the association of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) with ankle-brachial index (ABI), a measure of peripheral arterial disease (PAD), in a biethnic cohort of adults without known coronary heart disease or stroke. Methods: Participants included 1102 blacks (mean age, 63 years; 74% women) and 1013 non-Hispanic whites (mean age, 58 years; 59% women) belonging to sibships ascertained on the basis of hypertension. We measured plasma concentrations of sICAM-1 and sVCAM-1 using high-sensitivity immunoassays and ABI using a standard protocol; PAD was defined as ABI <0.9. We used generalized estimating equations to assess whether sICAM-1 and sVCAM-1 were associated with ABI and PAD, independently of conventional risk factors. Results: After adjustment for conventional risk factors, blacks with sICAM-1 and sVCAM-1 concentrations in the highest quartiles had lower ABIs than those in the lowest quartiles (mean ABI 1.02 vs 0.98, P = 0.007, vs 1.02 vs 0.99, P = 0.003, respectively). In multivariable logistic regression analysis, sICAM-1 and sVCAM-1 concentrations in the highest quartiles were each associated with a higher odds ratio of having PAD, compared with the lowest quartiles: odds ratio (95% CI): 5.2 (1.8–15.2) and 2.2 (1.0–4.8), respectively. In contrast, in non-Hispanic whites, sICAM-1 and sVCAM-1 concentrations were not associated with ABI or PAD. Conclusions: Higher sICAM-1 and sVCAM-1 concentrations were independently associated with lower ABI and PAD in blacks, but not in non-Hispanic whites.

Vascular reactivity may affect the stiffness characteristics of the arterial wall. We investigated the association between forearm microcirculatory and conduit artery function and measures of arterial stiffness in 527 asymptomatic non-Hispanic white adults without known cardiovascular disease. High-resolution ultrasonography of the brachial artery (ba) was performed to assess forearm microcirculatory function (ba blood flow velocity, local shear stress, and forearm vascular resistance at rest and during reactive hyperemia) and conduit artery function (ba flow-mediated dilatation [baFMD] and ba nitroglycerin-mediated dilatation [baNMD]). Arterial stiffness was assessed by cuff-derived brachial pulse pressure and aortic pulse wave velocity (aPWV) measured by applanation tonometry. In regression analyses that adjusted for heart rate, mean arterial pressure, height, cardiovascular risk factors, and hypertension medication and statin use, higher baseline ba systolic velocity and systolic shear stress were associated with greater pulse pressure ( P =0.0002 and P =0.006, respectively) and higher aPWV (each P <0.0001). During hyperemia, lower ba mean velocity and lower mean shear stress were associated with higher pulse pressure ( P =0.045 and P =0.036, respectively), whereas both systolic and mean velocity ( P <0.0001 and P =0.002, respectively) and systolic and mean shear stress ( P <0.0001 and P =0.003, respectively) were inversely associated with aPWV. baFMD was not associated with pulse pressure but was inversely associated with aPWV ( P =0.011). baNMD was inversely associated with pulse pressure ( P =0.0002) and aPWV ( P =0.008). Our findings demonstrate that impaired forearm microvascular function (in the form of elevated resting blood flow velocity and impaired flow reserve) and impaired brachial artery reactivity are associated with increased arterial stiffness.

Resistin, a recently discovered proinflammatory cytokine, has been variably associated with insulin resistance, inflammation, and renal dysfunction. We investigated the association of plasma resistin with estimated glomerular filtration rate and albuminuria in 1575 hypertensive adults without known coronary heart disease or stroke (857 blacks and 718 non-Hispanic whites). Resistin was measured by a solid phase sandwich immunoassay, estimated glomerular filtration rate was estimated from serum creatinine, and albuminuria was expressed as urine albumin:creatinine ratio. After adjustment for coronary heart disease risk factors (age, sex, body mass index, smoking history, systolic blood pressure, diabetes, and total and high-density lipoprotein cholesterol) and use of renin-angiotensin blockers and statins, higher plasma resistin levels were associated with lower estimated glomerular filtration rate in both ethnic groups (each P <0.0001); the association remained significant after further adjustment for a marker of insulin resistance (homeostasis model assessment for insulin resistance) and a marker of inflammation (plasma C-reactive protein) and was seen in subjects with and without diabetes (each P <0.0001) in both ethnic groups. Higher plasma resistin levels were associated with a higher urine albumin:creatinine ratio in black subjects with diabetes ( P <0.0001) and non-Hispanic white subjects with diabetes ( P =0.032), independent of coronary heart disease risk factors, hypertension medication use, and statin use; the association remained significant after additional adjustment for homeostasis model assessment for insulin resistance and C-reactive protein. In adults with hypertension, higher circulating resistin levels were associated with a lower estimated glomerular filtration rate and with increased urine albumin:creatinine ratio in the presence of concomitant diabetes. This association was independent of coronary heart disease risk factors and markers of insulin resistance and inflammation.