Michael P. Caulfield

Direct Determination of Lipoprotein Particle Sizes and Concentrations by Ion Mobility Analysis

BACKGROUND Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility-based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very-low-density lipoprotein (VLDL). METHODS After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2-540.0 A. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis. RESULTS Intra- and interassay coefficients of variation for LDL particle size were <1.0%. The intra- and interassay variation for LDL and HDL particle subfraction measurements was <20%. IM-measured non-HDL correlated well with apolipoprotein B (r = 0.92). CONCLUSIONS The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.

Lipoprotein(a) Concentrations, Rosuvastatin Therapy, and Residual Vascular Risk An Analysis From the JUPITER Trial (Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin)

Background— Lipoprotein(a) [Lp(a)] is a low-density lipoprotein–like particle largely independent of known risk factors and predictive of cardiovascular disease. Statins may offset the risk associated with elevated Lp(a), but it is unknown whether Lp(a) is a determinant of residual risk in the setting of low low-density lipoprotein cholesterol after potent statin therapy. Methods and Results— Baseline and on-treatment Lp(a) concentrations were assessed in 9612 multiethnic participants in the JUPITER trial (Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin) before and after random allocation to rosuvastatin 20 mg/d or placebo, with outcomes reported for whites (n=7746). Lp(a) concentrations (median [25th–75th percentile], in nmol/L) were highest in blacks (60 [34–100]), then Asians (38 [18–60]), Hispanics (24 [11–46]), and whites (23 [10–50]; P<0.001). Although the median change in Lp(a) with rosuvastatin and placebo was zero, rosuvastatin nonetheless resulted in a small but statistically significant positive shift in the overall Lp(a) distribution (P<0.0001). Baseline Lp(a) concentrations were associated with incident cardiovascular disease (adjusted hazard ratio per 1-SD increment in Ln[Lp(a)], 1.18; 95% confidence interval, 1.03–1.34; P=0.02). Similarly, on-statin Lp(a) concentrations were associated with residual risk of cardiovascular disease (adjusted hazard ratio, 1.27; 95% confidence interval, 1.01–1.59; P=0.04), which was independent of low-density lipoprotein cholesterol and other factors. Rosuvastatin significantly reduced incident cardiovascular disease among participants with baseline Lp(a) greater than or equal to the median (hazard ratio, 0.62; 95% confidence interval, 0.43–0.90) and Lp(a) less than the median (hazard ratio, 0.46; 95% confidence interval, 0.30–0.72), with no evidence of interaction. Similar results were obtained when analyses included nonwhites. Conclusion— Among white JUPITER participants treated with potent statin therapy, Lp(a) was a significant determinant of residual risk. The magnitude of relative risk reduction with rosuvastatin was similar among participants with high or low Lp(a). Clinical Trials Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00239681.

Ion Mobility Analysis of Lipoprotein Subfractions Identifies Three Independent Axes of Cardiovascular Risk

Objective—Whereas epidemiological studies show that levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) predict incident cardiovascular disease (CVD), there is limited evidence relating lipoprotein subfractions and composite measures of subfractions to risk for CVD in prospective cohort studies. Methods and Results—We tested whether combinations of lipoprotein subfractions independently predict CVD in a prospective cohort of 4594 initially healthy men and women (the Malmö Diet and Cancer Study, mean follow-up 12.2 years, 377 incident cardiovascular events). Plasma lipoproteins and lipoprotein subfractions were measured at baseline with a novel high-resolution ion mobility technique. Principal component analysis (PCA) of subfraction concentrations identified 3 major independent (ie, zero correlation) components of CVD risk, one representing LDL-associated risk, a second representing HDL-associated protection, and the third representing a pattern of decreased large HDL, increased small/medium LDL, and increased triglycerides. The last corresponds to the previously described “atherogenic lipoprotein phenotype.” Several genes that may underlie this phenotype—CETP, LIPC, GALNT2, MLXIPL, APOA1/A5, LPL—are suggested by SNPs associated with the combination of small/medium LDL and large HDL. Conclusion—PCA on lipoprotein subfractions yielded three independent components of CVD risk. Genetic analyses suggest these components represent independent mechanistic pathways for development of CVD.

Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: Total and free testosterone reference ranges

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 microL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y=1.02x+1.5, r(2)=0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.

Risk Profiles for Aortic Dissection and Ruptured or Surgically Treated Aneurysms: A Prospective Cohort Study

Background Community screening to guide preventive interventions for acute aortic disease has been recommended in high‐risk individuals. We sought to prospectively assess risk factors in the general population for aortic dissection (AD) and severe aneurysmal disease in the thoracic and abdominal aorta. Methods and Results We studied the incidence of AD and ruptured or surgically treated aneurysms in the abdominal (AAA) or thoracic aorta (TAA) in 30 412 individuals without diagnosis of aortic disease at baseline from a contemporary, prospective cohort of middle‐aged individuals, the Malmö Diet and Cancer study. During up to 20 years of follow‐up (median 16 years), the incidence rate per 100 000 patient‐years at risk was 15 (95% CI 11.7 to 18.9) for AD, 27 (95% CI 22.5 to 32.1) for AAA, and 9 (95% CI 6.8 to 12.6) for TAA. The acute and in‐hospital mortality was 39% for AD, 34% for ruptured AAA, and 41% for ruptured TAA. Hypertension was present in 86% of individuals who subsequently developed AD, was strongly associated with incident AD (hazard ratio [HR] 2.64, 95% CI 1.33 to 5.25), and conferred a population‐attributable risk of 54%. Hypertension was also a risk factor for AAA with a smaller effect. Smoking (HR 5.07, 95% CI 3.52 to 7.29) and high apolipoprotein B/A1 ratio (HR 2.48, 95% CI 1.73 to 3.54) were strongly associated with AAA and conferred a population‐attributable risk of 47% and 25%, respectively. Smoking was also a risk factor for AD and TAA with smaller effects. Conclusions This large prospective study identified distinct risk factor profiles for different aortic diseases in the general population. Hypertension accounted for more than half of the population risk for AD, and smoking for half of the population risk of AAA.

Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High-Throughput Clinical Laboratory

BACKGROUND Circulating insulin concentrations reflect the amount of endogenous insulin produced by the pancreas and can be monitored to check for insulin resistance. Insulin is commonly measured using immunochemiluminometric assays (ICMA). Unfortunately, differing crossreactivities of the various ICMA antibodies have led to variability in assay results. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches can provide a highly specific alternative to immunoassays. METHODS Insulin was extracted from patient serum and reduced to liberate the insulin B chain. Subsequent resolution of the peptide was achieved by LC coupled to triple-quadrupole MS. Selected-reaction monitoring of B-chain transitions was used for quantification. Recombinant human insulin was used as a calibrator and was compared against the National Institute for Biological Standards and Control (NIBSC) reference standard. Bovine insulin and a stable isotopic-labeled ((13)C/(15)N) human insulin B chain were used and compared as internal standards. RESULTS The LC-MS/MS assay described herein has been validated according to CLIA guidelines with a limit of detection of 1.8 μIU/mL (10.8 pmol/L) and a limit of quantitation of 3 μIU/mL (18.0 pmol/L). A correlation between the LC-MS/MS assay and a US Food and Drug Administration-approved ICMA was completed for patient samples and the resulting Deming regression revealed good agreement. A reference interval for the assay was established. CONCLUSIONS A simple, high-throughput, quantitative LC-MS/MS insulin assay traceable to the NIBSC standard has been successfully developed and validated.

Testosterone reference ranges in normally cycling healthy premenopausal women.

INTRODUCTION At present, there are no well-accepted reference ranges for serum testosterone concentrations in women. AIM The aim of this study was to determine the reference ranges for serum testosterone and sex hormone-binding globulin (SHBG) in premenopausal women with normal menstrual cycles. METHODS We measured serum total, free, and bioavailable testosterone and SHBG concentrations in 161 healthy, normally cycling women (18-49 years). Morning blood samples were collected during follicular, mid-cycle, and luteal phases of the menstrual cycle and analyzed using validated methods. Mean, median, and weighted average hormone levels across menstrual cycle phases as well as percentiles for a typical 30-year-old woman were determined. MAIN OUTCOME MEASURES Age-related serum levels of total, free, and bioavailable testosterone and SHBG levels in normally cycling premenopausal women. RESULTS Serum testosterone concentrations exhibited an age-related decline, whereas SHBG remained relatively stable across studied age ranges. Reference ranges for total, free, and bioavailable testosterone and SHBG were established using 5th and 95th percentiles. The estimated 5th and 95th percentiles for a 30-year-old woman were: testosterone, 15-46 ng/dL (520-1595 pmol/L); free testosterone, 1.2-6.4 pg/mL (4.16-22.2 pmol/L); calculated free testosterone, 1.3-5.6 pg/mL (4.5-19.4 pmol/L); bioavailable testosterone, 1.12-7.62 ng/dL (38.8-264.21 pmol/L); and SHBG 18-86 nmol/L. The variations of hormones and SHBG across menstrual cycle were consistent with previous literature. CONCLUSIONS Reference ranges for free, total, and bioavailable testosterone and SHBG were established in premenopausal women using validated immunoassays and an adequate number of subjects consistent with recommendations by the National Committee for Clinical Laboratory Standards. The increase in testosterone in the mid-cycle period is relatively small compared with the overall variability, so these reference ranges can be applied irrespective of the day in the menstrual cycle the sample has been taken.

Atherogenic Lipoprotein Subfractions Determined by Ion Mobility and First Cardiovascular Events After Random Allocation to High-Intensity Statin or Placebo: The JUPITER Trial

Background —Cardiovascular disease (CVD) can occur in individuals with low LDL-cholesterol (LDL-c). We investigated whether detailed measures of LDL subfractions and other lipoproteins can be used to assess CVD risk in a population with both low LDL-c and high C-reactive protein that was randomized to high-intensity statin or placebo. Methods and Results —In 11,186 JUPITER participants, we tested whether lipids, apolipoproteins, and ion mobility (IM)-measured particle concentrations at baseline and after random allocation to rosuvastatin 20 mg/d or placebo were associated with first CVD events (n=307) or CVD/all-cause death (n=522). In placebo-allocated participants, baseline LDL-c was not associated with CVD (adjusted HR per SD, 1.03, 95% CI 0.88-1.21). In contrast, associations with CVD events were observed for baseline non-HDL-cholesterol (non-HDL-c: 1.18, 1.01-1.38), apolipoprotein B (apoB: 1.28, 1.11-1.48), and IM-measured non-HDL particles (non-HDL-p: 1.19, 1.05-1.35) and LDL particles (LDL-p: 1.21, 1.07-1.37). Association with CVD events was also observed for several LDL and VLDL subfractions, but not for IM-measured HDL subfractions. In statin-allocated participants, CVD events were associated with on-treatment LDL-c, non-HDL-c, and apoB; these were also associated with CVD/all-cause death, as were several LDL and VLDL subfractions albeit with a pattern of association that differed from the baseline risk. Conclusions —In JUPITER, baseline LDL-c was not associated with CVD events, in contrast with significant associations for non-HDL-c and atherogenic particles: apoB and IM-measured non-HDL-p, LDL-p, and select subfractions of VLDL-p and LDL-p. During high-intensity statin therapy, on-treatment levels of LDL-c and atherogenic particles were associated with residual risk of CVD/all-cause death. Clinical Trial Registration Information —ClinicalTrials.gov. Identifier: NCT00239681.Background— Cardiovascular disease (CVD) can occur in individuals with low low-density lipoprotein (LDL) cholesterol (LDL-C). We investigated whether detailed measures of LDL subfractions and other lipoproteins can be used to assess CVD risk in a population with both low LDL-C and high C-reactive protein who were randomized to high-intensity statin or placebo. Methods and Results— In 11 186 Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) participants, we tested whether lipids, apolipoproteins, and ion mobility–measured particle concentrations at baseline and after random allocation to rosuvastatin 20 mg/d or placebo were associated with first CVD events (n=307) or CVD/all-cause death (n=522). In placebo-allocated participants, baseline LDL-C was not associated with CVD (adjusted hazard ratio [HR] per SD, 1.03; 95% confidence interval [CI], 0.88–1.21). In contrast, associations with CVD events were observed for baseline non–high-density lipoprotein (HDL) cholesterol (HR, 1.18; 95% CI, 1.01–1.38), apolipoprotein B (HR, 1.28; 95% CI, 1.11–1.48), and ion mobility–measured non-HDL particles (HR, 1.19; 95% CI, 1.05–1.35) and LDL particles (HR, 1.21; 95% CI, 1.07–1.37). Association with CVD events was also observed for several LDL and very-low-density lipoprotein subfractions but not for ion mobility–measured HDL subfractions. In statin-allocated participants, CVD events were associated with on-treatment LDL-C, non-HDL cholesterol, and apolipoprotein B; these were also associated with CVD/all-cause death, as were several LDL and very-low-density lipoprotein subfractions, albeit with a pattern of association that differed from the baseline risk. Conclusions— In JUPITER, baseline LDL-C was not associated with CVD events, in contrast with significant associations for non-HDL cholesterol and atherogenic particles: apolipoprotein B and ion mobility–measured non-HDL particles, LDL particles, and select subfractions of very-low-density lipoprotein particles and LDL particles. During high-intensity statin therapy, on-treatment levels of LDL-C and atherogenic particles were associated with residual risk of CVD/all-cause death. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00239681.

Classification of LDL Phenotypes by 4 Methods of Determining Lipoprotein Particle Size

Background Low-density lipoprotein cholesterol (LDL-C) lowering is the primary objective of patient management for cardiovascular disease. However, large numbers of patients who have achieved their LDL-C goal remain at risk for cardiovascular events. Low-density lipoprotein subfractions may provide insight into this residual risk. Thus, LDL subfraction standardization and consistency are critical to these efforts. Aim This study aimed to determine the agreement of the analytical results among 4 methods commonly used for LDL subfractionation, namely, segmented gradient gel electrophoresis (sGGE), ultracentrifugation-vertical auto profile (VAP), nuclear magnetic resonance (NMR), and ion mobility (IM). Methods Blood samples were collected from 228 apparently healthy adults and sent to 4 clinical reference laboratories for analysis. The LDL phenotype was reported as pattern A (larger, less dense particles) or pattern B (smaller, more dense particles), respectively, and was the primary measure of comparison. An intermediate pattern (A/B) was also reported for sGGE and VAP. Results We observed complete agreement in the LDL phenotype among the 4 methods in 64% of subjects and agreement among at least 3 of the 4 methods in 87% of subjects. Agreement among pairs of methods ranged from 73% to 98% depending on how differences in reporting of subjects with intermediate results were considered. When subjects having intermediate A/B pattern were excluded, sGGE and IM had the highest agreement (98%) of any pair of methods. Conclusions We found substantial agreement in the reported LDL phenotype among 4 LDL subfraction measurement technologies as performed by different clinical reference laboratories.

A randomized, double blind, placebo-controlled pilot trial of the safety and efficacy of atorvastatin in children with elevated low-density lipoprotein cholesterol (LDL-C) and type 1 diabetes.

Children with type 1 diabetes (T1D) and elevated LDL‐C have an increased risk for cardiovascular disease, a process that can begin in childhood.