The Biological Variability of Plasma Ceramides in Healthy Subjects.

BACKGROUND: Ceramides are bioactive lipid species that mediate numerous cell-signaling events. Elevated plasma ceramides concentration constitutes a risk factor for several pathologies. Multiple studies have affirmed the plasma concentrations of 4 specific ceramides (Cer16:0, Cer18:0, Cer24:0, and Cer24:1) can predict cardiovascular disease risk. Furthermore, these ceramides can be altered by many lipid-lowering therapies. Understanding the biological variability within an individual, and within a population, will further inform the clinical use of plasma ceramides as a biomarker. In this study, we aimed to define the intra- and interbiological variability of ceramides in a healthy reference population in a weekly and monthly manner. METHODS: Fasting plasma from 24 healthy adults was collected daily (5 days), weekly (4 weeks), and monthly (7 months). Ceramide concentrations were measured with liquid chromatography-mass spectrometry (LC-MS). For analysis, we used random-effects regression models to estimate variance components. RESULTS: The analytical variability was smaller compared to the biological variability overall. The greatest variation reported was between-subject variation for all ceramide species. The critical difference-reference change value (RCV) for within-subject variations monthly were 0.07 mcmol/L (Cer16:0), 0.04 mcmol/L (Cer18:0), 1.09 mcmol/L (Cer24:0), and 0.27 mcmol/L (Cer24:1). The index of individuality (IOI) of ceramides were 0.82 (Cer16:0), 0.96 (Cer18:0), 1.06 (Cer24:0), and 0.89 (Cer24:1). The most consistent ceramide species was Cer18:0 with the lowest within- and between-subject critical differences in weekly and monthly measurements. CONCLUSIONS: Overall, this study demonstrates that the variability of ceramide concentrations at different time points is minimal within individuals, allowing a single draw to be sufficient at least in a yearly time frame.

Biological variability of lipoprotein-associated phospholipase A2 activity in healthy individuals.

OBJECTIVES: Measuring lipoprotein-associated phospholipase A2 (Lp-PLA2) activity can aid in identifying individuals at higher risk of coronary heart disease. However, the biological variation of Lp-PLA2 activity and corresponding reference change value (RCV) is unknown which limits interpretation of results. In this study we aim to define the intra- and inter-individual variability of Lp-PLA2 activity in a healthy reference population. METHODS: A total of 24 healthy individuals (22-47years of age) were prospectively collected at several time points: daily for five days (after overnight fast), daily for three days (while non-fasting), weekly for four weeks (after overnight fast), and monthly for 6months (after overnight fast). Intra-individual and inter-individual variability was determined. The index of individuality (IoI) and reference change value (RCV) were calculated for each time period. RESULTS: Variability in Lp-PLA2 activity was not different in fasting versus non-fasting states and also did not change in daily versus monthly testing. The RCV for monthly Lp-PLA2 activity was found to be 17%. More than 90% of the variability was attributable to between person differences while analytic variability comprised <9% of the variability and within-person variability was low at <0.7%. The index of individuality for monthly testing was 0.30 CONCLUSIONS: In a healthy population, Lp-PLA2 activity displays low analytical and within-person variability and higher inter-individual variability. The change required to differentiate a true change in patient status was determined to be 17% for monthly measurements. The between individual variability and corresponding RCV for the activity assay are lower than previously reported results for the Lp-PLA2 mass assay.

Effects on lipoprotein particles of long-term dehydroepiandrosterone in elderly men and women and testosterone in elderly men.

CONTEXT: Although age-related declines in dehydroepiandrosterone sulfate (DHEAS) and testosterone are associated with cardiovascular risk, it remains to be determined whether replacement of these hormones improves cardiovascular risk factors. OBJECTIVE: This study sought to determine the effect of long-term replacement of dehydroepiandrosterone (DHEA) in elderly men and women and testosterone in elderly men on lipid and lipoprotein concentrations and particle sizes. METHODS: A 2-yr randomized, placebo-controlled, double-blind study was conducted in 87 elderly men with low levels of DHEAS and bioavailable testosterone and 57 elderly women with low levels of DHEAS. Among elderly men, 29 received DHEA (75 mg/d), 27 received testosterone (5 mg/d), and 31 received placebo. Among the elderly women, 27 received DHEA (50 mg/d), and 30 received placebo. Baseline lipoprotein profiles in the elderly were compared to healthy younger participants. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particle sizes and concentrations were quantified using nuclear magnetic resonance spectroscopy. RESULTS: The elderly had higher concentrations of total cholesterol, triglycerides, LDL cholesterol, total LDL particles, and small, dense LDL particles than the young. In men, neither DHEA nor testosterone affected LDL or HDL particle concentrations. In women, DHEA reduced HDL cholesterol [median difference (95% confidence intervals), -5.0 (-8.0, -2.0) mg/dl; P = 0.002] and the number of large HDL particles [-1.0 (-1.8, -0.2) micromol/liter; P = 0.003]. CONCLUSIONS: Long-term DHEA and testosterone had no significant effect on plasma lipoproteins in elderly men, but elderly women showed a lowering of the large HDL particles that may have potential adverse clinical implications.

Coronary artery endothelial dysfunction is positively correlated with low density lipoprotein and inversely correlated with high density lipoprotein subclass particles measured by nuclear magnetic resonance spectroscopy.

OBJECTIVE: The association between cholesterol and endothelial dysfunction remains controversial. We tested the hypothesis that lipoprotein subclasses are associated with coronary endothelial dysfunction. METHODS AND RESULTS: Coronary endothelial function was assessed in 490 patients between November 1993 and February 2007. Fasting lipids and nuclear magnetic resonance (NMR) lipoprotein particle subclasses were measured. There were 325 females and 165 males with a mean age of 49.8+/-11.6 years. Coronary endothelial dysfunction (epicardial constriction>20% or increase in coronary blood flow<50% in response to intracoronary acetylcholine) was diagnosed in 273 patients, the majority of whom (64.5%) had microvascular dysfunction. Total cholesterol and LDL-C (low density lipoprotein cholesterol) were not associated with endothelial dysfunction. One-way analysis and multivariate methods adjusting for age, gender, diabetes, hypertension and lipid-lowering agent use were used to determine the correlation between lipoprotein subclasses and coronary endothelial dysfunction. Epicardial endothelial dysfunction was significantly correlated with total (p=0.03) and small LDLp (LDL particles) (p<0.01) and inversely correlated with total and large HDLp (high density lipoprotein particles) (p<0.01). CONCLUSIONS: Epicardial, but not microvascular, coronary endothelial dysfunction was associated directly with LDL particles and inversely with HDL particles, suggesting location-dependent impact of lipoprotein particles on the coronary circulation.

Effect of dehydroepiandrosterone replacement on lipoprotein profile in hypoadrenal women.

CONTEXT: Levels of dehydroepiandrosterone (DHEA) and its sulfate form (DHEAS) are inversely associated with cardiovascular mortality in men but not women. Very little evidence is available on the impact of DHEA administration on lipoprotein profile in women. DHEAS levels are very low/undetectable in hypoadrenal women. OBJECTIVE: The objective of the study was to determine the impact of DHEA replacement on lipoprotein profile in hypoadrenal women. DESIGN AND SETTING: A double-blind, randomized, placebo-controlled, cross-over design study was conducted at the Mayo Clinic. PARTICIPANTS: Thirty-three hypoadrenal Caucasian women (mean +/- sd; age 50.3 +/- 15.2 yr, body mass index 26.6 +/- 4.4 kg/m(2)) took part in the study. INTERVENTION: Study participants were assigned to receive either a placebo or 50 mg/d of DHEA for 3 months each. Lipid levels and lipoprotein profile were analyzed using the Lipo Science Lipoprotein nuclear magnetic resonance system. MAIN OUTCOME MEASURES: Changes in various lipoprotein sizes and levels were measured. RESULTS: The DHEA period had higher plasma DHEAS levels than during placebo (<0.3 +/- 0.0 vs. 3.5 +/- 1.3 nmol/liter, P < 0.001). DHEA replacement significantly reduced total cholesterol (20.0 vs. -22, P = 0.02) and high-density lipoprotein (HDL) levels (2.0 vs. -6.0, P = 0.006) and tends to reduce triglyceride and total low-density lipoprotein levels. Although, DHEA replacement had no effect on low-density lipoprotein particle size, it significantly reduced larger HDL particles and to modest extent small HDL particles. CONCLUSIONS: Our study findings showed that oral DHEA administration in hypoadrenal women results in an unfavorable lipoprotein profile. The results warrant long-term studies to determine the impact of DHEA replacement on cardiovascular risk.

Response of novel biomarkers to BNP infusion in patients with decompensated heart failure: a multimarker paradigm.

Multibiomarker paradigms have been proposed to diagnose, define progression, and to monitor therapy of heart failure (HF) patients. The aim of this study was to evaluate the prognostic and therapy-monitoring potential of four novel biomarkers (copeptin, midregional proatrial natriuretic peptide (MR-proANP), neopterin, and procalcitonin) which have been shown to be elevated in the plasma of patients with HF and reported to have prognostic value. In a prospective study of 40 patients hospitalized for decompensated HF and who received nesiritide infusions as part of their care, blood was drawn before, during, and postinfusion and assayed for the novel biomarkers. B-type natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) which were previously measured and reported in this cohort were also included in the analyses. All biomarkers were elevated at baseline prior to nesiritide infusion, but copeptin, MR-proANP, and NT-proBNP demonstrated significant acute reductions in plasma levels in response to therapy. Copeptin levels were higher in posthospital nonsurvivors and by proportional hazards model were associated with an increased mortality risk (p = 0.04). Procalcitonin and neopterin added no incremental information on response to therapy or risk stratification. In contrast, copeptin and MR-proANP appear to have potential for monitoring acute responses to therapy. Only copeptin and BNP contributed to risk stratification in this cohort of advanced HF patients, but the conjoint use of BNP or NT-proBNP does not appear to impact the prognostic value of copeptin alone. These results are hypothesis generating to stimulate additional investigation.

Aspirin responsiveness in healthy volunteers measured with multiple assay platforms.

BACKGROUND: We evaluated the sensitivity, precision, and concordance of 4 assays designed to detect aspirin responsiveness or resistance. METHODS: Twenty-nine healthy laboratory volunteers took 80 mg aspirin for 7 days, and a subset of volunteers took 325 mg aspirin for an additional 7 days. We measured platelet function by light transmission aggregometry with arachidonic acid, PFA-100, and VerifyNow. PFA-100 and VerifyNow assays were performed in duplicate to assess method imprecision. Some volunteers had samples taken within 2-4 h of the final dose of aspirin and again within 20-24 h of the final dose. We measured urinary 11-dehydro-thromboxane B(2) at baseline and after 80 or 325 mg aspirin. RESULTS: No volunteers were nonresponsive to aspirin therapy as measured by the PFA-100. One of 29 participants demonstrated lack of response to aspirin as measured by VerifyNow and urinary 11-dehydro-thromboxane B(2); 2 of 29 demonstrated lack of response as measured by light transmission aggregometry. Imprecision was <10% for the PFA-100 and VerifyNow. Concordance was high (>90%) between all assays. Neither aspirin dose (80 vs 325 mg) nor timing between final dose of aspirin and blood draw (2-4 vs 20-24 h) affected any of the assays. CONCLUSIONS: Light transmission aggregometry, PFA-100, VerifyNow, and urinary 11-dehydro-thromboxane B(2) are all sensitive to the effects of aspirin in healthy individuals. Variables such as aspirin dose, timing between final dose of aspirin and blood collection, and imprecision do not affect the ability of the assays to detect aspirin effect on platelet function.

Lipoprotein-associated phospholipase A2 is an independent marker for coronary endothelial dysfunction in humans.

OBJECTIVE: The purpose of the current study was to determine whether lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with coronary endothelial dysfunction and is a predictor of endothelial dysfunction in humans. METHODS AND RESULTS: Patients (172) with no significant coronary artery disease (<30% stenosis) undergoing assessment of coronary endothelial function were studied. Endothelial function was assessed by the change in coronary blood flow and coronary artery diameter in response to intracoronary acetylcholine. Plasma concentrations of Lp-PLA2 were measured, and patients were divided into tertiles. Patients in tertiles 2 and 3 had a significantly lower change in coronary blood flow (63.8+/-73.2 and 32.0+/-71.7 versus 78.4+/-73.2%; P<0.001) and greater epicardial coronary artery vasoconstriction (-14.1+/-14.7 and -23.3+/-25.1 versus -9.5+/-15.2% mean diameter change; P<0.001) in response to acetylcholine. Patients with coronary endothelial dysfunction had significantly higher serum concentrations of Lp-PLA2 than those with normal endothelial function (246.2+/-71.6 versus 209+/-56.7 ng/mL; P=0.001). The odds ratio for coronary endothelial dysfunction in patients with Lp-PLA2 in the highest tertile was 3.3 (95% CI, 1.6 to 6.6). CONCLUSIONS: Lp-PLA2 is independently associated with coronary artery endothelial dysfunction and is a strong predictor of endothelial dysfunction in humans.

Automated spectrophotometric analysis of mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts.

BACKGROUND: Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial. METHOD: We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities. RESULTS: A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RCC was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%-18%. CONCLUSIONS: With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.

Electrophoretic measurement of lipoprotein(a) cholesterol in plasma with and without ultracentrifugation: comparison with an immunoturbidimetric lipoprotein(a) method.

OBJECTIVES: Elevated plasma lipoprotein(a) [Lp(a)] is a significant risk factor for vascular disease. Standardization of Lp(a) mass measurement is complicated by the heterogeneity of apolipoprotein(a) [apo(a)]. We investigated whether Lp(a) cholesterol measurement, which is not influenced by apo(a) size, is a viable alternative to measuring Lp(a) mass. DESIGN AND METHODS: Plasma Lp(a) cholesterol was measured electrophoretically, with and without ultracentrifugation, and results were compared to each other and to immunoturbidimetrically measured Lp(a) mass in 470 subjects. RESULTS: Ultracentrifuged and whole plasma Lp(a) cholesterol levels demonstrated high correlation (R = 0.964). All samples with detectable (>/=2.0 mg/dl) Lp(a) cholesterol had Lp(a) mass >30 mg/dl (the clinically relevant cutpoint), while 59 samples with Lp(a) mass >30 mg/dl did not have detectable Lp(a) cholesterol. CONCLUSIONS: Lp(a) cholesterol can be measured in whole plasma without interference from VLDL lipoproteins. The relative clinical merits of measuring Lp(a) cholesterol vs. Lp(a) mass or both in combination deserves investigation.