Lipoprotein-associated phospholipase A2, a novel cardiovascular inflammatory marker, in HIV-infected patients.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an emerging biomarker of cardiovascular disease. This study was conducted to describe the distribution of Lp-PLA2 in a cohort of human immunodeficiency virus (HIV)-infected adults and to determine associations between Lp-PLA2, cardiometabolic risk factors, and subclinical atherosclerosis in this population. METHODS: Lp-PLA2 was assessed in 341 (25% women, 52% white, 74% on highly active antiretroviral therapy [HAART]) participants of a cohort with detailed characterization of atherogenic risk factors, including surrogate markers of carotid and coronary atherosclerosis. RESULTS: Mean Lp-PLA2 mass was 313 ± 105 ng/mL and activity 173 ± 49 nmol/minute/mL. Seventy-five percent of participants had abnormal Lp-PLA2. Those in the highest Framingham Risk Score tertile had significantly higher Lp-PLA2 activity. Participants with abnormal carotid intima-media thickness (cIMT) had higher Lp-PLA2 mass and activity. Those with coronary artery calcium (CAC) scores >100 had significantly higher Lp-PLA2 mass than those with lower or nondetectable calcium. Those on HAART and protease inhibitor (PI)-based treatment had significantly higher Lp-PLA2 mass and activity than those who were treatment-naive or not on PIs. In multivariate regression, HAART and PI use were positively associated with Lp-PLA2 activity and mass after adjusting for age, race, sex, low-density and high-density lipoprotein cholesterol levels, triglyceride level, and smoking. Adding Lp-PLA2 activity tertiles to the model improved the predictive value for abnormal common cIMT, but not internal cIMT or CAC score. CONCLUSIONS: Lp-PLA2 is highly abnormal in HIV-infected patients and is associated with several cardiovascular and HIV treatment-specific risk factors. Lp-PLA2 may be used as an additional and more vascular specific biomarker for cardiovascular risk stratification in HIV-positive patients.

The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor of cardiovascular disease. Plasma Lp-PLA2 is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA2. We determined the plasma levels of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA2 concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA2 [apoB/Lp-PLA2⁻] was calculated by subtracting the apoB/Lp-PLA2 from total apoB. The apoB/Lp-PLA2 levels were 3.6-fold higher, while apoB/Lp-PLA2⁻ were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA2 and apoB/Lp-PLA2⁻ levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA2, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA2⁻. Considering that Lp-PLA2 is proatherogenic, the predominance of apoB/Lp-PLA2 particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease.

Metabolomic signatures of aggressive prostate cancer.

BACKGROUND: Current diagnostic techniques have increased the detection of prostate cancer; however, these tools inadequately stratify patients to minimize mortality. Recent studies have identified a biochemical signature of prostate cancer metastasis, including increased sarcosine abundance. This study examined the association of tissue metabolites with other clinically significant findings. METHODS: A state of the art metabolomics platform analyzed prostatectomy tissues (331 prostate tumor, 178 cancer-free prostate tissues) from two independent sites. Biochemicals were analyzed by gas chromatography-mass spectrometry and ultrahigh performance liquid chromatography-tandem mass spectrometry. Statistical analyses identified metabolites associated with cancer aggressiveness: Gleason score, extracapsular extension, and seminal vesicle and lymph node involvement. RESULTS: Prostate tumors had significantly altered metabolite profiles compared to cancer-free prostate tissues, including biochemicals associated with cell growth, energetics, stress, and loss of prostate-specific biochemistry. Many metabolites were further associated with clinical findings of aggressive disease. Aggressiveness-associated metabolites stratified prostate tumor tissues with high abundances of compounds associated with normal prostate function (e.g., citrate and polyamines) from more clinically advanced prostate tumors. These aggressive prostate tumors were further subdivided by abundance profiles of metabolites including NAD+ and kynurenine. When added to multiparametric nomograms, metabolites improved prediction of organ confinement (AUROC from 0.53 to 0.62) and 5-year recurrence (AUROC from 0.53 to 0.64). CONCLUSIONS: These findings support and extend earlier metabolomic studies in prostate cancer and studies where metabolic enzymes have been associated with carcinogenesis and/or outcome. Furthermore, these data suggest that panels of analytes may be valuable to translate metabolomic findings to clinically useful diagnostic tests.

Relationship of lipoprotein-associated phospholipase A2 mass and activity with incident vascular events among primary prevention patients allocated to placebo or to statin therapy: an analysis from the JUPITER trial.

BACKGROUND: Although lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) levels are associated with cardiovascular events, Lp-PLA(2) is physically linked to LDL cholesterol (LDL-C). Whether measures of Lp-PLA(2) mass or activity continue to predict risk after LDL-C reduction by statin therapy is uncertain. METHODS: Lp-PLA(2) mass concentration and activity were evaluated at baseline and after treatment in the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial comparing rosuvastatin 20 mg to placebo among 17 802 men and women without cardiovascular disease or diabetes at study entry. The relationships of Lp-PLA(2) mass and activity with risk of future vascular events were evaluated in the placebo and rosuvastatin groups. RESULTS: Before randomization, levels of Lp-PLA(2) mass and activity correlated moderately with each other and with LDL-C. The magnitude of these correlations increased after statin therapy. Rosuvastatin reduced Lp-PLA(2) mass by 33.8%, Lp-PLA(2) activity by 33.2%, and LDL-C by 48.7% (all P < 0.0001). Among those study participants allocated to placebo, increasing quartiles of Lp-PLA(2) activity (P(trend) = 0.04) but not Lp-PLA(2) mass (P(trend) = 0.92) were associated with incident cardiovascular events after adjustment for LDL-C and conventional risk factors. Comparable analyses conducted among those allocated to rosuvastatin revealed no significant relationship between Lp-PLA(2) levels and subsequent vascular events. The ability of rosuvastatin to reduce vascular events was not significantly modified by baseline Lp-PLA(2) level. CONCLUSIONS: Among JUPITER trial participants allocated to placebo, levels of Lp-PLA(2) activity, but not mass, were associated with cardiovascular risk. However, Lp-PLA(2) no longer predicted risk or modified clinical outcomes when participants were treated with rosuvastatin.

Lipoprotein associated phospholipase A2 concentration predicts total and cardiovascular mortality independently of established risk factors (The Ludwigshafen Risk and Cardiovascular Health Study).

BACKGROUND: Lipoprotein-associated phospholipase A2 (LpPLA2) is a lipoprotein-bound enzyme involved in inflammation and atherosclerosis. This cohort study investigates LpPLA2 concentration to predict cardiovascular and total mortality in patients scheduled for coronary angiography. METHODS: LpPLA2 concentration was determined in 2298 patients with and in 661 patients without angiographically confirmed coronary artery disease (CAD). During the median observation period of 8.0 years 686 patients died. RESULTS: In patients with tertiles of LpPLA2 of 307 - 475 ng/mL, or > or = 475 ng/mL unadjusted hazard ratios (HR) for total mortality were 1.47 (95% CI 1.21 - 1.80; p < 0.001), and 1.63 (95% CI 135 - 1.97; p < 0.001), respectively, compared to patients with LpPLA2 < or = 307 ng/mL. HRs for cardiovascular death were 1.33 (95% CI 1.04 - 1.71; p = 0.026), and 1.59 (95% CI 1.26 - 2.02; p < 0.001), respectively. After accounting for established risk factors and including angiographic CAD status and high sensitivity C-reactive protein (hsCRP), the 3rd tertile of LpPLA2 concentration predicted death from all causes with a HR of 1.40 (95% CI 1.15 - 1.71; p = 0.001) and cardiovascular death with a HR of 1.35 (95% CI 1.05 - 1.73; p = 0.018). LpPLA2 increased the risk of cardiovascular death significantly even in individuals with high hsCRP. In patients with hsCRP > 33 mg/L and LpPLA2 > 392 ng/mL the risk of cardiovascular death was almost two-fold higher compared to patients with low hsCRP and low LpPLA2 with a HR of 1.98 (95% CI 1.50 - 2.62; p < 0.001). CONCLUSIONS: LpPLA2 concentration predicts risk for total and cardiovascular mortality independently from established risk factors and indicates risk for cardiovascular death even in patients with high hsCRP levels.

Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis.

Plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and oxidized low density lipoprotein (oxLDL) have been identified as risk factors for cardiovascular disease. Lp-PLA(2) is the sole enzyme responsible for the hydrolysis of oxidized phospholipids on LDL particles in atherosclerotic plaques. We have studied the relationship between Lp-PLA(2) and oxLDL in carotid endarterectomy (CEA) tissues and in matched plasmas. In extracts from CEA anatomical segments, the levels of oxLDL were significantly associated with the levels of Lp-PLA(2) protein (r = 0.497) and activity (r = 0.615). OxLDL and Lp-PLA(2) mass/activity were most abundant in the carotid bifurcation and internal segments where plaque was most abundant. In extracts from CEA atheroma, the levels of oxLDL and Lp-PLA(2) were significantly correlated (r = 0.634). In matched plasma and atheroma extracts, the levels of Lp-PLA(2) were negatively correlated (r = - 0.578). The ratio of Lp-PLA(2) to oxLDL was higher in atheromatous tissue (277:1) than in normal tissue (135:1) and plasma (13:1). Immunohistochemical experiments indicated that in plaques, oxLDL and Lp-PLA(2) existed in overlapping but distinctly different distribution. Fluorescence microscopy showed both oxLDL and Lp-PLA(2) epitopes on the same LDL particle in plasma but not in plaque. These results suggest that the relationship between Lp-PLA(2) and oxLDL in the atherosclerotic plaque is different from that in the plasma compartment.

Field Test and Validation of the Multiplier Measles, Mumps, Rubella, and Varicella-Zoster Multiplexed Assay System in the Democratic Republic of the Congo by Using Dried Blood Spots.

Here we describe baseline validation studies and field performance of a research-use-only chemiluminescent multiplex serology panel for measles, mumps, rubella, and varicella-zoster virus used with dried blood spots in support of the 2013-2014 Democratic Republic of the Congo Demographic and Health Survey. Characterization of the panel using U.S. FDA-cleared commercial kits shows good concordance for measles, mumps, rubella, and varicella-zoster with average sensitivity across assays of 94.9% and an average specificity of 91.4%. As expected, performance versus available standards validated for plaque-reduction assays does not provide a 1:1 correspondence with international units and yet demonstrates excellent linearity (average Hill's slope = 1.02) and ∼4 logs of dynamic range. In addition, for the four assays, the multiplexed format allowed for inclusion of three positive and two negative controls for each sample. A prototype Dynex Multiplier chemiluminescent automated immunoassay instrument with a charge-coupled device camera provided a rugged and robust processing and data acquisition platform. Performance of a multiplex instrument for serological testing in a substantially resource-limited environment shows excellent reproducibility, minimal cross-reactivity, and a clear discrimination between specific assays and should be considered a viable option for future serosurveys.IMPORTANCE The critical evaluation of immunization programs is key to identifying areas of suboptimal vaccination coverage, monitoring activities, and aiding development of public health policy. For evaluation of vaccine effectiveness, direct antibody binding assay methods, including enzyme immunoassay, enzyme-linked fluorescence assays, and indirect immunofluorescence assay, are most commonly used for detection of IgG antibodies. However, despite their well-demonstrated, reliable performance, they can be labor-intensive and time-consuming and require separate assays for each individual marker. This necessitates increased sample volumes, processing time, and personnel, which may limit assessment to a few key targets in resource-limited settings, that is, low- and middle-income locations where funding for public health or general infrastructure that directly impacts public health is restricted, limiting access to equipment, infrastructure, and trained personnel. One solution is a multiplexed immunoassay, which allows for the detection of multiple analytes in a single reaction for increased efficiency and rapid surveillance of infectious diseases in limited-resource settings. Thus, the scope of the project precluded a full validation, and here we present abbreviated validation studies demonstrating adequate sensitivity, specificity, and reproducibility.

Differential effect of hypolipidemic drugs on lipoprotein-associated phospholipase A2.

OBJECTIVE: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor for incident atherosclerotic disease. We investigated the effect of 3 hypolipidemic drugs that exert their action through different mechanisms on plasma and lipoprotein-associated Lp-PLA2 activity and mass. METHODS AND RESULTS: In 50 patients with Type IIA dyslipidemia were administered rosuvastatin (10 mg daily), whereas in 50 Type IIA dyslipidemic patients exhibiting intolerance to previous statin therapy were administered ezetimibe as monotherapy (10 mg daily). Fifty patients with Type IV dyslipidemia were given micronised fenofibrate (200 mg daily). Low- and high-density lipoprotein (LDL and HDL, respectively) subclass analysis was performed electrophoretically, whereas lipoprotein subfractions were isolated by ultracentrifugation. Ezetimibe reduced plasma Lp-PLA2 activity and mass attributable to the reduction in plasma levels of all LDL subfractions. Rosuvastatin reduced enzyme activity and mass because of the decrease in plasma levels of all LDL subfractions and especially the Lp-PLA2 on dense LDL subfraction (LDL-5). Fenofibrate preferentially reduced the Lp-PLA2 activity and mass associated with the VLDL+IDL and LDL-5 subfractions. Among studied drugs only fenofibrate increased HDL-associated Lp-PLA2 (HDL-Lp-PLA2) activity and mass attributable to a preferential increase in Lp-PLA2 associated with the HDL-3c subfraction. CONCLUSIONS: Ezetimibe, rosuvastatin, and fenofibrate reduce Lp-PLA2 activity and mass associated with the atherogenic apoB-lipoproteins. Furthermore, fenofibrate improves the enzyme specific activity on apoB-lipoproteins and induces the HDL-Lp-PLA2. The clinical implications of these effects remain to be established.

A multiparametric panel for ovarian cancer diagnosis, prognosis, and response to chemotherapy.

PURPOSE: Our goal was to examine a panel of 11 biochemical variables, measured in cytosolic extracts of ovarian tissues (normal, benign, and malignant) by quantitative ELISAs for their ability to diagnose, prognose, and predict response to chemotherapy of ovarian cancer patients. EXPERIMENTAL DESIGN: Eleven proteins were measured (9 kallikreins, B7-H4, and CA125) in cytosolic extracts of 259 ovarian tumor tissues, 50 tissues from benign conditions, 35 normal tissues, and 44 tissues from nonovarian tumors that metastasized to the ovary. Odds ratios and hazard ratios and their 95% confidence interval were calculated. Time-dependent receiver operating characteristic curves for censored survival data were used to evaluate the performance of the biomarkers. Resampling was used to validate the performance. RESULTS: Most biomarkers effectively separated cancer from noncancer groups. A composite marker provided an area under the curve of 0.97 (95% confidence interval, 0.95-0.99) for discriminating normal and cancer groups. Univariately, hK5 and hK6 were positively associated with progression. After adjusting for clinical variables in multivariate analysis, both hK10 and hK11 significantly predicted time to progression. Increasing levels of hK13 were associated with chemotherapy response, and the predictive power of hK13 to chemotherapy response was improved by a panel of five biomarkers. CONCLUSIONS: The evidence shows that a group of kallikreins and multiparametric combinations with other biomarkers and clinical variables can significantly assist with ovarian cancer classification, prognosis, and response to platinum-based chemotherapy. In particular, we developed a multiparametric strategy for predicting ovarian cancer response to chemotherapy, comprising several biomarkers and clinical features.

B7-h4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer.

Using cDNA database mining strategies and real-time quantitative reverse transcription-PCR, we identified B7-H4 as a novel gene that is overexpressed in ovarian and breast cancer tissues when compared with normal tissues. The gene encodes a protein of 282 amino acids with a signal sequence, an immunoglobulin domain, and a COOH-terminal hydrophobic transmembrane domain. Immunohistochemistry experiments show plasma membrane staining in serous ovarian and breast cancer, confirming the tissue specificity and cell surface localization. We have developed a sensitive dual monoclonal antibody sandwich ELISA to analyze the level of B7-H4 protein in >2,500 serum samples, ascites fluids, and tissue lysates. High levels of B7-H4 protein were detected in ovarian cancer tissue lysates when compared with normal tissues. B7-H4 was present at low levels in all sera but showed an elevated level in serum samples from ovarian cancer patients when compared with healthy controls or women with benign gynecologic diseases. The median B7-H4 concentration in endometrioid and serous histotypes was higher than in mucinous histotypes, consistent with results of immunohistochemical staining. The multivariate logistic regression analysis of B7-H4 and CA125 measured in the same sample set resulted in an area under the curve (AUC) of 0.86 for all stages and 0.86 for stage I/II patients, which was significantly higher than the AUC for either marker alone. In early-stage patients, the sensitivity at 97% specificity increased from 52% for CA125 alone to 65% when used in combination with B7-H4. We conclude that B7-H4 is a promising new biomarker for ovarian carcinoma.

Biochemical differences in the mass and activity tests of lipoprotein-associated phospholipase A2 explain the discordance in results between the two assay methods.

OBJECTIVES: There are two platforms for the detection of Lp-PLA2 in sera or plasmas: by its enzymatic activity (PLAC® activity test) and by its mass concentration (PLAC® mass test). It has been long recognized that these two platforms are not correlated well. The underlying cause for this is therefore investigated by the biochemical characterization of the two PLAC tests. DESIGN & METHODS: Human sera with and without the treatment by detergent were fractionated by using a Superose-6 column in phosphate buffered saline and the phospholipid associated Lp-PLA2 was assessed by both PLAC mass and activity tests. The Lp-PLA2 values of the two PLAC tests were compared under such conditions. RESULTS: Fractionation of sera and plasmas indicates that the association of Lp-PLA2 with phospholipids, especially LDL and other large size phospholipid vesicles, may block the detection of the enzyme by antibodies in the immunoassay format under the conditions of the PLAC mass test. Inclusion of high concentration (>CMC, critical micelle concentration) of detergents in the assay buffer of PLAC mass test dissociates Lp-PLA2 from phospholipid vesicles and results in the full detection of all Lp-PLA2 in sera or plasmas for concentration. Such assay modification significantly improves the correlation between the PLAC mass and PLAC activity tests. CONCLUSIONS: PLAC mass test only detects a small portion of the total Lp-PLA2, mainly the Lp-PLA2 associated with HDL. This is the main cause of the discordance and poor correlation between the PLAC mass and activity tests. Our results demonstrate the PLAC activity test is more accurate in assessing the total level of circulating Lp-PLA2.

Lipoprotein-associated phospholipase A2 independently predicts the angiographic diagnosis of coronary artery disease and coronary death.

BACKGROUND: Whereas C-reactive protein (CRP) is a nonspecific marker of coronary artery disease (CAD) and cardiovascular (CV) events, Lp-PLA2 may be a nonvariable inflammatory biomarker. We evaluated the independent association of lipoprotein-associated phospholipase A2 (Lp-PLA2) to angiographic CAD and CV events adjusting for standard factors, lipids, and CRP. METHODS: Lipoprotein-associated phospholipase A2 (PLAC test, diaDexus, Inc, San Francisco, CA) and CRP were measured from samples donated by consecutive consenting patients (N = 1493) enrolled in the registry of the Intermountain Heart Collaborative Study. All patients underwent coronary angiography (1996-1998) for CAD determination and were followed for 6.7 +/- 0.5 years (range 5.7-7.9 years) for CV events (death [including all-cause, CAD, and non-CAD CV death], myocardial infarction, and cerebrovascular accident). RESULTS: Lipoprotein-associated phospholipase A2 weakly correlated with lipids (low-density lipoprotein: r = 0.22, P < .001; high-density lipoprotein: r = -0.13, P < .001), but not CRP (r = 0.03, P = .26). Increasing quartile (Q) of Lp-PLA2 predicted greater the presence of CAD (vs Q1) for Q2 (adjusted odds ratio [OR] 1.15, 95% CI 0.78-1.71, P = .48), for Q3 (OR 1.53, 95% CI 1.02-2.31, P = .042), and for Q4 (OR 2.44, 95% CI 1.58-3.79, P < .001), although CRP was also predictive (vs Q1, Q2: OR 1.47, P = .057; Q3: OR 1.93, P = .002; Q4: OR 3.43, P < .001). In Cox regression, Lp-PLA2 predicted CAD death (vs Q1; Q2: adjusted hazard ratio [HR] 1.27, 95% CI 0.58-2.78, P = .55; Q3: HR 2.18, 95% CI 1.04-4.57, P = .04; Q4: HR 1.73, 95% CI 0.84-3.61, P = .14). CONCLUSION: Lipoprotein-associated phospholipase A2 was confirmed to predict the presence of CAD, even among patients undergoing coronary angiography. Uniquely, Lp-PLA2 predicted the risk of CAD death, but not all-cause death, myocardial infarction, or cerebrovascular accident.

Lipoprotein-associated phospholipase A2: review and recommendation of a clinical cut point for adults.

Recently, several epidemiologic studies have demonstrated an association between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) concentration and risk of subsequent cardiovascular events. Several major commercial and reference laboratories across the United States are now offering Lp-PLA2 testing for clinical use to evaluate cardiovascular risk and as a guide to intensity of therapy in individuals at intermediate risk for developing coronary heart disease. Each laboratory has established its own cut points, or "decision values," for Lp-PLA2, which vary from the 50th to the 95th percentile values of individual populations tested at each site. Uniform reporting of cut points has not been achieved. The purpose of this manuscript is to recommend appropriate decision values for Lp-PLA2, endorsed by a consensus panel of laboratorians and clinicians from the major laboratories where the test is performed. These coauthors possess considerable experience with assessment of cardiovascular risk marker decision values in general and are familiar with the validation of the Lp-PLA2 immunoassay and the Lp-PLA2 clinical studies conducted thus far. An ideal risk marker, studied in an ideal population, might yield a consistent cut point associated with a sudden increase in cardiovascular risk. While acknowledging that additional studies will be required to test and refine the recommended decision value, this article reviews the most current information with which to provide guidance to practicing clinicians regarding Lp-PLA2 levels. Since several studies have demonstrated increased risk associated with the second and third tertiles vs. the first tertile for Lp-PLA2, the 50th percentile cut point (235 ng/mL) is recommended as a conservative cut point associated with increased risk for cardiovascular disease. This cut point is not proposed as a treatment target, but rather as a level above which clinicians should consider a patient to be at higher risk for cardiovascular events, independent of established risk factors, high- and low-density lipoprotein cholesterol, and high-sensitivity C-reactive protein.

The effect of statin therapy on lipoprotein associated phospholipase A2 levels.

Lipoprotein associated phospholipase A2 (Lp-PLA2), a biomarker of oxidation and inflammation, has been associated with increased coronary heart disease (CHD) risk. To date, data examining the effect of HMG CoA reductase inhibitors on Lp-PLA2 are few. We evaluated the effect of pravastatin 40 mg daily versus placebo on Lp-PLA2 levels among 481 subjects free of cardiovascular disease (pravastatin N=246 and placebo N=235) who participated in the Pravastatin Inflammation/CRP Study (PRINCE). After 12 weeks, Lp-PLA2 levels decreased by 22.1% among pravastatin treated participants and by 7.8% among those randomized to placebo (p<0.001). These results were similar in all subgroups evaluated according to age, blood pressure, lipid parameters, diabetic status, smoking status, aspirin use, body mass index and gender. There were correlations between change in Lp-PLA2 levels and baseline Lp-PLA2 levels (r=-0.63, p<0.001), total cholesterol change (r=-0.26, p<0.001), LDL-C change (r=-0.32, p<0.001) and C-reactive protein (CRP) change (r=-0.13, p=0.05). Multivariate linear regression models that assessed the relationship between the log difference in Lp-PLA2 at 12 weeks and treatment revealed a beta-coefficient of 0.15 for the treatment variable (p<0.01). However, adjustment for change in LDL-C substantially attenuated the beta-coefficient associated with treatment to 0.07 (P<0.005) and after additional control for other potential confounders, the effect of treatment was no longer significant. Thus, Lp-PLA2 levels were significantly reduced at 12 weeks by pravastatin, an effect that was significantly related to LDL cholesterol reduction accounting for about 6% of the variability in this response. Moreover, pravastatin induced reduction in Lp-PLA2 was no longer significant after taking the latter into account.

Metabolomics and its Application to the Development of Clinical Laboratory Tests for Prostate Cancer.

INTRODUCTION: There is a critical need to develop clinical laboratory assays that provide risk assessment for men at elevated risk for prostate cancer, and once diagnosed, could further identify those men with clinically significant disease. METHODS: Recent advancements in analytical instrumentation have enabled mass spectrometry-based metabolomics methodologies. Further advancements in chromatographic techniques have facilitated high throughput, quantitative assays for a broad spectrum of biochemicals. RESULTS: Screening metabolomics techniques have been applied to biospecimens from large cohorts of men comparing those individuals with prostate cancer to those with no evidence of malignancy. Work beginning in tissues has identified biochemical profiles that correlate with disease and disease severity, including tumor grade and stage. Some of these metabolic abnormalities, such as dramatic elevations in sarcosine, have been found to translate into biological fluids, especially blood and urine, which can be sampled in a minimally invasive manner. DISCUSSION: The differential abundances of these tumor-associated metabolites have been found to improve the performance of clinical prognostic/diagnostic tools. CONCLUSION: The outlook is bright for metabolomic technology to address clinical diagnostic needs for prostate cancer patient management. Early validation of specific clinical tests provides a preview of further successes in this area. Metabolomics has shown its utility to complement and augment traditional clinical approaches as well as emerging genomic, transcriptomic and proteomic methodologies, and is expected to play a key role in the precision medicine-based management of the prostate cancer patient.

A novel test for IGT utilizing metabolite markers of glucose tolerance.

The oral glucose tolerance test (OGTT) is the only method to diagnose patients having impaired glucose tolerance (IGT), but its use has diminished considerably in recent years. Metabolomic profiling studies have identified a number of metabolites whose fasting levels are associated with dysglycemia and type 2 diabetes. These metabolites may serve as the basis of an alternative test for IGT. Using the stable isotope dilution technique, quantitative assays were developed for 23 candidate biomarker metabolites. These metabolites were measured in fasting plasma samples taken just prior to an OGTT from 1623 nondiabetic subjects: 955 from the Relationship between Insulin Sensitivity and Cardiovascular Disease Study (RISC Study; 11.7% IGT) and 668 subjects from the Diabetes Mellitus and Vascular Health Initiative (DMVhi) cohort from the DEXLIFE project (11.8% IGT). The associations between metabolites, anthropometric, and metabolic parameters and 2hPG values were assessed by Pearson correlation coefficients and Random Forest classification analysis to rank variables for their ability to distinguish IGT from normal glucose tolerance (NGT). Multivariate logistic regression models for estimating risk of IGT were developed and evaluated using AUCs calculated from the corresponding ROC curves. A model based on the fasting plasma levels of glucose, α-hydroxybutyric acid, ß-hydroxybutyric acid, 4-methyl-2-oxopentanoic acid, linoleoylglycerophosphocholine, oleic acid, serine and vitamin B5 was optimized in the RISC cohort (AUC = 0.82) and validated in the DMVhi cohort (AUC = 0.83). A novel, all-metabolite-based test is shown to be a discriminate marker of IGT. It requires only a single fasted blood draw and may serve as a more convenient surrogate for the OGTT or as a means of identifying subjects likely to be IGT.

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis.

Kidney cancer [or renal cell carcinoma (RCC)] is known as "the internist's tumor" because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel-Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the ß-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Together, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC.

Lp-PLA2: an emerging biomarker of coronary heart disease.

Cardiovascular disease is the leading cause of death in most industrialized countries. However, the diagnosis and management of coronary heart disease is far from optimal. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase, is an enzyme that hydrolyses oxidized phospholipids and is primarily associated with low-density lipoprotein. Discussed in this review is the accumulating evidence supporting the view that Lp-PLA2 is a potential biomarker of coronary heart disease and plays and an important proinflammatory role in the progression of atherosclerosis. A new ELISA method for the quantitative measurement of Lp-PLA2 mass in human plasma developed by diaDexus, Inc. is presented. Furthermore, potential clinical applications of Lp-PLA2 mass measurements are proposed.

Lipoprotein-associated phospholipase A2 is associated with atherosclerotic stroke risk: the Northern Manhattan Study.

BACKGROUND: Lipoprotein-associated phospholipase A2 (LpPLA2) levels are associated with stroke, though whether this extends to all populations and stroke subtypes is unknown. METHODS: Serum samples from stroke-free community participants in the Northern Manhattan Study were assayed for LpPLA2 mass and activity. Participants were followed annually for stroke. Cox-proportional-hazard models were fitted to estimate hazard-ratios and 95% confidence intervals (HR, 95% CI) for the association of LpPLA2 levels with ischemic stroke (IS), after adjusting for demographic and medical risk factors. RESULTS: Serum samples were available in 1946 participants, of whom 151 (7.8%) experienced a first IS during median follow-up 11 years. Mean age was 69 (SD 10), 35.6% were men, 20% non-Hispanic Whites, 22% non-Hispanic Blacks, and 55% Hispanics. LpPLA2 mass and activity levels were not associated with overall IS risk. LpPLA2 mass but not activity levels were associated with strokes due to large artery atherosclerosis (LAA; adjusted HR per SD 1.55, 95% CI 1.17-2.04). There was a dose-response relationship with LAA (compared to first quartile, 2nd quartile HR = 1.43, 95% CI 0.23-8.64; 3rd quartile HR = 4.47, 95% CI 0.93-21.54; 4th quartile HR = 5.07, 95% CI 1.07-24.06). The associations between LpPLA2-mass and LAA-stroke risk differed by race-ethnicity (p = 0.01); LpPLA2-mass was associated with increased risk of LAA among non-Hispanic Whites (adjusted HR per SD 1.44, 95% CI 0.98-2.11), but not other race-ethnic groups. CONCLUSION: LpPLA2-mass levels were associated with risk of atherosclerotic stroke among non-Hispanic White participants, but not in other race-ethnic groups in the cohort. Further study is needed to confirm these race-ethnic differences and the reasons for them.