Development of a fast liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in murine microdialysate.

The continuous measurement of multiple neurotransmitters in microdialysate of freely moving mice to study neurochemical changes in specific brain regions requires a rapid and very sensitive quantitative analytical method. The quantitative analysis of 11 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (L-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), acetylcholine (ACh), choline (Ch), and γ-aminobutyric acid (GABA), was performed using a biphenyl column coupled to an API-QTrap 3200 (AB SCIEX) mass spectrometer in positive electrospray ionization mode. To the microdialysate samples, 0.5 ng of isotopically labeled standard was added for analyte quantification. A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous analysis of monoamines, their precursor, and metabolites, as well as ACh, Ch, and GABA in murine microdialysate within 7.0 min. The limit of detection in artificial CSF ranged from 0.005 ng/mL (ME) to 0.75 ng/mL (NE and GABA). A comprehensive pre-analytical protocol was validated. Recovery was between 87 and 117% for neurotransmitter concentrations from 0.6 to 45 ng/mL with an inter-day accuracy of below 20%. Basal neurotransmitter values were determined in the striatum of mice over a time period of 3 h. This LC-MS/MS method, including a short and gentle sample preparation, is suitable for simultaneous measurements of neurotransmitters in murine cerebral microdialysate and enables the determination of basal neurotransmitter levels in specific brain regions to detect disease-related and drug-induced neurochemical changes.Graphical abstract.

Integration of Genome-Wide SNP Data and Gene-Expression Profiles Reveals Six Novel Loci and Regulatory Mechanisms for Amino Acids and Acylcarnitines in Whole Blood.

Profiling amino acids and acylcarnitines in whole blood spots is a powerful tool in the laboratory diagnosis of several inborn errors of metabolism. Emerging data suggests that altered blood levels of amino acids and acylcarnitines are also associated with common metabolic diseases in adults. Thus, the identification of common genetic determinants for blood metabolites might shed light on pathways contributing to human physiology and common diseases. We applied a targeted mass-spectrometry-based method to analyze whole blood concentrations of 96 amino acids, acylcarnitines and pathway associated metabolite ratios in a Central European cohort of 2,107 adults and performed genome-wide association (GWA) to identify genetic modifiers of metabolite concentrations. We discovered and replicated six novel loci associated with blood levels of total acylcarnitine, arginine (both on chromosome 6; rs12210538, rs17657775), propionylcarnitine (chromosome 10; rs12779637), 2-hydroxyisovalerylcarnitine (chromosome 21; rs1571700), stearoylcarnitine (chromosome 1; rs3811444), and aspartic acid traits (chromosome 8; rs750472). Based on an integrative analysis of expression quantitative trait loci in blood mononuclear cells and correlations between gene expressions and metabolite levels, we provide evidence for putative causative genes: SLC22A16 for total acylcarnitines, ARG1 for arginine, HLCS for 2-hydroxyisovalerylcarnitine, JAM3 for stearoylcarnitine via a trans-effect at chromosome 1, and PPP1R16A for aspartic acid traits. Further, we report replication and provide additional functional evidence for ten loci that have previously been published for metabolites measured in plasma, serum or urine. In conclusion, our integrative analysis of SNP, gene-expression and metabolite data points to novel genetic factors that may be involved in the regulation of human metabolism. At several loci, we provide evidence for metabolite regulation via gene-expression and observed overlaps with GWAS loci for common diseases. These results form a strong rationale for subsequent functional and disease-related studies.

Cholesterol esterification in plasma as a biomarker for liver function and prediction of mortality.

BACKGROUND: Advanced stages of liver cirrhosis lead to a dramatically increased mortality. For valid identification of these patients suitable biomarkers are essential. The most important biomarkers for liver function are bilirubin and prothrombin time expressed as International Normalized Ratio (INR). However, the influence of several anticoagulants on the prothrombin time limits its diagnostic value. Aim of this study was the evaluation of cholesterol esterification (CE) fraction (esterified cholesterol vs. total cholesterol) as an alternative biomarker for liver synthesis and mortality prediction. Under physiological conditions the CE fraction in blood is closely regulated by lecithin-cholesterol acyltransferase (LCAT) which is produced in the liver. METHODS: One hundred forty-two patients with liver disease clinically considered for orthotopic liver transplant for different indications were enrolled in the study. One patient was excluded because of the intake of a direct oral factor Xa inhibitor which has a strong impact on prothrombin time. RESULTS: Results of CE fraction were in good agreement with INR (R2 = 0.73; p < 0.001). In patients who died or survived within three months mean CE fraction was 56% vs. 74% (p < 0.001) and mean INR was 2.0 vs. 1.3 (p < 0.001), respectively. The predictive value of CE fraction for three-month mortality risk was higher compared to INR (p = 0.04). Results for one-year mortality were comparable. CONCLUSIONS: The cholesterol esterification fraction is a valid biomarker for liver synthesis and allows reliable prediction of mortality. In contrast to INR, it is independent of anticoagulation and other analytical limitations of coagulation tests.

Prediagnostic immunoglobulin E levels and risk of chronic lymphocytic leukemia, other lymphomas and multiple myeloma-results of the European Prospective Investigation into Cancer and Nutrition.

Previous epidemiological studies suggest an inverse association between allergies, marked by elevated immunoglobulin (Ig) E levels, and non-Hodgkin lymphoma (NHL) risk. The evidence, however, is inconsistent and prospective data are sparse. We examined the association between prediagnostic total (low: <20; intermediate: 20-100; high >100 kU/l) and specific IgE (negative: <0.35; positive ≥0.35 kU/I) concentrations against inhalant antigens and lymphoma risk in a study nested within the European Prospective Investigation into Cancer and Nutrition cohort. A total of 1021 incident cases and matched controls of NHL, multiple myeloma (MM) and Hodgkin lymphoma with a mean follow-up time of 7 years were investigated. Multivariate-adjusted odds ratios (ORs) with 95% confidence intervals (CI) were calculated by conditional logistic regression. Specific IgE was not associated with the risk of MM, B-cell NHL and B-cell NHL subtypes. In contrast, total IgE levels were inversely associated with the risk of MM [high level: OR = 0.40 (95% CI = 0.21-0.79)] and B-cell NHL [intermediate level: OR = 0.68 (95% CI = 0.53-0.88); high level: OR = 0.62 (95% CI = 0.44-0.86)], largely on the basis of a strong inverse association with chronic lymphocytic leukemia [CLL; intermediate level: OR = 0.49 (95% CI = 0.30-0.80); high level: OR = 0.13 (95% CI = 0.05-0.35)] risk. The inverse relationship for CLL remained significant for those diagnosed 5 years after baseline. The findings of this large prospective study demonstrated significantly lower prediagnostic total IgE levels among CLL and MM cases compared with matched controls. This corresponds to the clinical immunodeficiency state often observed in CLL patients prior to diagnosis. No support for an inverse association between prediagnostic levels of specific IgE and NHL risk was found.

Prediagnostic plasma testosterone, sex hormone-binding globulin, IGF-I and hepatocellular carcinoma: etiological factors or risk markers?

Elevated prediagnostic testosterone and insulin-like growth factor I (IGF-I) concentrations have been proposed to increase risk of hepatocellular carcinoma (HCC). However, the metabolism of these hormones is altered as a consequence of liver damage and they may have clinical utility as HCC risk markers. A case-control study was nested within the European Prospective Investigation into Cancer and Nutrition cohort and included 125 incident HCC cases and 247 individually matched controls. Testosterone, sex hormone-binding globulin (SHBG) and IGF-I were analyzed by immunoassays. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by conditional logistic regression. The area under the receiver operating curves (AUC) was calculated to assess HCC predictive ability of the tested models. After adjustments for epidemiological variables (body mass index, smoking, ethanol intake, hepatitis and diabetes) and liver damage (a score based on albumin, bilirubin, aspartate aminotransaminase, alanine aminotransaminase, gamma-glutamyltransferase and alkaline phosphatase concentrations), only SHBG remained significantly associated with risk [OR for top versus bottom tertile of 3.86 (1.32-11.3), p(trend) = 0.009]. As a single factor SHBG had an AUC of 0.81 (0.75-0.86). A small, but significant increase in AUC was observed when SHBG was added to a model including the liver damage score and epidemiological variables (from 0.89 to 0.91, p = 0.02) and a net reclassification of 0.47% (0.45-0.48). The observed associations of HCC with prediagnostic SHBG, free testosterone and IGF-I concentrations are in directions opposite to that expected under the etiological hypotheses. SHBG has a potential to be tested as prediagnostic risk marker for HCC.

Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma.

BACKGROUND: Liquid biobanking is an important tool for laboratory diagnostics in routine settings and clinical studies. However, the current knowledge about adequate storage conditions for different classes of biomarkers is incomplete and, in part, contradictory. Here, we performed a comprehensive study on the effects of different storage conditions on the stability of various biomarkers in human serum and plasma. METHODS: Serum and citrated plasma were aliquoted and stored at 4 °C, -20 °C, -80 °C, and <-130 °C for 0, 7, 30, and 90 days, respectively (5-10 pools/condition). Additionally, frozen aliquots were temporarily exposed to higher temperatures during storage to simulate removing individual samples. Stability was tested for 32 biomarkers from 10 different parameter classes (electrolytes, enzymes, metabolites, inert proteins, complement factors, ketone bodies, hormones, cytokines, coagulation factors, and sterols). RESULTS: Biobanking at -80 °C and <-130 °C for up to 90 days did not lead to substantial changes (defined as >3 interassay coefficients of variation and p<0.01) of any biomarker concentration. In contrast, storage at 4 °C and -20 °C induced substantial changes in single biomarker concentrations in most classes. Such substantial changes were increases (<20%) in electrolytes, metabolites, and proteins, and decreases (<96%) in enzymes, ketone bodies, cytokines, and coagulation factors. Biomarker stability was minimally affected by occasional short-term thermal exposure. CONCLUSIONS: Based on these results, we provide recommendations for storage conditions of up to 90 days for several biomarkers. Generally, storage at ≤-80 °C for at least 90 days including occasional short-term thermal exposure is an excellent storage condition for most biomarkers.

Does the Quantification of Δ9-Tetrahydrocannabinolic Acid A in Serum/Plasma Provide Any Additional Information About Consumption Pattern from Drivers Under the Influence of Cannabis?

Introduction: Δ9-tetrahydrocannabinolic acid A (THCA-A) is one of the main ingredients of cannabis plants and is converted to the psychoactive substance Δ9-tetrahydrocannabinol (THC) by decarboxylation during heating above ∼90°C. During the consumption of cannabis, a varying proportion of THCA-A is absorbed into the body. Therefore, the quantification of THCA-A in serum/plasma might provide additional information on consumption behavior in driving under the influence of cannabis cases. Materials and Methods: In this study, an already established gas-chromatography mass-spectrometry (GC-MS) method for the quantification of THC, 11-OH-THC, and THC-COOH in serum and plasma samples was extended to include THCA-A. This validated method was then applied to 1228 routinely achieved serum/plasma samples from drivers suspected of cannabis consumption in Western Saxony. Two different grouping systems for chronic/occasional consumption, one system for acute/subacute consumption, Huestis formulas, and the cannabis influence factor (CIF) were used for evaluation. Results: Method validation showed appropriate results for forensic toxicological routine analysis. Limit of detection and lower limit of quantification (LLOQ) for THCA-A were 0.3 and 1.0 ng/mL, respectively. Reproducibility was <11% and accuracy ranged between 104% and 107%. THCA-A was stable in native samples at least for 2 weeks at room temperature or 4°C as well as 1 month at -20°C. Freeze-thaw stability for three cycles and processed sample stability over 3 days was proven. A total of 865 cases with a THC concentration above the German analytical cutoff of 1 ng/mL as well as the analytical LLOQs of 0.9 and 2.5 ng/mL for 11-OH-THC and THC-COOH, respectively, were included in further statistical analysis. In 407 (47.1%) of these samples, THCA-A was quantifiable. Different statistical analyses indicated a correlation between THCA-A and THC concentrations in cases of chronic and acute consumption. In addition, an increase of chronic and acute cases with increasing THCA-A concentrations was observed. However, no correlation between THCA-A and CIF was found. Discussion: These data show that THCA-A might be an additional indicative marker to provide information about consumption frequency and acuteness. Additional studies with known consumption frequencies and times are required to verify these findings.

Plasma 25-hydroxyvitamin D and the risk of breast cancer in the European prospective investigation into cancer and nutrition: a nested case-control study.

Experimental evidence suggests that vitamin D might play a role in the development of breast cancer. Although the results of case-control studies indicate that circulating 25-hydroxyvitamin D [25(OH)D] is inversely associated with the risk of breast cancer, the results of prospective studies are inconsistent. A case-control study embedded in the European Prospective Investigation into Cancer and Nutrition (EPIC) was carried out comprising 1,391 incident breast cancer cases and 1,391 controls. Multivariable conditional logistic regression models did not reveal a significant overall association between season-standardized 25(OH)D levels and the risk of breast cancer (ORQ4-Q1 [95% CI]: 1.07 [0.85-1.36], ptrend = 0.67). Moreover, 25(OH)D levels were not related to the risks of estrogen receptor positive tumors (ORQ4-Q1 [95% CI]: 0.97 [0.67-1.38], ptrend = 0.90) and estrogen receptor negative tumors (ORQ4-Q1 [95% CI]: 0.97 [0.66-1.42], ptrend = 0.98). In hormone replacement therapy (HRT) users, 25(OH)D was significantly inversely associated with incident breast cancer (ORlog2 [95% CI]: 0.62 [0.42-0.90], p = 0.01), whereas no significant association was found in HRT nonusers (ORlog2 [95% CI]: 1.14 [0.80-1.62], p = 0.48). Further, a nonsignificant inverse association was found in women with body mass indices (BMI) < 25 kg/m(2) (ORlog2 [95% CI]: 0.83 [0.67-1.03], p = 0.09), as opposed to a borderline significant positive association in women with BMI ≥ 25 kg/m(2) (ORlog2 [95% CI]: 1.30 [1.0-1.69], p = 0.05). Overall, prediagnostic levels of circulating 25(OH)D were not related to the risk of breast cancer in the EPIC study. This result is in line with findings in the majority of prospective studies and does not support a role of vitamin D in the development of breast cancer.

Hormonal, metabolic, and inflammatory profiles and endometrial cancer risk within the EPIC cohort--a factor analysis.

A "Western" lifestyle characterized by physical inactivity and excess weight is associated with a number of metabolic and hormonal dysregulations, including increased circulating estrogen levels, hyperinsulinemia, hyperglycemia, and chronic inflammation. The same hormonal and metabolic axes might mediate the association between this lifestyle and the development of endometrial cancer. Using data collected within the European Prospective Investigation into Cancer and Nutrition (EPIC), a prospective cohort study carried out in 10 European countries during 1992-2000, we conducted a factor analysis to delineate important components that summarize the variation explained by a set of biomarkers and to examine their association with endometrial cancer risk. Prediagnostic levels of testosterone, androstenedione, dehydroepiandrosterone sulfate, sex hormone-binding globulin, estrone, estradiol, C-peptide, insulin-like growth factor-binding proteins 1 and 2, adiponectin, high- and low-density lipoprotein cholesterol, glucose, triglycerides, tumor necrosis factor (TNF) α, soluble TNF receptors 1 and 2, C-reactive protein, interleukin-6, and interleukin-1 receptor antagonist were measured in 233 incident endometrial cancer cases and 446 matched controls. Factor analysis identified 3 components associated with postmenopausal endometrial cancer risk that could be labeled "insulin resistance/metabolic syndrome," "steroids," and "inflammation" factors. A fourth component, "lipids," was not significantly associated with endometrial cancer. In conclusion, besides the well-known associations of risk with sex hormones and insulin-regulated physiological axes, our data further support the hypothesis that inflammation factors play a role in endometrial carcinogenesis.

Evaluation of a novel commercial assay for the determination of cyclosporine A, tacrolimus, sirolimus, and everolimus by liquid chromatography-tandem mass spectrometric assay.

OBJECTIVE: The aim of this study was the evaluation of the first commercially available in-vitro diagnostic (IVD)- mass spectrometric immunosuppressant assay from Chromsystems (MassTox Immunosuppressants ONEMINUTE Test) and the comparison to a routinely used online solid phase extraction liquid chromatography-tandem mass spectrometric assay method for the measurement of cyclosporine A, everolimus, sirolimus, and tacrolimus in patient whole blood samples. METHODS: An API 4000 [tandem mass spectrometer (AB SCIEX)] combined with a CTC Pal autosampler (CTC Analytics AG) and a Shimadzu ultra-fast liquid chromatography (UFLC) system were applied for the direct liquid chromatography-tandem mass spectrometric assay analysis using electrospray ionization in positive ion mode. Isotope-labeled internal standards were used for the commercial assay. Within- and between-day variation, accuracy, and limits of quantification were determined. Samples from external international proficiency testing schemes were measured to assess the accuracy of the commercial assay. About 100 patient samples were reanalyzed with the commercial test kit, and the results were compared with our in-house results. RESULTS: The limit of quantification for the commercial assay was 0.5 ng/mL for everolimus, sirolimus, and tacrolimus and 5 ng/mL for cyclosporine A. The coefficient of variation for all immunosuppressants was lower than 7% (within day) and 12% (between days) for all 5 concentration levels. Accuracy ranged between 82% and 111% for quality control samples and between 89% and 112% for samples from the external quality assurance program. Both methods showed a very good agreement (r > 0.91) in patient samples over the whole concentration range for all immunosuppressants. CONCLUSION: The commercial immunosuppressant assay from Chromsystems represents a standardized IVD-certified alternative to our in-house developed assay.

Quantification of (9R)- and (9S)-hexahydrocannabinol (HHC) via GC-MS in serum/plasma samples from drivers suspected of cannabis consumption and immunological detection of HHC and related substances in serum, urine, and saliva.

The semisynthetic cannabinoid hexahydrocannabinol (HHC) is currently getting a lot of media attention because the legal status in many countries is not clearly specified. In this study, a GC-MS method for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) was extended to (9R)- and (9S)-HHC. The applicability was proven by serum/plasma samples from drivers suspected of cannabis consumption. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.15 and 0.25 ng/mL, respectively. Within-run imprecision was <6.5% and between-run imprecision was <10.0%. Inter-injection stability, processed sample stability (3 days), freeze-thaw stability (three cycles), and storage stability (1 week room temperature; 1 month 4°C, -20°C) could be proven. Both HHC diastereomers could be detected in 17 (5.3%) out of 321 analyzed samples from traffic controls in Western Saxony. The mean ratio between (9R)- and (9S)-HHC was 1.99 (CV = 14.6%). Quantification resulted in concentrations between

Sensitive and simultaneous quantification of 16 neurotransmitters and metabolites in murine microdialysate by fast liquid chromatography-tandem mass spectrometry.

The sensitive and simultaneous measurement of multiple neurotransmitters in microdialysate (MD) of freely moving mice is a prerequisite to study neurochemical imbalances in specific brain regions. The quantitative analysis of 16 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (l-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), homovanillinic acid (HVA), acetylcholine (ACh), deoxy carnitine (iso-ACh), choline (Ch), and É£-aminobutyric acid (GABA), adenosine (ADE), glutamine (Gln), and glutamic acid (Glu) was achieved within a chromatographic separation time of 6.5 min by the application of a biphenyl column coupled to an API-QTrap 5500 (AB SCIEX) mass spectrometer. Optimized chromatographic separation as well as high sensitivity allow the simultaneous analysis and precise quantification of 16 neurotransmitters and metabolites in artificial cerebrospinal fluid (CSF). Sample preparation procedure consisted of simply adding isotopically labeled internal standard solution to the microdialysis sample. The limits of detection in aCSF ranged from 0.025 pg (Ch) to 9.75 pg (Gln) and 85.5 pg (HVA) on column. Recoveries were between 83 and 111% for neurotransmitter concentrations from 0.6 to 45 ng/ml or 200 ng/ml with a mean intra-day and inter-day coefficient of variation of 7.6% and 11.2%, respectively. Basal extracellular concentrations of the following analytes: 5-HT, 5-HIAA, ME, DA, 3-MT, HVA, ACh, iso-ACh, Ch, GABA, ADE, Gln, and Glu were determined in the striatum of mice with a MD flow rate of 0.5 µl/min. This LC-MS/MS method leads to an accurate quantification of ACh and its isobaric structure iso-ACh, which were detected in the MD samples at ratios of 1:8.6. The main advantage of the high sensitivity is the miniaturization of the MD protocol with short sample collection times and volumes down to 5 µl, which makes this method suitable for pharmacological intervention and optogenetic studies to detect neurochemical changes in vivo.

An atlas of genome-wide gene expression and metabolite associations and possible mediation effects towards body mass index.

Investigating the cross talk of different omics layers is crucial to understand molecular pathomechanisms of metabolic diseases like obesity. Here, we present a large-scale association meta-analysis of genome-wide whole blood and peripheral blood mononuclear cell (PBMC) gene expressions profiled with Illumina HT12v4 microarrays and metabolite measurements from dried blood spots (DBS) characterized by targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) in three large German cohort studies with up to 7706 samples. We found 37,295 associations comprising 72 amino acids (AA) and acylcarnitine (AC) metabolites (including ratios) and 8579 transcripts. We applied this catalogue of associations to investigate the impact of associating transcript-metabolite pairs on body mass index (BMI) as an example metabolic trait. This is achieved by conducting a comprehensive mediation analysis considering metabolites as mediators of gene expression effects and vice versa. We discovered large mediation networks comprising 27,023 potential mediation effects within 20,507 transcript-metabolite pairs. Resulting networks of highly connected (hub) transcripts and metabolites were leveraged to gain mechanistic insights into metabolic signaling pathways. In conclusion, here, we present the largest available multi-omics integration of genome-wide transcriptome data and metabolite data of amino acid and fatty acid metabolism and further leverage these findings to characterize potential mediation effects towards BMI proposing candidate mechanisms of obesity and related metabolic diseases. KEY MESSAGES: Thousands of associations of 72 amino acid and acylcarnitine metabolites and 8579 genes expand the knowledge of metabolome-transcriptome associations. A mediation analysis of effects on body mass index revealed large mediation networks of thousands of obesity-related gene-metabolite pairs. Highly connected, potentially mediating hub genes and metabolites enabled insight into obesity and related metabolic disease pathomechanisms.

Serum enterolactone and postmenopausal breast cancer risk by estrogen, progesterone and herceptin 2 receptor status.

Lignans are a group of estrogenic compounds present in plants. Several epidemiological studies proposed that lignans may protect against breast cancer by exerting anticarcinogenic activity. Levels of enterolactone were determined in serum samples of 1,250 cases and 2,164 controls from a large population-based case-control study. We assessed the association between serum enterolactone and postmenopausal breast cancer risk using conditional logistic regression accounting for potential risk and confounding factors. Fractional polynomials were used to determine the function that best fitted the data. Moreover, we assessed heterogeneity by estrogen/progesterone/herceptin (ER/PR/HER2) status of the tumor. Additionally, a meta-analysis with seven further studies addressing enterolactone concentrations and breast cancer risk was performed. Postmenopausal breast cancer risk decreased with increasing serum enterolactone levels [highest compared to lowest quintile: [odds ratio = 0.65; 95% confidence interval (CI) 0.52-0.83, p(trend) = < 0.0001]. A significant inverse association for ER+/PR+ as well as ER-/PR- tumors was observed, with a significantly stronger association for ER-/PR- tumors (p(heterogeneity) = 0.03). The association for ER-/PR- tumors did not differ by expression of HER2 (p(heterogeneity) = 0.3). The meta-analysis yielded a significant reduced pooled risk estimate of: 0.66; 95% CI: 0.55-0.77) comparing the highest to the lowest quantiles of enterolactone levels. We found strong evidence for a significant inverse association between serum enterolactone and postmenopausal breast cancer risk, which was stronger for ER-PR- than for ER+PR+ tumors but not differential by further expression of HER2. The overall evidence together with other studies supports an inverse association between higher serum enterolactone levels and postmenopausal breast cancer risk.

The association of circulating adiponectin levels with pancreatic cancer risk: a study within the prospective EPIC cohort.

Excess body weight and type 2 diabetes mellitus, risk factors of pancreatic cancer, are characterized by decreased levels of adiponectin. In addition to anti-inflammatory and anti-proliferative actions, adiponectin has an important role in regulating glucose metabolism, i.e., decreasing circulating blood glucose levels. Prospectively, hyperglycemia has been associated with risk of pancreatic cancer. The aim of this study was to investigate the association of pre-diagnostic adiponectin levels with pancreatic cancer risk. We conducted a case-control study nested within European Prospective Investigation into Cancer and Nutrition. Blood samples of 452 pancreatic cancer cases and 452 individually matched controls were analyzed by immunoassays. Multivariate conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Overall, adiponectin showed no association with pancreas cancer risk; however, among never smokers, higher circulating levels of adiponectin were associated with a reduction in pancreatic cancer risk (OR = 0.44 [95% CI 0.23-0.82] for highest vs. lowest quartile), whereas among current smokers there was no significant association (OR = 1.59 [95% CI 0.67-3.76] for highest vs. lowest quartile; p-trend = 0.530; p-interaction = 0.309). In our study, lower adiponectin concentrations may be associated with the development of pancreatic cancer among never smokers, whereas the only other prospective study being conducted so far showed a decrease in risk among male smokers. Therefore, further studies are needed to clarify the role of adiponectin in pancreatic cancer development.

A metabolomic approach to identify the link between sports activity and atheroprotection.

AIMS: Physical activity (PA) is a mainstay of cardiovascular prevention. This study aimed to identify metabolic mediators of PA that protect against the development of atherosclerosis. METHODS AND RESULTS: A total of 2160 participants in the LIFE heart study were analysed with data on PA and vascular phenotyping. In a targeted metabolomic approach, 61 metabolites (amino acids and acylcarnitines) were measured using liquid chromatography-tandem mass spectrometry. We investigated the interactions between PA, metabolites and markers of atherosclerosis in order to uncover possible mediation effects. Intended sports activity, but no daily PA, was associated with a lower degree of atherosclerosis, odds ratio (OR) for total atherosclerotic burden of 0.76 (95% confidence interval 0.62-0.94), carotid artery plaque OR 0.79 (0.66-0.96), and peripheral artery disease OR 0.74 (0.56-0.98). Twelve amino acids, free carnitine, five acylcarnitines were associated with sports activity. Of these, eight metabolites were also associated with the degree of atherosclerosis. In the mediation analyses, a cluster of amino acids (arginine, glutamine, pipecolic acid, taurine) were considered as possible mediators of atheroprotection. In contrast, a group of members of the carnitine metabolism (free carnitine, acetyl carnitine, octadecenoyl carnitine) were associated with inactivity and higher atherosclerotic burden. CONCLUSION: Our metabolomic approach, which is integrated into a mediation model, provides transformative insights into the complex metabolic processes involved in atheroprotection. Metabolites with antioxidant and endothelial active properties are believed to be possible mediators of atheroprotection. The metabolomic mediation approach can support the understanding of complex diseases in order to identify targets for prevention and therapy.

Tumor necrosis factor (TNF)-α, soluble TNF receptors and endometrial cancer risk: the EPIC study.

Chronic inflammation has been hypothesized to play a role in endometrial cancer development. Tumor necrosis factor-α (TNF-α), one of the major pro-inflammatory cytokines, has also been implicated in endometrial physiology. We conducted a case-control study nested within the European prospective investigation into cancer and nutrition (EPIC) to examine the association of TNF-α and its two soluble receptors (sTNFR1 and sTNFR2) with endometrial cancer risk. Two-hundred-seventy cases and 518 matched controls were analyzed using conditional logistic regression. All statistical tests were two-sided. We observed an increased risk of endometrial cancer among women in the highest versus lowest quartile of TNF-α (odds ratio [OR]: 1.73, 95% CI: 1.09-2.73, P(trend) = 0.01), sTNFR1 (OR: 1.68, 95% CI: 0.99-2.86, P(trend) = 0.07) and sTNFR2 (OR: 1.53, 95%CI: 0.92-2.55, P(trend) = 0.03) after adjustment for body-mass-index, parity, age at menopause and previous postmenopausal hormone therapy use. Further adjustments for estrogens and C-peptide had minor effect on risk estimates. Our data show that elevated prediagnostic concentrations of TNF-α and its soluble receptors are related to a higher risk of endometrial cancer, particularly strong in women diagnosed within 2 years of blood donation. This is the first study of its kind and therefore deserves replication in further prospective studies.

Physical activity and endogenous sex hormones in postmenopausal women: to what extent are observed associations confounded or modified by BMI?

OBJECTIVES: To investigate associations between physical activity and endogenous sex hormones after menopause with a special focus on confounding and effect modification by body mass index (BMI). METHODS: A cross-sectional study among 1,260 postmenopausal women was conducted. Generalized linear models were used to compare levels of total leisure-time physical activity, sports activities, bicycling, and walking with levels of sex hormones and sex-hormone-binding-globulin (SHBG). RESULTS: Higher sports activity levels were significantly associated with lower levels of estrone and total and free testosterone in multivariate adjusted models. After additional adjustment for BMI, associations with estrone and free testosterone were attenuated; the association with total testosterone remained unchanged. No physical activity variable was significantly related to total and free estradiol, androstenedione, or SHBG. We did not observe effect modification by BMI. CONCLUSIONS: Sports activities may lead to lower levels of estrone and testosterone in postmenopausal women. While effects on estrone and free testosterone seem to be largely mediated by BMI, effects on total testosterone appear to be mainly independent of BMI. The BMI-independent effects on these hormones (especially on total testosterone) could at least partly explain why physical activity has been frequently reported to be preventive for postmenopausal breast cancer, even after accounting for BMI.

Free cholesterol, cholesterol precursor and plant sterol levels in atherosclerotic plaques are independently associated with symptomatic advanced carotid artery stenosis.

BACKGROUND AND AIMS: Circulating sterols result either from cholesterol (CH) synthesis or intestinal uptake. They are mainly esterified and can be oxygenated. Sterols accumulate in atherosclerotic plaques whereby their clinical impact is uncertain. Here, we determined associations between circulating and plaque sterol levels in patients with advanced carotid artery stenosis in respect to a prior ischemic event and statin treatment. METHODS: Free and esterified CH, CH precursors and plant sterols as well as oxysterols were quantified by liquid chromatography-tandem mass spectrometry in 63 consecutive patients undergoing carotid endarterectomy. RESULTS: CH, CH precursors, plant sterols and oxysterols accumulated in carotid artery plaques. Absolute circulating sterol levels were not predictive for their corresponding plaque levels. After normalisation to CH, plant sterol but not oxysterol levels correlated between plasma and plaques. Among the circulating sterols, oxysterols occurred proportionally less in plaques. Furthermore, CH and plant sterols were less esterified in plaques than in plasma. Patients who experienced a prior ischemic event (n = 29) and asymptomatic patients had, except for lanosterol, comparable circulating sterol levels. In contrast, the absolute plaque levels of free CH, CH precursors and plant sterols as well as oxysterols were increased in symptomatic compared to asymptomatic patients. These differences remained significant for free CH, precursors and 3 out of 4 analyzed plant sterols after adjustment to the most influencing covariates - statin treatment, type 2 diabetes and age. CONCLUSIONS: Increased absolute plaque levels of free CH, precursors and plant sterols predict an ischemic event in patients with advanced carotid artery stenosis.

Exogenous and endogenous hormones, mammographic density and breast cancer risk: can mammographic density be considered an intermediate marker of risk?

Elevated mammographic density measures are a well-established, relatively strong risk factor for breast cancer development. A systematic review of prospective cohort studies and cross-sectional studies strikingly establishes parallels between the associations of combined postmenopausal estrogen and progestin replacement therapy with, on the one hand, mammographic densities and, on the other hand, breast cancer risk. Other parallel observations were the inverse associations of both mammographic density and breast cancer risk with the selective estrogen receptor modulator tamoxifen, and direct associations with prolactin. Paradoxically, however, high mammographic density has been found associated with higher risks of both estrogen- and progesterone-receptor positive (ER+/ PR+) and negative (ER-/PR-) breast cancers, while hormone replacement therapy (HRT) use, but also circulating (blood) levels of androgens, estrogens, and prolactin appear to be associated more specifically to the risk of ER+ tumors. The effects of aromatase inhibitors and gonadotropin-releasing hormone agonists on breast density, as well as on breast cancer risk, still require further investigation. Regarding circulating levels of insulin-like growth factor (IGF)-I or IGFBP-3, studies did not show fully consistent relationships with mammographic density measures and breast cancer risk. In view of these various findings, it is impossible, at present, to propose mammographic density measures as an intermediate risk-related phenotype, integrating the effects of exogenous and/or endogenous hormones on the risk of developing breast cancer.