Phospholipid Removal for Enhanced Chemical Exposomics in Human Plasma.

The challenge of chemical exposomics in human plasma is the 1000-fold concentration gap between endogenous substances and environmental pollutants. Phospholipids are the major endogenous small molecules in plasma, thus we validated a chemical exposomics protocol with an optimized phospholipid-removal step prior to targeted and non-targeted liquid chromatography high-resolution mass spectrometry. Increased injection volume with negligible matrix effect permitted sensitive multiclass targeted analysis of 77 priority analytes; median MLOQ = 0.05 ng/mL for 200 µL plasma. In non-targeted acquisition, mean total signal intensities of non-phospholipids were enhanced 6-fold in positive (max 28-fold) and 4-fold in negative mode (max 58-fold) compared to a control method without phospholipid removal. Moreover, 109 and 28% more non-phospholipid molecular features were detected by exposomics in positive and negative mode, respectively, allowing new substances to be annotated that were non-detectable without phospholipid removal. In individual adult plasma (100 µL, n = 34), 28 analytes were detected and quantified among 10 chemical classes, and quantitation of per- and polyfluoroalkyl substances (PFAS) was externally validated by independent targeted analysis. Retrospective discovery and semi-quantification of PFAS-precursors was demonstrated, and widespread fenuron exposure is reported in plasma for the first time. The new exposomics method is complementary to metabolomics protocols, relies on open science resources, and can be scaled to support large studies of the exposome.

Adipokines are possible risk markers for aortic stenosis requiring surgery.

OBJECTIVES: Aortic stenosis (AS) is the most prevalent valvular heart disease among adults. The adipocyte-derived hormones, leptin and adiponectin, have profound metabolic actions. We examined whether these adipokines are independently associated with future aortic valve replacement (AVR). DESIGN: In this longitudinal case-control study, we identified 336 cases who had undergone AVR due to AS, and who had previously participated in population-based health surveys. Two referents were matched to each case and leptin and adiponectin concentrations were analysed from stored baseline survey samples. Uni- and multivariable logistic regression analyses were used to estimate the risk of future AVR. An additional cohort was identified for validation including 106 cases with AVR and 212 matched referents. RESULTS: Median age (interquartile range (IQR)) in years at survey was 59.9 (10.4) and at surgery 68.3 (12.7), and 48% were women. An elevated concentration of leptin was not associated with future AVR (odds ratio [95% confidence interval]) (1.10 [0.92-1.32]), although leptin was associated with a higher risk in patients with coronary artery disease (CAD) having more than 5 years between survey and AVR (1.41 [1.08-1.84]). Adiponectin was not associated with higher risk for future AVR (0.95 [0.82-1.11]), although after stratification for age, higher levels were associated with reduced risk for AVR in persons aged ≥60 years at surgery (0.79 [0.64-0.98]). In the validation study, leptin was associated with future AVR whereas adiponectin was not. None of the associations remained significant after adjustment for body mass index (BMI). CONCLUSIONS: The adipokine leptin may promote the development of AS.

Longitudinal changes in concentrations of persistent organic pollutants (1986-2016) and their associations with type 2 diabetes mellitus.

BACKGROUND: Positive associations have been reported between persistent organic pollutants (POPs) and type 2 diabetes mellitus (T2DM); however, causality has not been established. Over the last decades, environmental exposure to legacy POPs has decreased, complicating epidemiological studies. In addition, physiological risk factors for T2DM may also influence POP concentrations, contributing to a complex network of factors that could impact associations with T2DM. Longitudinal studies on this topic are lacking, and few have assessed prospective and cross-sectional associations between repeated POP measurements and T2DM in the same individuals, which may shed light on causality. OBJECTIVES: To compare longitudinal trends in concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in T2DM cases and controls, and to examine prospective and cross-sectional associations between PCBs, OCPs and T2DM at different time-points before and after T2DM diagnosis in cases. METHODS: We conducted a longitudinal, nested case-control study (1986-2016) of 116 T2DM cases and 139 controls from the Tromsø Study. All participants had three blood samples collected before T2DM diagnosis in cases, and up to two samples thereafter. We used linear mixed-effect models to assess temporal changes of POPs within and between T2DM cases and controls, and logistic regression models to investigate the associations between different POPs and T2DM at different time-points. RESULTS: PCBs, trans-nonachlor, cis-nonachlor, oxychlordane, cis-heptachlor epoxide, p,p'-DDE, and p,p'-DDT declined more slowly in cases than controls, whereas ß-HCH and HCB declined similarly in both groups. Most POPs showed positive associations between both pre- and post-diagnostic concentrations and T2DM, though effect estimates were imprecise. These associations were most consistent for cis-heptachlor epoxide. DISCUSSION: The observed positive associations between certain POPs and T2DM may be because of higher POP concentrations within prospective T2DM cases, due to slower temporal declines as compared to controls.

Pre- and post-diagnostic blood profiles of chlorinated persistent organic pollutants and metabolic markers in type 2 diabetes mellitus cases and controls; a pilot study.

OBJECTIVE: Several risk factors for type 2 diabetes mellitus (T2DM) are also associated with blood concentrations of persistent organic pollutants (POPs), and factors related to the disease may affect POP concentrations, and subsequent associations between POPs and T2DM. The purpose of this pilot study was to investigate the change in concentrations of lipids, hormones and POPs pre- and post-diagnosis in T2DM cases compared to healthy controls and their associations with T2DM. METHODS: We measured POPs, lipids, and thyroid and steroid hormones in plasma from 44 female cases collected prior to (pre-diagnostic) and following (post-diagnostic) T2DM diagnosis, and in 44 healthy female age-matched controls. We compared cross-sectional differences and longitudinal changes within and between matched cases and controls with t-tests and multivariable linear regression models. Associations between POP concentrations and T2DM were investigated using conditional logistic regression. RESULTS: Between the pre- and post-diagnostic measurement, cases developed more favorable lipid profiles and the longitudinal changes in lipid-normalized concentrations of non-dioxin-like polychlorinated biphenyls (PCBs), dioxin-like PCBs, beta-hexachlorocyclohexane (HCH), HCB, and 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (p,p'-DDE) differed significantly between cases and controls. The longitudinal changes in POPs were mainly driven by changes in bodyweight, total lipids and T2DM status. Cases had significantly higher pre-diagnostic concentrations of POPs and triglycerides, and lower concentrations of high-density lipoprotein cholesterol and free thyroxin than controls. Pre-diagnostic POP concentrations were not significantly associated with incident T2DM, whereas several post-diagnostic POP concentrations were significantly positively associated with prevalent T2DM. CONCLUSIONS: This pilot study suggests that factors related to T2DM affect blood concentrations of POPs and may partly explain the positive associations between POPs and T2DM.

Unexpected Acetylation of Endogenous Aliphatic Amines by Arylamine N-Acetyltransferase NAT2.

N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamine N-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamine N-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselective N8 -acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.

Proteomic Biomarkers for Incident Aortic Stenosis Requiring Valvular Replacement.

BACKGROUND: Aortic valve stenosis (AS) is the most common indication for cardiac valve surgery; untreated AS is linked to high mortality. The etiological background of AS is unknown. Previous human studies were typically based on case-control studies. Biomarkers identified in prospective studies could lead to novel mechanistic insights. METHODS: Within a large population survey with blood samples obtained at baseline, 334 patients were identified who later underwent surgery for AS (median age [interquartile range], 59.9 [10.4] years at survey and 68.3 [12.7] at surgery; 48% female). For each case, 2 matched referents were allocated. Plasma was analyzed with the multiplex proximity extension assay for screening of 92 cardiovascular candidate proteins. Conditional logistic regression models were used to assess associations between each protein and AS, with correction for multiple testing. A separate set of 106 additional cases with 212 matched referents was used in a validation study. RESULTS: Six proteins (growth differentiation factor 15, galectin-4, von Willebrand factor, interleukin 17 receptor A, transferrin receptor protein 1, and proprotein convertase subtilisin/kexin type 9) were associated with case status in the discovery cohort; odds ratios ranged from 1.25 to 1.37 per SD increase in the protein signal. Adjusting the multivariable models for classical cardiovascular risk factors at baseline yielded similar results. Subanalyses of case-referent triplets (n=133) who showed no visible coronary artery disease at the time of surgery in the index person supported associations between AS and growth differentiation factor 15 (odds ratio, 1.40; 95% confidence interval, 1.10-1.78) and galectin-4 (odds ratio, 1.27; 95% confidence interval, 1.02-1.59), but these associations were attenuated after excluding individuals who donated blood samples within 5 years before surgery. In triplets (n=201), which included index individuals with concurrent coronary artery disease at the time of surgery, all 6 proteins were robustly associated with case status in all sensitivity analyses. In the validation study, the association of all but 1 (interleukin 17 receptor A) of these proteins were replicated in patients with AS with concurrent coronary artery disease but not in patients with AS without coronary artery disease. CONCLUSIONS: We provide evidence that 5 proteins were altered years before AS surgery and that the associations seem to be driven by concurrent atherosclerotic disease.

Blood levels of cadmium and lead in relation to breast cancer risk in three prospective cohorts.

Cadmium and lead have been classified as carcinogens by the International Agency for Research on Cancer. However, their associations with breast cancer risk are unknown despite their persistence in the environment and ubiquitous human exposure. We examined associations of circulating levels of cadmium and lead with breast cancer risk in three case-control studies nested within the Cancer Prevention Study-II (CPS-II) LifeLink Cohort, European Prospective Investigation into Cancer and Nutrition - Italy (EPIC-Italy) and the Northern Sweden Health and Disease Study (NSHDS) cohorts. Metal levels were measured in stored erythrocytes from 1,435 cases and 1,433 controls using inductively coupled plasma-mass spectrometry. Summary relative risks (RR) and 95% confidence intervals (CI) were calculated using random-effects models with each study result weighted by the within- and between-study variances. I2 values were calculated to estimate proportion of between study variation. Using common cut-points, cadmium levels were not associated with breast cancer risk in the CPS-II cohort (continuous RR = 1.01, 95% CI 0.76-1.34), but were inversely associated with risk in the EPIC- Italy (continuous RR = 0.80, 95% CI 0.61-1.03) and NSHDS cohorts (continuous RR = 0.73, 95% CI 0.54-0.97). The inverse association was also evident in the meta-analysis (continuous RR = 0.84, 95% CI 0.69-1.01) with low between-study heterogeneity. Large differences in lead level distributions precluded a meta-analysis of their association with breast cancer risk; no associations were found in the three studies. Adult cadmium and lead levels were not associated with higher risk of breast cancer in our large meta-analysis.

Tea and coffee consumption in relation to DNA methylation in four European cohorts.

Lifestyle factors, such as food choices and exposure to chemicals, can alter DNA methylation and lead to changes in gene activity. Two such exposures with pharmacologically active components are coffee and tea consumption. Both coffee and tea have been suggested to play an important role in modulating disease-risk in humans by suppressing tumour progression, decreasing inflammation and influencing estrogen metabolism. These mechanisms may be mediated by changes in DNA methylation. To investigate if DNA methylation in blood is associated with coffee and tea consumption, we performed a genome-wide DNA methylation study for coffee and tea consumption in four European cohorts (N = 3,096). DNA methylation was measured from whole blood at 421,695 CpG sites distributed throughout the genome and analysed in men and women both separately and together in each cohort. Meta-analyses of the results and additional regional-level analyses were performed. After adjusting for multiple testing, the meta-analysis revealed that two individual CpG-sites, mapping to DNAJC16 and TTC17, were differentially methylated in relation to tea consumption in women. No individual sites were associated with men or with the sex-combined analysis for tea or coffee. The regional analysis revealed that 28 regions were differentially methylated in relation to tea consumption in women. These regions contained genes known to interact with estradiol metabolism and cancer. No significant regions were found in the sex-combined and male-only analysis for either tea or coffee consumption.

Haploinsufficiency of UNC13D increases the risk of lymphoma.

BACKGROUND: Experimental models have demonstrated that immune surveillance by cytotoxic lymphocytes can protect from spontaneous neoplasms and cancer. In humans, defective lymphocyte cytotoxicity is associated with the development of hemophagocytic lymphohistiocytosis, a hyperinflammatory syndrome. However, to the best of the authors' knowledge, the degree to which human lymphocyte cytotoxicity protects from cancer remains unclear. In the current study, the authors examined the risk of lymphoma attributable to haploinsufficiency in a gene required for lymphocyte cytotoxicity. METHODS: The authors exploited a founder effect of an UNC13D inversion, which abolishes Munc13-4 expression and causes hemophagocytic lymphohistiocytosis in an autosomal recessive manner. Within 2 epidemiological screening programs in northern Sweden, an area demonstrating a founder effect of this specific UNC13D mutation, all individuals with a diagnosis of lymphoma (487 patients) and matched controls (1844 controls) were assessed using polymerase chain reaction for carrier status. RESULTS: Among 487 individuals with lymphoma, 15 (3.1%) were heterozygous carriers of the UNC13D inversion, compared with 18 controls (1.0%) (odds ratio, 3.0; P = .002). It is interesting to note that a higher risk of lymphoma was attributed to female carriers (odds ratio, 3.7; P = .004). CONCLUSIONS: Establishing a high regional prevalence of the UNC13D inversion, the authors have reported an overrepresentation of this mutation in individuals with lymphoma. Therefore, the results of the current study indicate that haploinsufficiency of a gene required for lymphocyte cytotoxicity can predispose patients to lymphoma, suggesting the importance of cytotoxic lymphocyte-mediated surveillance of cancer. Furthermore, the results of the current study suggest that female carriers are more susceptible to lymphoma.

Immune marker changes and risk of multiple myeloma: a nested case-control study using repeated pre-diagnostic blood samples.

Biomarkers reliably predicting progression to multiple myeloma (MM) are lacking. Myeloma risk has been associated with low blood levels of monocyte chemotactic protein-3 (MCP-3), macrophage inflammatory protein-1 alpha (MIP-1α), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), fractalkine, and transforming growth factor-alpha (TGF-α). In this study, we aimed to replicate these findings and study the individual dynamics of each marker in a prospective longitudinal cohort, thereby examining their potential as markers of myeloma progression. For this purpose, we identified 65 myeloma cases and 65 matched cancer-free controls each with two donated blood samples within the Northern Sweden Health and Disease Study. The first and repeated samples from myeloma cases were donated at a median 13 and 4 years, respectively, before the myeloma was diagnosed. Known risk factors for progression were determined by protein-, and immunofixation electrophoresis, and free light chain assays. We observed lower levels of MCP-3, VEGF, FGF-2, and TGF-α in myeloma patients than in controls, consistent with previous data. We also observed that these markers decreased among future myeloma patients while remaining stable in controls. Decreasing trajectories were noted for TGF-α (P=2.5 × 10-4) indicating progression to MM. Investigating this, we found that low levels of TGF-α assessed at the time of the repeated sample were independently associated with risk of progression in a multivariable model (hazard ratio = 3.5; P=0.003). TGF-α can potentially improve early detection of MM.

Joint Analysis of Metabolite Markers of Fish Intake and Persistent Organic Pollutants in Relation to Type 2 Diabetes Risk in Swedish Adults.

BACKGROUND: There is conflicting evidence regarding the association between fish intake and type 2 diabetes (T2D) incidence, possibly owing to measurement errors in self-reported intake and coexposure to persistent organic pollutants (POPs) present in fish. OBJECTIVE: The aim of this study was to identify plasma metabolites associated with fish intake and to assess their association with T2D risk, independently of POPs, in Swedish adults. METHODS: In a case-control study nested in the Swedish Västerbotten Intervention Programme, fasting plasma samples from 421 matched T2D case-control pairs of men and women aged 30-60 y at baseline and 10-y follow-up samples from a subset of 149 pairs were analyzed using untargeted metabolomics. Moreover, 16 plasma POPs were analyzed for the 149 pairs who had repeated samples available. Fish-related plasma metabolites were identified using multivariate modelling and partial correlation analysis. Reproducibility of metabolites and metabolite patterns, derived via principal component analysis (PCA), was assessed by intraclass correlation. A unique component of metabolites unrelated to POPs was dissected by integrating metabolites and POPs using 2-way orthogonal partial least squares regression. ORs of T2D were estimated using conditional logistic regression. RESULTS: We identified 31 metabolites associated with fish intake that had poor to good reproducibility. A PCA-derived metabolite pattern strongly correlated with fish intake (ρ = 0.37, P < 0.001) but showed no association with T2D risk. Integrating fish-related metabolites and POPs led to a unique metabolite component independent of POPs, which tended to be inversely associated with T2D risk (OR: 0.75; 95% CI: 0.54, 1.02, P = 0.07). This component mainly consisted of metabolites reflecting fatty fish intake. CONCLUSIONS: Our results suggest that fatty fish intake may be beneficial for T2D prevention, after removing the counteractive effects of coexposure to POPs in Swedish adults. Integrating metabolite markers and POP exposures appears a promising approach to advance the understanding of associations between fish intake and T2D incidence.

Chlorinated persistent organic pollutants and type 2 diabetes - A population-based study with pre- and post- diagnostic plasma samples.

BACKGROUND: Persistent organic pollutants (POPs) have been associated with type 2 diabetes (T2D), but causality is uncertain. OBJECTIVE: Within longitudinal population-based data from northern Sweden, we assessed how POPs associated with T2D prospectively and cross-sectionally, and further investigated factors related to individual changes in POP concentrations. METHODS: For 129 case-controls pairs matched by age, sex and date of sampling, plasma concentrations of hexachlorobenzene (HCB), dichlorodiphenyl-dichloroethylene (p,p'-DDE), dioxin-like (DL) polychlorinated biphenyl congeners (PCB-118 and PCB-156), and non-dioxin like (NDL-PCB: PCB-74, -99, -138 -153, -170, -180, -183 and PCB-187) were analyzed twice (baseline and follow-up, 9-20 years apart). The cases received their T2D diagnose between baseline and follow-up. Prospective (using baseline data) and cross-sectional (using follow-up data) odds ratios (ORs) for T2D on lipid standardized POPs (HCB, p,p'-DDE, ∑DL-PCBs, ∑NDL-PCBs) were estimated using conditional logistic regression, adjusting for body mass index (BMI) and plasma lipids. The influence of BMI, weight-change, and plasma lipids on longitudinal changes in POP concentrations were evaluated among non-diabetic individuals (n = 306). RESULTS: POPs were associated with T2D in both the prospective and cross-sectional assessments. Of a standard deviation increase in POPs, prospective ORs ranged 1.42 (95% CI: 0.99, 2.06) for ∑NDL-PCBs to 1.55 (95% CI: 1.01, 2.38) for HCB (p < 0.05 only for HCB), and cross-sectional ORs ranged 1.62 (95% CI: 1.13; 2.32) for p,p'-DDE to 2.06 (95% CI: 1.29, 3.28) for ∑DL-PCBs (p < 0.05 for all POPs). In analyses of non-diabetic individuals, higher baseline BMI, decreased weight and decreased plasma lipid concentrations were associated with a slower decrease of POPs. Cases had, besides a higher BMI, reduced cholesterol and weight gain at follow-up compared to controls, which can explain the higher ORs in the cross-sectional assessments. DISCUSSION: The association between POPs and T2D was confirmed, but an indication that individuals body fat history might influence POP-T2D associations weakens the epidemiological support for a causal association. It also warrants studies based on other exposure metrics than biomonitoring. In addition, we note that a cross-sectional design overestimates the ORs if T2D cases have successfully intervened on weight and/or blood lipids, as changes in these factors cause changes in POPs.

Methodological issues in a prospective study on plasma concentrations of persistent organic pollutants and pancreatic cancer risk within the EPIC cohort.

BACKGROUND: The use of biomarkers of environmental exposure to explore new risk factors for pancreatic cancer presents clinical, logistic, and methodological challenges that are also relevant in research on other complex diseases. OBJECTIVES: First, to summarize the main design features of a prospective case-control study -nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort- on plasma concentrations of persistent organic pollutants (POPs) and pancreatic cancer risk. And second, to assess the main methodological challenges posed by associations among characteristics and habits of study participants, fasting status, time from blood draw to cancer diagnosis, disease progression bias, basis of cancer diagnosis, and plasma concentrations of lipids and POPs. Results from etiologic analyses on POPs and pancreatic cancer risk, and other analyses, will be reported in future articles. METHODS: Study subjects were 1533 participants (513 cases and 1020 controls matched by study centre, sex, age at blood collection, date and time of blood collection, and fasting status) enrolled between 1992 and 2000. Plasma concentrations of 22 POPs were measured by gas chromatography - triple quadrupole mass spectrometry (GC-MS/MS). To estimate the magnitude of the associations we calculated multivariate-adjusted odds ratios by unconditional logistic regression, and adjusted geometric means by General Linear Regression Models. RESULTS: There were differences among countries in subjects' characteristics (as age, gender, smoking, lipid and POP concentrations), and in study characteristics (as time from blood collection to index date, year of last follow-up, length of follow-up, basis of cancer diagnosis, and fasting status). Adjusting for centre and time of blood collection, no factors were significantly associated with fasting status. Plasma concentrations of lipids were related to age, body mass index, fasting, country, and smoking. We detected and quantified 16 of the 22 POPs in more than 90% of individuals. All 22 POPs were detected in some participants, and the smallest number of POPs detected in one person was 15 (median, 19) with few differences by country. The highest concentrations were found for p,p'-DDE, PCBs 153 and 180 (median concentration: 3371, 1023, and 810 pg/mL, respectively). We assessed the possible occurrence of disease progression bias (DPB) in eight situations defined by lipid and POP measurements, on one hand, and by four factors: interval from blood draw to index date, tumour subsite, tumour stage, and grade of differentiation, on the other. In seven of the eight situations results supported the absence of DPB. CONCLUSIONS: The coexistence of differences across study centres in some design features and participant characteristics is of relevance to other multicentre studies. Relationships among subjects' characteristics and among such characteristics and design features may play important roles in the forthcoming analyses on the association between plasma concentrations of POPs and pancreatic cancer risk.

Arterial hypertension and diastolic blood pressure associate with aortic stenosis.

OBJECTIVES: Due to age-related differences in aortic valve structure, it is likely that the pathophysiology of aortic stenosis (AS) and associated risk factors differ between age groups. Here we prospectively studied the influence of traditional cardiovascular risk factors on AS development requiring surgery among patients without concomitant coronary artery disease (CAD) and stratified for age. DESIGN: This study included 322 patients, who had prior to surgery for AS participated in population-based surveys, and 131 of them had no visible CAD upon preoperative coronary angiogram. For each case, we selected four referents matched for age, gender, and geographic area. To identify predictors for surgery, we used multivariable conditional logistic regression with a model including arterial hypertension (or measured blood pressure and antihypertensive medication), cholesterol levels, diabetes, body mass index (BMI), and smoking. RESULTS: In patients without CAD, future surgery for AS was associated with arterial hypertension and elevated levels of diastolic blood pressure in patients younger than 60 years at surgery (odds ratio [95% confidence interval]), (3.40 [1.45-7.93] and 1.60 [1.09-2.37], respectively), and with only impaired fasting glucose tolerance in patients 60 years or older at surgery (3.22 [1.19-8.76]). CONCLUSION: Arterial hypertension and elevated diastolic blood pressure are associated with a risk for AS requiring surgery in subjects below 60 years of age. Strict blood pressure control in this group is strongly advocated to avoid other cardiovascular diseases correlated to hypertension. If hypertension and elevated diastolic blood pressure are risk factors for developing AS requiring surgery need further investigations. Notably, elevated fasting glucose levels were related to AS requiring surgery in older adults without concomitant CAD.

Mild impairment of renal function (shrunken pore syndrome) is associated with increased risk for future surgery for aortic stenosis.

Recently, a new approach was proposed to detect mild impairment in renal function: a reduced ratio between estimated glomerular filtration rate (eGFR) calculated by cystatin C and eGFR calculated by creatinine. We aimed to evaluate if this ratio is associated with aortic stenosis (AS) requiring surgery. We identified 336 patients that first participated in population surveys and later underwent surgery for AS (median age [interquartile range] 59.8 [10.3] years at survey and 68.3 [12.7] at surgery, 48% females). For each patient, two matched referents were allocated. Cystatin C and creatinine were determined in stored plasma. eGFRcystatin C and eGFRcreatinine and their ratio were estimated. Conditional logistic regression analyses were used to estimate the risk (odds ratio (OR) with [95% confidence interval (CI)]) related to one (ln) standard deviation increase in the ratio between eGFRcystatin C and eGFRcreatinine. A high ratio was associated with lower risk for AS requiring surgery (OR [95% CI]) (OR 0.84 [0.73-0.97]), especially in women (0.74 [0.60-0.92] vs. 0.93 [0.76-1.13] in men). After further stratification for coronary artery disease (CAD), the association remained in women with CAD but not in women without CAD (0.60 [0.44-0.83] and 0.89 [0.65-1.23], respectively). In conclusion, a high ratio between eGFRcystatin C and eGFRcreatinine was associated with lower risk for surgery for AS, especially in women. Mild impairment of renal function is thus associated with future risk for AS requiring surgery.

Passive personal air sampling of dust in a working environment-A pilot study.

The aim of this study was to make a preliminary evaluation of the University of North Carolina passive aerosol sampler (UNC sampler) for personal air sampling of particles. Nine personal air samplings of respirable fraction were conducted in an open-pit mine, with pairwise UNC samplers and a respirable cyclone mounted on the chest of workers. UNC samples were analyzed with scanning electron microscopy (SEM) and to some extent energy dispersive X-ray spectroscopy (EDS). Respirable cyclone filter samples were weighed. Correlations and particle elemental compositions were described. Microscopic imaging of the collection surface showed that the particles were heterogeneously deposited across the surface of the UNC sampler. Collected particles were shaped as gravel particles and the resulting particle size distribution in air showed a peak at ca. 3 µm aerodynamic diameter, similarly to what has previously been reported from the same mine. The elemental composition indicated mineral origin. All correlations between the airborne mass concentrations from UNC samplers and respirable cyclones (Pearson = 0.54 and Spearman = 0.43) and between pairs of parallel UNC samplers (Pearson = 0.55 and Spearman = 0.67) were weak. The UNC sampler mass concentrations were approximately 30 times higher than those measured with the respirable cyclone. In conclusion, the UNC sampler, when used for personal sampling in a mine, provides a reasonable particle size distribution and the deposited particles appeared to be of mineral origin and not from textile or skin but the approximately 30-fold overestimation of mass concentrations when comparing with respirable cyclone sampling indicates that further improvements are necessary. Positioning of the sampler may be critical and moving the UNC sampler from the chest to e.g. the top of a helmet might be an improvement. Grounding of the sampler in order to avoid static electricity might also be useful. The UNC sampler should continue to be researched for personal sampling, as passive sampling might become a useful alternative to more laborious sampling techniques.

Plasma metabolites associated with type 2 diabetes in a Swedish population: a case-control study nested in a prospective cohort.

AIMS/HYPOTHESIS: The aims of the present work were to identify plasma metabolites that predict future type 2 diabetes, to investigate the changes in identified metabolites among individuals who later did or did not develop type 2 diabetes over time, and to assess the extent to which inclusion of predictive metabolites could improve risk prediction. METHODS: We established a nested case-control study within the Swedish prospective population-based Västerbotten Intervention Programme cohort. Using untargeted liquid chromatography-MS metabolomics, we analysed plasma samples from 503 case-control pairs at baseline (a median time of 7 years prior to diagnosis) and samples from a subset of 187 case-control pairs at 10 years of follow-up. Discriminative metabolites between cases and controls at baseline were optimally selected using a multivariate data analysis pipeline adapted for large-scale metabolomics. Conditional logistic regression was used to assess associations between discriminative metabolites and future type 2 diabetes, adjusting for several known risk factors. Reproducibility of identified metabolites was estimated by intra-class correlation over the 10 year period among the subset of healthy participants; their systematic changes over time in relation to diagnosis among those who developed type 2 diabetes were investigated using mixed models. Risk prediction performance of models made from different predictors was evaluated using area under the receiver operating characteristic curve, discrimination improvement index and net reclassification index. RESULTS: We identified 46 predictive plasma metabolites of type 2 diabetes. Among novel findings, phosphatidylcholines (PCs) containing odd-chain fatty acids (C19:1 and C17:0) and 2-hydroxyethanesulfonate were associated with the likelihood of developing type 2 diabetes; we also confirmed previously identified predictive biomarkers. Identified metabolites strongly correlated with insulin resistance and/or beta cell dysfunction. Of 46 identified metabolites, 26 showed intermediate to high reproducibility among healthy individuals. Moreover, PCs with odd-chain fatty acids, branched-chain amino acids, 3-methyl-2-oxovaleric acid and glutamate changed over time along with disease progression among diabetes cases. Importantly, we found that a combination of five of the most robustly predictive metabolites significantly improved risk prediction if added to models with an a priori defined set of traditional risk factors, but only a marginal improvement was achieved when using models based on optimally selected traditional risk factors. CONCLUSIONS/INTERPRETATION: Predictive metabolites may improve understanding of the pathophysiology of type 2 diabetes and reflect disease progression, but they provide limited incremental value in risk prediction beyond optimal use of traditional risk factors.

Pre-diagnostic blood immune markers, incidence and progression of B-cell lymphoma and multiple myeloma: Univariate and functionally informed multivariate analyses.

Recent prospective studies have shown that dysregulation of the immune system may precede the development of B-cell lymphomas (BCL) in immunocompetent individuals. However, to date, the studies were restricted to a few immune markers, which were considered separately. Using a nested case-control study within two European prospective cohorts, we measured plasma levels of 28 immune markers in samples collected a median of 6 years before diagnosis (range 2.01-15.97) in 268 incident cases of BCL (including multiple myeloma [MM]) and matched controls. Linear mixed models and partial least square analyses were used to analyze the association between levels of immune marker and the incidence of BCL and its main histological subtypes and to investigate potential biomarkers predictive of the time to diagnosis. Linear mixed model analyses identified associations linking lower levels of fibroblast growth factor-2 (FGF-2 p = 7.2 × 10-4 ) and transforming growth factor alpha (TGF-α, p = 6.5 × 10-5 ) and BCL incidence. Analyses stratified by histological subtypes identified inverse associations for MM subtype including FGF-2 (p = 7.8 × 10-7 ), TGF-α (p = 4.08 × 10-5 ), fractalkine (p = 1.12 × 10-3 ), monocyte chemotactic protein-3 (p = 1.36 × 10-4 ), macrophage inflammatory protein 1-alpha (p = 4.6 × 10-4 ) and vascular endothelial growth factor (p = 4.23 × 10-5 ). Our results also provided marginal support for already reported associations between chemokines and diffuse large BCL (DLBCL) and cytokines and chronic lymphocytic leukemia (CLL). Case-only analyses showed that Granulocyte-macrophage colony stimulating factor levels were consistently higher closer to diagnosis, which provides further evidence of its role in tumor progression. In conclusion, our study suggests a role of growth-factors in the incidence of MM and of chemokine and cytokine regulation in DLBCL and CLL.

Epigenome-wide association study of adiposity and future risk of obesity-related diseases.

BACKGROUND: Obesity is an established risk factor for several common chronic diseases such as breast and colorectal cancer, metabolic and cardiovascular diseases; however, the biological basis for these relationships is not fully understood. To explore the association of obesity with these conditions, we investigated peripheral blood leucocyte (PBL) DNA methylation markers for adiposity and their contribution to risk of incident breast and colorectal cancer and myocardial infarction. METHODS: DNA methylation profiles (Illumina Infinium® HumanMethylation450 BeadChip) from 1941 individuals from four population-based European cohorts were analysed in relation to body mass index, waist circumference, waist-hip and waist-height ratio within a meta-analytical framework. In a subset of these individuals, data on genome-wide gene expression level, biomarkers of glucose and lipid metabolism were also available. Validation of methylation markers associated with all adiposity measures was performed in 358 individuals. Finally, we investigated the association of obesity-related methylation marks with breast, colorectal cancer and myocardial infarction within relevant subsets of the discovery population. RESULTS: We identified 40 CpG loci with methylation levels associated with at least one adiposity measure. Of these, one CpG locus (cg06500161) in ABCG1 was associated with all four adiposity measures (P = 9.07×10-8 to 3.27×10-18) and lower transcriptional activity of the full-length isoform of ABCG1 (P = 6.00×10-7), higher triglyceride levels (P = 5.37×10-9) and higher triglycerides-to-HDL cholesterol ratio (P = 1.03×10-10). Of the 40 informative and obesity-related CpG loci, two (in IL2RB and FGF18) were significantly associated with colorectal cancer (inversely, P < 1.6×10-3) and one intergenic locus on chromosome 1 was inversely associated with myocardial infarction (P < 1.25×10-3), independently of obesity and established risk factors. CONCLUSION: Our results suggest that epigenetic changes, in particular altered DNA methylation patterns, may be an intermediate biomarker at the intersection of obesity and obesity-related diseases, and could offer clues as to underlying biological mechanisms.