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Analysis of essential and non-essential trace elements in urine has emerged as a valuable tool for assessing occupational and environmental exposures, diagnosing nutritional status and guiding public health and health care intervention. Our study focused on the analysis of trace elements in urine samples from the Multi-Ethnic Study of Atherosclerosis (MESA), a precious resource for health research with limited sample volumes. Here we provide a comprehensive and sensitive method for the analysis of 18 elements using only 100 μL of urine. Method sensitivity, accuracy, and precision were assessed. The analysis by inductively coupled plasma mass spectrometry (ICP-MS) included the measurement of antimony (Sb), arsenic (As), barium (Ba), cadmium (Cd), cesium (Cs), cobalt (Co), copper (Cu), gadolinium (Gd), lead (Pb), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se), strontium (Sr), thallium (Tl), tungsten (W), uranium (U), and zinc (Zn). Further, we reported urinary trace element concentrations by covariates including gender, ethnicity/race, smoking and location. The results showed good accuracy and sensitivity of the ICP-MS method with the limit of detections rangings between 0.001 μg L−1for U to 6.2 μg L−1for Zn. Intra-day precision for MESA urine analysis varied between 1.4% for Mo and 26% for Mn (average 6.4% for all elements). The average inter-day precision for most elements was <8.5% except for Gd (20%), U (16%) and Mn (19%) due to very low urinary concentrations. Urinary mean concentrations of non-essential elements followed the order of Sr > As > Cs > Ni > Ba > Pb > Cd > Gd > Tl > W > U. The order of urinary mean concentrations for essential trace elements was Zn > Se > Mo > Cu > Co > Mn. Non-adjusted mean concentration of non-essential trace elements in urine from MESA participants follow the order Sr > As > Cs > Ni > Ba > Pb > Cd > Gd > Tl > W > U. The unadjusted urinary mean concentrations of essential trace elements decrease from Zn > Se > Mo > Cu > Co > Mn.

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IMPORTANCE: American Indian communities experience a high burden of coronary heart disease (CHD). Strategies are needed to identify individuals at risk and implement preventive interventions. OBJECTIVE: To investigate the association of blood DNA methylation (DNAm) with incident CHD using a large number of methylation sites (cytosine-phosphate-guanine [CpG]) in a single model. DESIGN, SETTING, AND PARTICIPANTS: This prospective study, including a discovery cohort (the Strong Heart Study [SHS]) and 4 additional cohorts (the Women’s Health Initiative [WHI], the Framingham Heart Study [FHS], the Atherosclerosis Risk in Communities Study ([ARIC]–Black, and ARIC-White), evaluated 12 American Indian communities in 4 US states; African American women, Hispanic women, and White women throughout the US; White men and White women from Massachusetts; and Black men and women and White men and women from 4 US communities. A total of 2321 men and women (mean [SD] follow-up, 19.1 [9.2] years) were included in the SHS, 1874 women (mean [SD] follow-up, 15.8 [5.9] years) in the WHI, 2128 men and women (mean [SD] follow-up, 7.7 [1.8] years) in the FHS, 2114 men and women (mean [SD] follow-up, 20.9 [7.2] years) in the ARIC-Black, and 931 men and women (mean [SD] follow-up, 20.9 [7.2] years) in the ARIC-White. Data were collected from May 1989 to December 2018 and analyzed from February 2019 to May 2021. EXPOSURE: Blood DNA methylation. MAIN OUTCOME AND MEASURE: Using a high-dimensional time-to-event elastic-net model for the association of 407 224 CpG sites with incident CHD in the SHS (749 events), this study selected the differentially methylated CpG positions (DMPs) selected in the SHS and evaluated them in the WHI (531 events), FHS (143 events), ARIC-Black (350 events), and ARIC-White (121 events) cohorts. RESULTS: The median (IQR) age of participants in SHS was 55 (49-62) years, and 1359 participants (58.6%) were women. Elastic-net models selected 505 DMPs associated with incident CHD in the SHS beyond established risk factors, center, blood cell counts, and genetic principal components. Among those DMPs, 33 were commonly selected in 3 or 4 of the other cohorts and the pooled hazard ratios from the standard Cox models were significant at P &lt; .05 for 10 of the DMPs. For example, the hazard ratio (95% CI) for CHD comparing the 90th and 10th percentiles of differentially methylated CpGs was 0.86 (0.78-0.95) for cg16604233 (tagged to COL11A2) and 1.23 (1.08-1.39) for cg09926486 (tagged to FRMD5). Some of the DMPs were consistent in the direction of the association; others showed associations in opposite directions across cohorts. Untargeted independent elastic-net models of CHD showed distinct DMPs, genes, and network of genes in the 5 cohorts. CONCLUSIONS AND RELEVANCE: In this multi-cohort study, blood-based DNAm findings supported an association between a complex blood epigenomic signature and CHD that was largely different across populations.

Objective: To determine the cost-effectiveness of 2 strategies for post-discharge suicide prevention, an Enhanced Contact intervention based on repeated in-person and telephone contacts, and an individual 2-month long problem-solving Psychotherapy program, in comparison to facilitated access to outpatient care following a suicide attempt.Methods: We conducted a cost-effectiveness analysis based on a decision tree between January and December 2019. Comparative effectiveness estimates were obtained from an observational study conducted between 2013 and 2017 in Madrid, Spain. Electronic health care records documented resource use (including extra-hospital emergency care, mortality, inpatient admission, and disability leave). Direct cost data were derived from Madrid’s official list of public health care prices. Indirect cost data were derived from Spain’s National Institute of Statistics.Results: Both augmentation strategies were more cost-effective than a single priority outpatient appointment considering reasonable thresholds of willingness to pay. Under the base-case scenario, Enhanced Contact and Psychotherapy incurred, respectively, €2,340 and 6,260 per averted attempt, compared to a single priority appointment. Deterministic and probabilistic sensitivity analyses showed both augmentation strategies to remain cost-effective under several scenarios. Enhanced Contact was slightly cost minimizing in comparison to Psychotherapy (base-case scenario: €−196 per averted attempt).Conclusions: Two post-discharge suicide prevention strategies based on Enhanced Contact and Psychotherapy were cost-effective in comparison to a single priority appointment. Increasing contacts between suicide attempters and mental health-care providers was slightly cost minimizing compared to psychotherapy.

Background: Elevated adiposity is often posited by medical and public health researchers to be a risk factor associated with cardiovascular disease, diabetes, and other diseases. These health challenges are now thought to be reflected in epigenetic modifications to DNA molecules, such as DNA methylation, which can alter gene expression. Methods: Here we report the results of three Epigenome Wide Association Studies (EWAS) in which we assessed the differential methylation of DNA (obtained from peripheral blood) associated with three adiposity phenotypes (BMI, waist circumference, and impedance-measured percent body fat) among American Indian adult participants in the Strong Heart Study. Results: We found differential methylation at 8264 CpG sites associated with at least one of our three response variables. Of the three adiposity proxies we measured, waist circumference had the highest number of associated differentially methylated CpGs, while percent body fat was associated with the lowest. Because both waist circumference and percent body fat relate to physiology, we focused interpretations on these variables. We found a low degree of overlap between these two variables in our gene ontology enrichment and Differentially Methylated Region analyses, supporting that waist circumference and percent body fat measurements represent biologically distinct concepts. Conclusions: We interpret these general findings to indicate that highly significant regions of the genome (DMR) and synthesis pathways (GO) in waist circumference analyses are more likely to be associated with the presence of visceral/abdominal fat than more general measures of adiposity. Our findings confirmed numerous CpG sites previously found to be differentially methylated in association with adiposity phenotypes, while we also found new differentially methylated CpG sites and regions not previously identified.

Fibrinogen plays an essential role in blood coagulation and inflammation. Circulating fibrinogen levels may be determined based on interindividual differences in DNA methylation at cytosine-phosphate-guanine (CpG) sites and vice versa.

Background:Epidemiologic evidence supports lead and cadmium as CVD risk factors. In the Trial to Assess Chelation Therapy (TACT), edetate disodium infusions lowered CVD risk in patients with a prior myocardial infarction. The impact of repeated chelation on established biomarkers of metal body burden, post-chelation lead and pre-chelation urine cadmium, is unknown.Objective:To assess metals in urine samples collected at the first and at 10 or more infusions with edetate disodium. We hypothesized that post-chelation urine lead and pre-chelation urine cadmium would be lower after 10 infusions compared to the first infusion. Other metals were exploratory.Methods:13 patients with coronary artery disease (CAD) received a median of 3 g of edetate disodium over 3h per infusion and a median of 51 infusions (range 22-61). We collected pre and post-chelation urine samples at infusions 1 and ≥10. Metals were analyzed with ICPMS and controlled for urine dilution. Pre and post-chelation geometric means (GM) of urine metals at the ≥10 vs. first infusion were compared using paired T-tests.Results:Mean (SD) age was 75 (11) years, 77% participants were men, 46% were past smokers, 92% had diabetes, and 92% had peripheral artery disease. Comparing infusions ≥10 to 1 (Table), the GM (μg/g creatinine) dropped from 0.45 to 0.03 (94% reduction; p=0.005) for pre-chelation lead and from 14.80 to 2.91 (80% reduction; p=0.04) for post-chelation lead. For pre and post-chelation cadmium, the corresponding figures were 0.34 to 0.12 (64% reduction; p=0.17) and 2.81 to 2.35 (16% reduction; p=0.48), respectively.Conclusions:TACT treatment may decrease total body burden of lead, further supporting lead is a modifiable CVD risk factor. Data are insufficient to inform on changes in the burden of cadmium or other metals. Ongoing, larger studies will assess the effect of repeated chelation on toxic metal burden and incident cardiovascular disease.

Background:Biological aging assessed by both leukocyte telomere length (LTL) and DNA methylation (DNAm) has been associated with CVD and its risk factors. Moreover, LTL is epigenetically regulated. We hypothesized that LTL-associated epigenetic changes are associated with risk of CVD in the community.Objective:To test whether LTL-associated loci are associated with incident CVD, independent of standard risk factors in multi-ethnic cohorts.Method:We evaluated 3,628 participants with complete LTL and DNAm data in four prospective cohorts, including 1,531 American Indians from the Strong Heart Study (SHS, mean age 56, 60% women), 821 non-Hispanic Whites (NHW) from the Framingham Heart Study (FHS, mean age 60, 51% women), 471 NHW, 150 Hispanics, and 162 African Americans from the Multi-ethnic Study of Atherosclerosis (MESA, mean age 70 , 55% women), and 471 NHW and 342 African Americans from the Women’s Health Initiative (WHI, mean age 65 , all women). LTL was quantified by qPCR (SHS, MESA) or Southern blot (FHS, WHI). DNAm was assayed by Illumina EPIC (SHS) or 450K (FHS, MESA and WHI) arrays. We imputed 450K to 850K using random forest algorithms. CVD events included fatal and nonfatal MI, CHD, heart failure, stroke, peripheral artery diseases, and cardiovascular deaths. Cohort-specific EWAS was conducted to identify CpGs associated with LTL, adjusting for age, sex, race/ethnicity, smoking, alcohol, BMI, site, cell proportion, and batch. Multiple testing was Bonferroni-corrected (genome-wide P< 2.4 x10-7). Results across studies were combined by random-effects meta-analysis. To examine whether LTL-associated epigenetic loci are associated with CVD risk, we used a weighted methylation score to predict incident CVD by Cox regression, adjusting for age, sex, site, smoking, alcohol, BMI, glucose, SBP, LDL-C, and total cholesterol.Results:We ascertained 2,001 CVD events, including 986 in the SHS (average follow-up 15.2 years), 208 in FHS (average follow-up 7.7 years), 74 in MESA (average follow-up 4.9 years), and 733 in WHI (average follow-up 12.2 years). Meta-EWAS identified 22 CpGs (mapped to 17 unique genes) associated with LTL. Of these, 19 loci (15 negatively and 4 positively associated with LTL) had consistent directionality of association across four cohorts. The most significant genes harboring altered CpG sites included TLL2(cg10549018, P=2.42 x 10-12) and TPST1 (cg10691866, P=8.6 x 10-10). A higher composite methylation score, which reflects longer LTL (P<0.0001), was significantly associated with a reduced risk of CVD in the SHS (HR=0.16, 95% CI: 0.07–0.37), FHS (HR=0.08, 95% CI: 0.01–0.40), and WHI (HR=0.32, 95%CI: 0.13–0.82), but not MESA (HR=0.47, 95% CI: 0.04–5.09).Conclusion:Altered DNA methylation at 19 CpG loci was significantly associated with LTL. Their combined effects may predict a reduced risk of CVD. The observed associations warrant further investigation.

Background:In the US, American Indians suffer a disproportionate burden of CHD compared to other racial/ethnic groups. Additional strategies are needed to identify individuals at risk.Objectives:Investigate the association of blood DNA methylation (DNAm) with incident CHD in the Strong Heart Study and the prediction ability of DNAm beyond traditional risk factors. We maximized prediction ability using Bayesian Hierarchical Cox (BHCox) and Survival Random Forest models, which allow large numbers of CpGs in a single model, instead of considering them individually.Methods:Among 2325 men and women 45-74 years old in 1989-1991, 557 CHD events were identified over 20 years of follow-up. DNAm was measured in 790,026 CpGs, pre-processed and corrected for batch effects. We ran adjusted BHCox models for subsets of CpGs selected from similarly adjusted Cox models for individual CpGs. Prediction ability of CpGs in BHCox was further evaluated with Survival Random Forest, which is robust to overfitting. We also conducted a targeted analysis using Cox regression.Results:26 CpGs associated with CVD in previous studies were nominally associated with incident CHD in a targeted analysis. The cross-validated C index for the model with traditional risk factors was 0.703. In BHCox, further entering 30K CpGs in a single model, resulted in 231 CpGs being significantly associated with incident CHD, with a cross-validation C statistic of 0.855. In Survival Random Forest, further entering the 231 CpGs from the BHCox model resulted in a cross-validation C statistic of 0.771 and 182 CpGs with variable importance (VIMP) greater than zero. The top CpGs in BHCox were also VIMP>0 in random forest models and were located in SLC24A1(calcium exchanger), SHBG(sex hormone binding globulin), and LINC00346(non-protein coding RNA 346).Conclusions:We found novel CpG sites prospectively associated with incident CHD and confirmed signals from previous studies. DNAm might help to identify individuals at high risk of developing CHD.