Within-person Reproducibility Research Articles

Abstract B072: Reproducibility of plasma metabolome over 1 year in a population-based study of Black breast cancer survivors

Abstract Background: African American/Black women have higher rates of breast cancer (BrCa) mortality and morbidity than White women. The metabolomics approach using blood samples from epidemiological studies has the potential to elucidate pathways or uncover biomarkers for adverse BrCa outcomes. Therefore, understanding the within-person reproducibility of the blood metabolome of BrCa survivors and factors that influence metabolite levels over time is crucial. However, the stability of blood metabolome among Black BrCa survivors is unknown. Objective: This study aimed to estimate the within-person reproducibility of metabolites over 1 year via global untargeted metabolomic profiling of plasma samples collected in the Women’s Circle of Health Follow-Up Study (WCHFS), a population-based longitudinal study of Black BrCa survivors in New Jersey. The study also aims to identify factors associated with reproducibility. Methods: We estimated the metabolite reproducibility among 61 Black women with BrCa diagnosed between 2012 and 2017 in WCHFS. Plasma samples were collected once for each participant at ~2 and 3 years post-diagnosis, respectively, and analyzed at Metabolon via ultrahigh performance liquid chromatography-tandem mass spectrometry. Using a mixed-effects model, we calculated the intraclass correlation coefficients (ICC) for each metabolite by dividing the between-person variance by the total variance (i.e., within- and between-person variances). ICCs were compared by age, obesity status, estrogen receptor (ER) status, and seasons, fasting status and time interval between two visits when blood samples were collected. Statistical significance was determined using the Wilcoxon test. Results: Of the 857 named metabolites identified after excluding those undetectable in ≥80% of samples or with a coefficient of variance≥0.25, the median ICC was 0.58 (interquartile range [IQR]: 0.44-0.70). 16.6% had high within-person reproducibility (ICC≥0.75), spanning across all metabolite classes, while 65.6% had an ICC within 0.4-0.75, and the remaining 17.9% had an ICC<0.4. Significant differences in ICCs were noted by participants’ age (median ICC [IQR; same below]: 0.56 [0.40 - 0.68] for younger women, i.e. ≤ 55.6 years vs. 0.61 [0.44 - 0.73] for older women), obesity status (0.55 [0.40 - 0.68] for non-obese vs. 0.62 [0.46 - 0.75] for obese), and ER status (0.56 [0.41 - 0.68] for ER+ vs. 0.62 [0.45 - 0.79] for ER-). The reproducibility also differed by fasting status (0.63 [0.45 - 0.77] for fasting vs. 0.53 [0.39 - 0.67] for non-fasting), but not the seasons or time interval between blood collections. Conclusion: The within-person reproducibility of plasma metabolites over 1 year among Black breast cancer survivors was generally reasonable, suggesting a single-time-point measurement could be useful in evaluating associations between metabolites and BrCa outcomes. However, the reproducibility varied across metabolites and was influenced by participant characteristics and certain sample collection conditions, which need consideration in future analysis or design. Citation Format: Bo Qin, Madhir Vyas, Steven C. Moore, Xiaoyang Su, Eileen White, Christine B. Ambrosone, Kitaw Demissie, Chi-Chen Hong, Elisa V. Bandera. Reproducibility of plasma metabolome over 1 year in a population-based study of Black breast cancer survivors [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B072.

Within-person reproducibility of proteoforms related to inflammation and renal dysfunction.

Protein biomarkers and microheterogeneity have attracted increasing attention in epidemiological and clinical research. Knowledge of within-person reproducibility over time is paramount to determine whether a single measurement accurately reflects an individual’s long-term exposure. Yet, research investigating within-person reproducibility for proteoforms is limited. We investigated the reproducibility of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9), and the renal function marker cystatin C (CnC) using a novel immuno-MALDI-TOF MS assay. Reproducibility, expressed as intraclass correlation coefficient (ICC), was calculated for 16 proteoforms using plasma samples of the Western Norway B Vitamin Intervention Trial (WENBIT) cohort collected 1–3 y apart from 295 stable angina pectoris (SAP) patients and 16 weeks apart from 38 subjects of the Intervention with Omega Fatty Acids in High-risk Patients with Hypertriglyceridemic Waist (OMEGA) trial with abdominal obesity but no other documented co-morbidities. ICCs for inflammatory markers were lower in WENBIT (CRP: 0.51, SAAt: 0.38, S100At: 0.31) compared to OMEGA subjects (CRP: 0.71, SAAt: 0.73, S100At: 0.48), while comparable for CnCt (WENBIT: 0.69, OMEGA: 0.67). Excluding SAP patients with elevated inflammation (CRP > 10 µg/ml) increased the ICC of SAAt to 0.55. Reduction of the time interval from 3 to 1 y in WENBIT group increased ICCs for all proteoforms. With a few exceptions ICCs did not differ between proteoforms of the same biomarker. ICCs were highest in OMEGA subjects with fair-to-good reproducibility for all markers. Reproducibility of SAA and S100A8/9 proteoforms in the WENBIT cohort was related to inflammation. This work will inform future clinical and epidemiological research which relies on single time point biomarker assessment to investigate inflammation and renal function.

Reproducibility of serum cytokines in an elderly population

BackgroundIt is important to assess the temporal reproducibility of circulating cytokines for their utility in epidemiological studies. However, existing evidence is limited and inconsistent, especially for the elderly population.MethodsSixty-five elderly (mean age = 77.89 ± 6.14 years) subjects were randomly selected from an existing prospective cohort study. Levels of 41 cytokines in 195 serum samples, collected at three separate visits that were up to 15.26 years apart, were measured by the Luminex technology. The temporal reproducibility of cytokines was estimated by the intraclass correlation coefficient (ICC) calculated using a mixed-effects model. In addition, data analyses were stratified by the median (4.49 years) of time intervals across sample collection. Sensitivity analyses were performed when excluding subjects with undetectable samples.ResultsA total of 23 cytokines were detectable in more than 60% of samples. Fair to good (ICC = 0.40 to 0.75) and excellent (ICC > 0.75) reproducibility was found in 10 (Eotaxin, VEGF, FGF-2, G-CSF, MDC, GM-CSF, TGFα, IP-10, MIP-1β, IL-1RA) and 5 (GRO, IFNγ, IL-17, PDGF-AA, IL-4) cytokines, respectively. The results were not changed dramatically in the stratification and sensitivity analyses.ConclusionsSerum levels of the selected 15 cytokines measured with Luminex technology displayed fair to excellent within-person temporal reproducibility among elderly population.

Trends and Patterns of Phthalates and Phthalate Alternatives Exposure in Pregnant Women from Mexico City during 2007-2010.

Phthalates are associated with several adverse health outcomes, but few studies have evaluated phthalate exposures in Mexican populations, particularly pregnant women. Between 2007 and 2011, 948 pregnant women from Mexico City were recruited as part of the PROGRESS cohort. We quantified 17 metabolites of phthalates and phthalate alternatives in urine samples collected during the second and third trimesters and examined temporal trends of metabolite concentrations, within-person reproducibility, and relations of individual metabolites with sociodemographic, lifestyle, and occupational factors. Concentrations of mono-2-ethyl-5-carboxypentyl terephthalate, a metabolite of the alternative phthalate di-2-ethylhexyl terephthalate, increased monotonically from 2007 to 2010 (31% per year; 95% confidence interval = 23 and 39%). We observed moderate to high correlations among metabolites collected at the same visit, but there was high variability between second and third trimester phthalate metabolite concentrations (intraclass correlation coefficients = 0.17-0.35). In general, higher socioeconomic status was associated with higher phthalate concentrations. Some metabolites were associated with maternal age and education, but no consistent patterns were observed. Women working in the home and those who worked in administration had higher concentrations of several phthalate metabolites relative to students, professionals, and those in customer service. Biomonitoring efforts are warranted to investigate present and future exposure trends and patterns.

Pre-Analytical and Within-Person Reproducibility of Nutritional Metabolomics over 2 Years in Elders at Risk for Dementia (P18-121-19)

ObjectivesNutritional metabolomics to objectively assess dietary intake in aging permit the opportunity to circumvent measurement errors that accompany subjective means of dietary assessment. At the same time, they may offer insights into mechanisms of action and metabolic disturbances that are actionable targets for modulation through diet in hopes of disease prevention and treatment. However, prior to more broad deployment the pre-analytical and temporal variation over time should be documented in order to design and interpret epidemiological studies properly. We quantified and examined 155 nutrient biomarkers and metabolites selected for their potential relevance to dementia. MethodsBlood samples from three time points, spanning a 2-year period, were obtained from older adults participating in the NIA-Layton Oregon Alzheimer’s Disease Center’s Nutrition and Brain Aging Study (NBAS). Blood samples were batched randomly across three time points for quantification of blood amino acids, minerals, water and fat-soluble micronutrients, lipids, one carbon, and kynurenine pathway metabolites using a variety of methods including, tandem mass spectrometry. Pre-analytical coefficients of variation (CV) were calculated for all the biomarkers and intraclass correlation coefficients (ICC) were calculated to evaluate the within-person reproducibility in a subset of 137 participants. ResultsThe mean baseline age of the analytic sample (n = 137) was 85.6 (± 8.3, 57 - 101 years), 70% are female, 21% carry the ApoEe4 allele and MMSE was 28.3 (± 1.78). The pre-analytical CVs ranged from 0.9% to 55.0% and the ICC ranged from 0 to 0.87 (25%-tile/median/75%-tile 0.41/0.54/0.66). Twenty four % had ICC < 0.40, 66% had ICC 0.40 −0.75 and 10% had ICC > 0.75. ConclusionsThe pre-analytical and within-person reproducibility of nutritional metabolomics in aging ranges widely. The majority can reliably estimate average concentrations over a 2 year period from a single time point and the biomarkers with ICC’s above 0.40 can be used for correction of measurement error and those below 0.40 should consider multiple samples per subject and exploring the methodological and biological explanation for the variation over time. Funding SourcesNestle Institute of Health Sciences, Hinda and Arthur Marcus Institute for Aging Research, NIA-Layton Aging & Alzheimer’s Disease Center (P30AGO8017).

Polyclonal human antibodies against glycans bearing red meat-derived non-human sialic acid N-glycolylneuraminic acid are stable, reproducible, complex and vary between individuals: Total antibody levels are associated with colorectal cancer risk.

BackgroundN-glycolylneuraminic acid (Neu5Gc) is a non-human red-meat-derived sialic acid immunogenic to humans. Neu5Gc can be metabolically incorporated into glycan chains on human endothelial and epithelial surfaces. This represents the first example of a “xeno-autoantigen”, against which circulating human “xeno-autoantibodies” can react. The resulting inflammation (“xenosialitis”) has been demonstrated in human-like Neu5Gc-deficient mice and contributed to carcinoma progression via antibody-mediated inflammation. Anti-Neu5Gc antibodies have potential as biomarkers for diseases associated with red meat consumption such as carcinomas, atherosclerosis, and type 2 diabetes.MethodsELISA assays measured antibodies against Neu5Gc or Neu5Gc-glycans in plasma or serum samples from the Nurses’ Health Studies, the Health Professionals Follow-up Study, and the European Prospective Investigation into Cancer and Nutrition, including inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over 1–3 years in archived samples. We also assessed associations between antibody levels and coronary artery disease risk (CAD) or red meat intake. A glycan microarray was used to detected antibodies against multiple Neu5Gc-glycan epitopes. A nested case-control study design assessed the association between total anti-Neu5Gc antibodies detected in the glycan array assay and the risk of colorectal cancer (CRC).ResultsELISA assays showed a wide range of anti-Neu5Gc responses and good inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over time, but these antibody levels did not correlate with CAD risk or red meat intake. Antibodies against Neu5Gc alone or against individual Neu5Gc-bearing epitopes were also not associated with colorectal cancer (CRC) risk. However, a sialoglycan microarray study demonstrated positive association with CRC risk when the total antibody responses against all Neu5Gc-glycans were combined. Individuals in the top quartile of total anti-Neu5Gc IgG antibody concentrations had nearly three times the risk compared to those in the bottom quartile (Multivariate Odds Ratio comparing top to bottom quartile: 2.98, 95% CI: 0.80, 11.1; P for trend = 0.02).ConclusionsFurther work harnessing the utility of these anti-Neu5Gc antibodies as biomarkers in red meat-associated diseases must consider diversity in individual antibody profiles against different Neu5Gc-bearing glycans. Traditional ELISA assays for antibodies directed against Neu5Gc alone, or against specific Neu5Gc-glycans may not be adequate to define risk associations. Our finding of a positive association of total anti-Neu5Gc antibodies with CRC risk also warrants confirmation in larger prospective studies.

Adjusting urinary chemical biomarkers for hydration status during pregnancy

One way of assessing a population’s exposure to environmental chemicals is by measuring urinary biomarker concentrations, which can vary depending on the hydration status of the individual. The physiological changes that occur during pregnancy can impact the hydration adjustment approaches, such as calculating the individual’s urinary flow rate (UFR), or adjusting concentrations using specific gravity (SG) or creatinine. A total of 1260 serial spot urine samples were collected from 80 women, averaging 32.4 years of age, throughout and shortly after pregnancy. The relationship between each approach was examined and time of day and across pregnancy differences were tested using linear mixed models. The correlation between the calculated excretion rate and each of the adjustment techniques was examined on a selection of seven phthalate metabolites. Based on the linear mixed model results, we found that UFR and creatinine excretion rates differed systematically across the population, with respect to body mass index (BMI) and time. SG differed with respect to BMI, but there were no systematic time trends. SG had the highest within-person reproducibility, according to the intraclass correlation coefficient (ICC). The excretion rate of each of the phthalates was most strongly correlated with the SG-standardized concentration. This analysis showed that SG showed a slightly better within-person reproducibility and the least amount of systematic variation when compared to creatinine adjustment. Therefore, SG correction appears to be a favorable approach for correcting for the hydration status of the pregnant women from this cohort.

Performance of plasma trigonelline as a marker of coffee consumption in an epidemiologic setting

Coffee is a widely consumed beverage, and studies suggest that drinking coffee has beneficial health effects. The phytohormone trigonelline is present in large amounts in coffee beans, and circulating concentrations of trigonelline have been shown to be positively related to dietary intake of coffee and to increase significantly after the consumption of a bolus dose of coffee. We cross-sectionally investigated the utility of plasma trigonelline as a marker of coffee consumption in an epidemiologic setting. We secondarily investigated if coffee intake is related to plasma concentrations of vitamin B-3 (niacin) forms. In a Norwegian cohort of 3503 participants, we combined questionnaire data on the number of cups of coffee consumed per day with plasma trigonelline to evaluate trigonelline as a marker of coffee intake. The suitability of plasma trigonelline to discriminate those not consuming from those consuming coffee was investigated by receiver operating characteristic (ROC) analysis. Plasma collected at 2 time points 1 y apart was used to determine the within-person reproducibility of trigonelline. We found that plasma trigonelline concentrations increased strongly with increasing amounts of coffee consumed. ROC analysis showed that trigonelline had an area under the curve of 0.92 (95% CI: 0.90, 0.94) for distinguishing coffee abstainers from coffee drinkers. Plasma trigonelline had a good within-person reproducibility (0.66; 95% CI: 0.64, 0.68) for samples collected 1 y apart. The amount of coffee consumed was not associated with plasma concentrations of the niacin vitamers nicotinamide and N1-methylnicotinamide. Plasma trigonelline performs well as a marker of coffee intake. Data used in this study were derived from the clinical trial registered at www.clinicaltrials.gov as NCT00354081.

Reproducibility of serum oxysterols and lanosterol among postmenopausal women: Results from EPIC-Heidelberg

BackgroundCirculating oxysterols have been proposed as biological markers of disease risk. However, within-person reproducibility of circulating oxysterols over time is not well established. MethodsWe evaluated the one-year reproducibility of 11 oxysterols and lanosterol among 30 postmenopausal women with repeat blood samples in the European Prospective Investigation into Cancer and Nutrition (EPIC) - Heidelberg, Germany cohort. Liquid chromatography–mass spectrometry (LC/MS) was performed to quantify serum concentrations of 22R–hydroxycholesterol, 25-hydroxycholesterol, 24S-hydroxycholesterol, 27-hydroxycholesterol, 22S-hydroxycholeterol, 24,25-epoxycholesterol, 5α,6β-dihydroxycholestanol, 7α-hydroxycholesterol, 5β,6β-epoxycholesterol, 5α,6α-epoxycholesterol, 24-dihydrolanosterol, and lanosterol. We evaluated Spearman correlations and intraclass correlation coefficients (ICCs) between quantifiable concentrations measured in repeat samples taken one-year apart to estimate within-person reproducibility. ResultsSpearman correlations (ICCs) over one year ranged from 0 (ICC=0.10) for 5β,6β-epoxycholesterol and 0.10 (ICC=0.20) for 5α,6α-epoxycholesterol, representing low within-person stability, to 0.81 (ICC=0.75) for 27-hydroxycholesterol and 0.86 (ICC=0.91) for 24S–hydroxycholesterol, representing relatively high within-person stability. Correlations between oxysterols and lanosterol ranged from 0.01 between 24S-hydroxycholesterol and lanosterol to 0.70 between 5α,6α-epoxycholesterol and 5β,6β-epoxycholesterol. ConclusionsOur results demonstrate that for 27-hydroxycholesterol, 24S–hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol and lanosterol, a single serum measurement can reliably estimate average levels over a one-year period. Circulating oxysterols are of increasing interest in epidemiologic studies of chronic disease risk including cancer and cardiovascular disease. Our data suggest that within-person stability of oxysterols differs depending on the individual oxysterol evaluated. We identified four oxysterols and lanosterol as stable over time to inform the use of circulating oxysterols in epidemiologic studies.

Prospective Associations of Systemic and Urinary Choline Metabolites with Incident Type 2 Diabetes.

Several compounds in the choline oxidation pathway are associated with insulin resistance and prevalent diabetes; however, prospective data are scarce.We explored the relationships between systemic and urinary choline-related metabolites and incident type 2 diabetes in an observational prospective study among Norwegian patients. We explored risk associations by logistic regression among 3621 nondiabetic individuals with suspected stable angina pectoris, of whom 3242 provided urine samples. Reclassification of patients was investigated according to continuous net reclassification improvement (NRI >0). After median (25th to 75th percentile) follow-up of 7.5 (6.4-8.7) years, 233 patients (6.4%) were registered with incident type 2 diabetes. In models adjusted for age, sex, and fasting status, plasma betaine was inversely related to new-onset disease [odds ratio (OR) per 1 SD, 0.72; 95% CI, 0.62-0.83; P < 0.00001], whereas positive associations were observed for urine betaine (1.25; 1.09-1.43; P = 0.001), dimethylglycine (1.22; 1.06-1.40; P = 0.007), and sarcosine (1.30; 1.13-1.49; P < 0.001). The associations were maintained in a multivariable model adjusting for body mass index, hemoglobin A1c, urine albumin-to-creatinine ratio, estimated glomerular filtration rate, C-reactive protein, HDL cholesterol, and medications. Plasma betaine and urine sarcosine, the indices most strongly related to incident type 2 diabetes, improved reclassification [NRI >0 (95% CI) 0.33 (0.19-0.47) and 0.16 (0.01-0.31), respectively] and showed good within-person reproducibility. Systemic and urinary concentrations of several choline metabolites were associated with risk of incident type 2 diabetes, and relevant biomarkers may improve risk prediction.

Within-person reproducibility of red blood cell mercury over a 10- to 15-year period among women in the Nurses' Health Study II.

Most epidemiologic studies of methylmercury (MeHg) health effects rely on a single measurement of a MeHg biomarker to assess long-term exposures. Long-term reproducibility data are, therefore, needed to assess the reliability of a single measure to reflect long-term exposures. In this study, we assessed within-person reproducibility of red blood cell (RBC) mercury (Hg), a marker of methyl-mercury, over 10-15 years in a sample of 57 women. Fifty-seven women from the Nurses' Health Study II provided two blood samples 10-15-years apart (median: 12 years), which were analyzed for mercury levels in the red blood cells (B-Hg*). To characterize within-person reproducibility, we estimated correlation and intraclass correlation coefficients (r and ICC) across the two samples. Further, we compared different prediction models, including variables on fish and seafood consumption, for B-Hg* at the first sample, using leave-one-out cross-validation to assess predictive ability. Overall, we observed strong correlations over 10-15 years (r=0.69), as well as a high ICC (0.67; 95% CI: 0.49, 0.79). Fish and seafood consumption reported concurrently with the first B-Hg* sample accounted for 26.8% of the variability in that B-Hg*, giving a correlation of r=0.52. Despite decreasing B-Hg* levels over time, we observed strong correlations and high ICC estimates across B-Hg* measured 10-15 years apart, suggesting good relative within-person stability over time. Our results indicate that a single measurement of B-Hg* likely is adequate to represent long-term exposures.

Most Blood Biomarkers Related to Vitamin Status, One-Carbon Metabolism, and the Kynurenine Pathway Show Adequate Preanalytical Stability and Within-Person Reproducibility to Allow Assessment of Exposure or Nutritional Status in Healthy Women and Cardiovascular Patients1–3

Knowledge of stability during sample transportation and changes in biomarker concentrations within person over time are paramount for proper design and interpretation of epidemiologic studies based on a single measurement of biomarker status. Therefore, we investigated stability and intraindividual vs. interindividual variation in blood concentrations of biomarkers related to vitamin status, one-carbon metabolism, and the kynurenine pathway. Whole blood (EDTA and heparin, n = 12) was stored with an icepack for 24 or 48 h, and plasma concentrations of 38 biomarkers were determined. Stability was calculated as change per hour, intraclass correlation coefficient (ICC), and simple Spearman correlation. Within-person reproducibility of biomarkers was expressed as ICC in samples collected 1–2 y apart from 40 postmenopausal women and in samples collected up to 3 y apart from 551 patients with stable angina pectoris. Biomarker stability was similar in EDTA and heparin blood. Most biomarkers were essentially stable, except for choline and total homocysteine (tHcy), which increased markedly. Within-person reproducibility in postmenopausal women was excellent (ICC > 0.75) for cotinine, all-trans retinol, cobalamin, riboflavin, α-tocopherol, Gly, pyridoxal, methylmalonic acid, creatinine, pyridoxal 5′-phosphate, and Ser; was good to fair (ICC of 0.74–0.40) for pyridoxic acid, kynurenine, tHcy, cholecalciferol, flavin mononucleotide, kynurenic acid, xanthurenic acid, 3-hydroxykynurenine, sarcosine, anthranilic acid, cystathionine, homoarginine, 3-hydroxyanthranilic acid, betaine, Arg, folate, total cysteine, dimethylglycine, asymmetric dimethylarginine, neopterin, symmetric dimethylarginine, and Trp; and poor (ICC of 0.39–0.15) for methionine sulfoxide, Met, choline, and trimethyllysine. Similar reproducibilities were observed in patients with coronary heart disease. Thus, most biomarkers investigated were essentially stable in cooled whole blood for up to 48 h and had a sufficient within-person reproducibility to allow one-exposure assessment of biomarker status in epidemiologic studies. The Western Norway B Vitamin Intervention Trial was registered at clinicaltrials.gov> as NTC00354081.

Reproducibility of Metabolomic Profiles among Men and Women in 2 Large Cohort Studies

Rigorous studies are necessary to demonstrate suitability of metabolomics platforms to profile metabolites in archived plasma within epidemiologic studies of human disease, for which attenuation of effect estimates due to measurement error is a key concern. Using a liquid chromatography-tandem mass spectrometry platform, we quantified 257 metabolites from archived plasma to evaluate metabolite interassay reproducibility, reproducibility with delayed processing, and within-person reproducibility over time. Interassay reproducibility was assessed with CVs from 60 duplicate plasma samples donated by participants in the Nurses' Health Study and Health Professionals Follow-up Study, and 20 QC pool plasma replicates. Metabolite reproducibility over a 24- to 48-h processing delay (n = 48 samples) and within-person reproducibility over 1-2 years (n = 80 samples) were assessed using Spearman and intraclass correlation coefficients (ICCs). CVs were <20% for 92% of metabolites and generally were similar by plasma anticoagulant type (heparin or EDTA) and fasting time. Approximately 75% of metabolites were reproducible over delays in processing of blood samples (Spearman correlation or ICC ≥ 0.75, comparing immediate and 24-h delayed processing). Carbohydrates and purine/pyrimidine derivatives were most adversely affected by the processing delay. Ninety percent of metabolites were reproducible over 1-2 years within individuals (Spearman correlation or ICC ≥ 0.4). For potential use in epidemiologic studies, the majority of plasma metabolites had low CVs and were reproducible over a 24-h processing delay and within individuals over 1-2 years. Certain metabolites, such as carbohydrates and purine/pyrimidine derivatives, may be challenging to evaluate if samples have delayed processing.

Within-person reproducibility of urinary bisphenol A and phthalate metabolites over a 1 to 3 year period among women in the Nurses’ Health Studies: a prospective cohort study

BackgroundAssociations of bisphenol A and phthalates with chronic disease health outcomes are increasingly being investigated in epidemiologic studies. The majority of previous studies of within-person variability in urinary bisphenol A and phthalate metabolite concentrations have focused on reproducibility over short time periods. Long-term reproducibility data are needed to assess the potential usefulness of these biomarkers for prospective studies, particularly those examining risk of diseases with long latency periods. Low within-person reproducibility may attenuate relative risk estimates and reduce statistical power to detect associations with disease. Therefore, we assessed within-person reproducibility of bisphenol A, eight phthalate metabolites, and phthalic acid in spot urine samples over 1 to 3 years among women enrolled in two large cohort studies.MethodsWomen in the Nurses’ Health Study and Nurses’ Health Study II provided two spot urine samples, 1 to 3 years apart (n = 80 women for analyses of bisphenol A; n = 40 women for analyses of phthalate metabolites; n = 34 women for analyses of phthalic acid). To measure within-person reproducibility, we calculated Spearman rank correlation coefficients and intraclass correlation coefficients for creatinine-adjusted concentrations of bisphenol A, phthalate metabolites, and phthalic acid.ResultsOver 1 to 3 years, within-person variability of bisphenol A was high relative to total variability (intraclass correlation coefficient = 0.14) and rankings of bisphenol A levels between time-points were weakly correlated (Spearman correlation = 0.19). Seven of the eight phthalate metabolites and phthalic acid demonstrated moderate within-person stability over time (Spearman correlation or intraclass correlation coefficient = 0.39-0.55). Restricting analyses to first-morning urine samples did not alter results.ConclusionsSingle measurements of bisphenol A in spot urine samples were highly variable within women over 1 to 3 years, indicating that investigation of associations between a single urinary bisphenol A measurement and disease risk may be challenging in epidemiologic studies. The majority of urinary phthalate metabolites and phthalic acid appeared moderately reproducible within women over time, suggesting single measurements may be useful in epidemiologic studies, although observed relative risks can be substantially attenuated.

Abstract 35: Within-person reproducibility of urinary bisphenol A and phthalate metabolites among women in the Nurses' Health Studies.

Abstract The purpose of our study was to examine the long-term within-person reproducibility of bisphenol A (BPA) and 8 phthalate metabolites. Associations of BPA and phthalates with health are increasingly being investigated in epidemiologic studies, although existing studies are largely cross-sectional. Most previous studies of within-person variability in urinary BPA and phthalate metabolite concentrations have focused on stability over 3 months or less. Long-term reproducibility data are needed to assess the potential usefulness of these biomarkers for predicting risk of disease onset in prospective studies. We measured BPA and 8 phthalate metabolites in spot urine samples donated 1 to 3 years apart by women in the Nurses' Health Study and Nurses' Health Study II (n=80 for analyses of BPA; n=40 for phthalate metabolites); 86% of samples were first-morning urines. Specific phthalate metabolites were mono-ethyl phthalate (MEP), mono-isobutyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono-benzyl phthalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydrohexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono-(2-ethyl-5carboxypentyl) phthalate (MECPP). To measure within-person reproducibility, we calculated Spearman rank correlation coefficients and intraclass correlations (ICCs) for creatinine-adjusted BPA and phthalate metabolite concentrations; ICCs were estimated using natural log-transformed values. Additional analyses were restricted to first-morning urine samples. Distributions (medians [5th to 95th percentiles]) of creatinine-adjusted analyte concentrations (ng/mg creatinine) were: 3 (1-6) for BPA, 141 (21-895) for MEP, 4 (1-12) for MiBP, 37 (10-177) for MnBP, 14 (4-97) for MBzP, 5 (2-26) for MEHP, 32 (9-163) for MEHHP, 26 (7-136) for MEOHP, and 43 (16-215) for MECPP. Over 1 to 3 years, within-person variability of BPA was high relative to total variability (ICC=0.14) and rankings of BPA levels between time-points were weakly correlated (Spearman correlation=0.19). All phthalate metabolites, except MEHP, demonstrated moderate within-person stability over time (Spearman correlation or ICC=0.39-0.55). Restricting analyses to first-morning urine samples did not alter results. Overall, we found that single measurements of BPA in spot urine samples were highly variable within women over 1 to 3 years. However, the majority of urinary phthalate metabolites appeared moderately reproducible within women over time. Our results suggest that investigation of associations between a single urinary BPA measurement and disease risk likely will be challenging in epidemiologic studies, while single measurements of urinary phthalate metabolites are reasonable measures of long-term exposure. Further, reproducibility data could be used to correct for within-person variability that results in the attenuation of estimates of relative risk. Citation Format: Mary Townsend, Adrian Franke, Xingnan Li, Frank Hu, A. Heather Eliassen. Within-person reproducibility of urinary bisphenol A and phthalate metabolites among women in the Nurses' Health Studies. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 35.

Reproducibility of ad libitum energy intake with the use of a computerized vending machine system

Accurate assessment of energy intake is difficult but critical for the evaluation of eating behavior and intervention effects. Consequently, methods to assess ad libitum energy intake under controlled conditions have been developed. Our objective was to evaluate the reproducibility of ad libitum energy intake with the use of a computerized vending machine system. Twelve individuals (mean + or - SD: 36 + or - 8 y old; 41 + or - 8% body fat) consumed a weight-maintaining diet for 3 d; subsequently, they self-selected all food with the use of a computerized vending machine system for an additional 3 d. Mean daily energy intake was calculated from the actual weight of foods consumed and expressed as a percentage of weight-maintenance energy needs (%WMEN). Subjects repeated the study multiple times during 2 y. The within-person reproducibility of energy intake was determined through the calculation of the intraclass correlation coefficients (ICCs) between visits. Daily energy intake for all subjects was 5020 + or - 1753 kcal during visit 1 and 4855 + or - 1615 kcal during visit 2. There were no significant associations between energy intake and body weight, body mass index, or percentage body fat while subjects used the vending machines, which indicates that intake was not driven by body size or need. Despite overconsumption (%WMEN = 181 + or - 57%), the reproducibility of intake between visits, whether expressed as daily energy intake (ICC = 0.90), %WMEN (ICC = 0.86), weight of food consumed (ICC = 0.87), or fat intake (g/d; ICC = 0.87), was highly significant (P < 0.0001). Although ad libitum energy intake exceeded %WMEN, the within-person reliability of this intake across multiple visits was high, which makes this a reproducible method for the measurement of ad libitum intake in subjects who reside in a research unit. This trial was registered at clinicaltrials.gov as NCT00342732.

Reproducibility of fifteen urinary estrogens and estrogen metabolites over a 2- to 3-year period in premenopausal women.

Endogenous estrogens play an integral role in the etiology of breast, endometrial, and, possibly, ovarian cancers. Estrogen metabolism yields products that are potentially both estrogenic and genotoxic, yet individual metabolic patterns are just beginning to be explored in epidemiologic studies. Within the Nurses' Health Study II, we examined reproducibility of 15 urinary estrogens and estrogen metabolites (EM) among 110 premenopausal women with three luteal-phase urine samples collected over 3 years. EM were measured by a recently developed high-performance liquid chromatography-tandem mass spectrometry (LC-MS(2)) method with high sensitivity, specificity, and precision. We assessed Spearman correlations and intraclass correlation coefficients (ICC) across the three samples. Correlations between urinary estrone or estradiol and EM were only modest (r = 0.1-0.5). The 2- and 4-hydroxylation pathways were highly correlated (r = 0.9) but weakly inversely correlated with the 16-hydroxylation pathway (r = -0.2). Within-woman reproducibility over time was fairly high for the three pathways, with ICCs ranging from 0.52 (16-hydroxylation pathway) to 0.72 (2-hydroxylation pathway). ICCs were similarly high for 2-catechols and the individual catechols (ICCs = 0.58-0.72). Individual and grouped methylated 2-catechols had fairly high ICCs (0.51-0.62), but methylated 4-catechols had low ICCs (0.14-0.27). These data indicate that, in general, urinary EM levels vary substantially among individuals compared with intraindividual variability. Within-person reproducibility over time for most EM measures is comparable to or better than that for well-vetted biomarkers such as plasma cholesterol and, in postmenopausal women, estradiol.

T-Cell Assays for Tuberculosis Infection: Deriving Cut-Offs for Conversions Using Reproducibility Data

BackgroundAlthough interferon-gamma release assays (IGRA) are promising alternatives to the tuberculin skin test, interpretation of repeated testing results is hampered by lack of evidence on optimal cut-offs for conversions and reversions. A logical start is to determine the within-person variability of T-cell responses during serial testing.Methodology/Principal FindingsWe performed a pilot study in India, to evaluate the short-term reproducibility of QuantiFERON-TB Gold In Tube assay (QFT) among 14 healthcare workers (HCWs) who underwent 4 serial QFT tests on day 0, 3, 9 and 12. QFT ELISA was repeated twice on the same sets of specimens. We assessed two types of reproducibility: 1) test-retest reproducibility (between-test variability), and 2) within-person reproducibility over time. Test-retest reproducibility: with dichotomous test results, extremely high concordance was noticed between two tests performed on the same sets of specimens: of the 56 samples, the test and re-test results agreed for all but 2 individuals (κ = 0.94). Discordance was noted in subjects who had IFN-γ values around the cut-off point, with both increases and decreases noted. With continuous IFN-γ results, re-test results tended to produce higher estimates of IFN-γ than the original test. Within-person reproducibility: when continuous IFN-γ data were analyzed, the within-person reproducibility was moderate to high. While persons with negative QFT results generally stayed negative, positive results tended to vary over time. Our data showed that increases of more than 16% in the IFN-γ levels are statistically improbable in the short-term.ConclusionsConservatively assuming that long-term variability might be at least twice higher than short-term, we hypothesize that a QFT conversion requires two conditions to be met: 1) change from negative to positive result, and 2) at least 30% increase in the baseline IFN-γ response. Larger studies are needed to confirm our preliminary findings, and determine the conversion thresholds for IGRAs.