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The thousands of features commonly observed when performing untargeted metabolomics with quadrupole time-of-flight (QTOF) and Orbitrap mass spectrometers often correspond to only a few hundred unique metabolites of biological origin, which is in the range of what can be assayed in a single targeted metabolomics experiment by using a triple quadrupole (QqQ) mass spectrometer. A major benefit of performing targeted metabolomics with QqQ mass spectrometry is the affordability of the instruments relative to high-resolution QTOF and Orbitrap platforms. Optimizing targeted methods to profile hundreds of metabolites on a QqQ mass spectrometer, however, has historically been limited by the availability of authentic standards, particularly for "unknowns" that have yet to be structurally identified. Here, we report a strategy to develop multiple reaction monitoring (MRM) methods for QqQ instruments on the basis of high-resolution spectra, thereby enabling us to use data from untargeted metabolomics to design targeted experiments without the need for authentic standards. We demonstrate that using high-resolution fragmentation data alone to design MRM methods results in the same quantitative performance as when methods are optimized by measuring authentic standards on QqQ instruments, as is conventionally done. The approach was validated by showing that Orbitrap ID-X data can be used to establish MRM methods on a Thermo TSQ Altis and two Agilent QqQs for hundreds of metabolites, including unknowns, without a dependence on standards. Finally, we highlight an application where metabolite profiling was performed on an ID-X and a QqQ by using the strategy introduced here, with both data sets yielding the same result. The described approach therefore allows us to use QqQ instruments, which are often associated with targeted metabolomics, to profile knowns and unknowns at a comprehensive scale that is typical of untargeted metabolomics.

Background Many CpGs become hyper or hypo-methylated with age. Multiple methods have been developed by Horvath et al. to estimate DNA methylation (DNAm) age including Pan-tissue, Skin & Blood, PhenoAge, and GrimAge. Pan-tissue and Skin & Blood try to estimate chronological age in the normal population whereas PhenoAge and GrimAge use surrogate markers associated with mortality to estimate biological age and its departure from chronological age. Here, we applied Horvath’s four methods to calculate and compare DNAm age in 499 subjects with type 1 diabetes (T1D) from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study using DNAm data measured by Illumina EPIC array in the whole blood. Association of the four DNAm ages with development of diabetic complications including cardiovascular diseases (CVD), nephropathy, retinopathy, and neuropathy, and their risk factors were investigated. Results Pan-tissue and GrimAge were higher whereas Skin & Blood and PhenoAge were lower than chronological age (p < 0.0001). DNAm age was not associated with the risk of CVD or retinopathy over 18–20 years after DNAm measurement. However, higher PhenoAge (β = 0.023, p = 0.007) and GrimAge (β = 0.029, p = 0.002) were associated with higher albumin excretion rate (AER), an indicator of diabetic renal disease, measured over time. GrimAge was also associated with development of both diabetic peripheral neuropathy (OR = 1.07, p = 9.24E−3) and cardiovascular autonomic neuropathy (OR = 1.06, p = 0.011). Both HbA1c (β = 0.38, p = 0.026) and T1D duration (β = 0.01, p = 0.043) were associated with higher PhenoAge. Employment (β = − 1.99, p = 0.045) and leisure time (β = − 0.81, p = 0.022) physical activity were associated with lower Pan-tissue and Skin & Blood, respectively. BMI (β = 0.09, p = 0.048) and current smoking (β = 7.13, p = 9.03E−50) were positively associated with Skin & Blood and GrimAge, respectively. Blood pressure, lipid levels, pulse rate, and alcohol consumption were not associated with DNAm age regardless of the method used. Conclusions Various methods of measuring DNAm age are sub-optimal in detecting people at higher risk of developing diabetic complications although some work better than the others.

Background: The Glycemia Reduction Approaches in Diabetes: A Comparative Effectiveness (GRADE) study has enrolled a racially and ethnically diverse population with type 2 diabetes, performed extensive phenotyping, and randomly assigned the participants to one of four second-line diabetes medications. The continuous glucose monitoring (CGM) substudy has been added to determine whether there are racial/ethnic differences in the relationship between average glucose (AG) and hemoglobin A1c (HbA1c). CGM will also be used to compare time in target range, glucose variability, and the frequency and duration of hypoglycemia across study groups. Methods: The observational CGM substudy will enroll up to 1800 of the 5047 GRADE study participants from the four treatment groups, including as many as 450 participants from each of 4 racial/ethnic minority groups to be compared: Hispanic White, non-Hispanic White, non-Hispanic African American, and non-Hispanic Other. CGM will be performed for 2 weeks in proximity to a GRADE annual visit, during which an oral glucose tolerance test will be performed and HbA1c and glycated albumin measured. Indicators of interindividual variation in red blood cell turnover, based on specialized erythrocyte measurements, will also be measured to explore the potential causes of interindividual HbA1c variations. Conclusions: The GRADE CGM substudy will provide new insights into whether differences exist in the relationship between HbA1c and AG among different racial/ethnic groups and whether glycemic profiles differ among frequently used diabetes medications and their potential clinical implications. Understanding such differences is important for clinical care and adjustment of diabetes medications in patients of different races or ethnicities.

Objective To compare concentrations of antimullerian hormone (AMH) in women with and without type 1 diabetes. Design Cross-sectional analysis of longitudinal studies, adjusting for repeated measures. Setting Not applicable. Patient(s) Women aged 30–45 years who had not undergone oophorectomy, hysterectomy, or natural menopause at the time of AMH measurement were included (n = 376 in the Michigan Bone Health and Metabolism Study and n=321 in the Epidemiology of Interventions and Complications Study). Linear mixed regression was used to evaluate whether AMH concentrations differed by diabetes status, adjusting for repeated measurements of AMH within individual women, body mass index, smoking status, and oral contraceptive use. Intervention(s) None. Main Outcome Measure(s) Concentrations of AMH. Result(s) In unadjusted comparisons, women with and without diabetes had similar median AMH values before 35 years of age, although women with type 1 diabetes had a lower proportion of women with elevated AMH concentrations (≥5.0 ng/dL). After adjustment for covariates and multiple observations per woman, log AMH concentrations were significantly lower among women with type 1 diabetes compared with women without diabetes (β-coefficient −1.27, 95% confidence interval [−2.18, −0.36] in fully adjusted models) before 35 years of age. Conclusion(s) Before 35 years of age, women with type 1 diabetes have lower AMH levels than women without diabetes. Further investigation is needed to determine the etiologies of this difference and how it may contribute to reproductive disorders among women with type 1 diabetes.

Low testosterone concentrations have been reported to be associated with increased risk of congestive heart failure, but the mechanisms are unclear. Our objective was to examine the relationship between endogenous testosterone and measures of cardiac mass and function among men with type 1 diabetes.Secondary analysis of a prospective observational study.Men (n = 508) in the Epidemiology of Diabetes Interventions and Complications (EDIC) study, the observational follow-up of the Diabetes Control and Complications Trial (DCCT).Testosterone assessed by liquid chromatography mass spectrometry at EDIC year 10 and cardiac magnetic resonance imaging (CMR) measures at EDIC years 14/15. Linear regression models were used to assess the relationship between testosterone, sex hormone binding globulin (SHBG) and left ventricular (LV) mass, volume, ejection fraction and cardiac index before and after adjustment for age, randomization arm, alcohol and cigarette use, macroalbuminuria, haemoglobin A1c, insulin dose, body mass index, lipids, blood pressure, use of antihypertensive medications and microvascular complications.In fully adjusted models, total testosterone concentrations were significantly associated with LV mass (P = 0·014), end-diastolic volume (P = 0·002), end-systolic volume (P = 0·012) and stroke volume (P = 0·022), but not measures of LV function after adjustment for cardiac risk factors. Bioavailable testosterone was associated with LV mass, but not volume or function, while SHBG was associated with volume, but not mass or function.Among men with type 1 diabetes, higher total testosterone was associated with higher LV mass and volume, but not with function. The clinical significance of this association remains to be established.

Background: Complete blood counts (CBCs) are commonly obtained in large multicenter studies. We assessed the stability of 10 parameters after short-term (up to 30 days) frozen storage. Methods: We compared CBC measurements from fresh samples (n = 53) with samples stored for up to 30 days at −70 °C. We calculated the CVs and intraclass correlation coefficients. Results: Mean values of most parameters, with the exception of hemoglobin and platelet count, were significantly different by 15 days of storage. White blood cell count (CV, 38.3%; 95% CI, 31.3%–46.2%) and red cell distribution width (CV, 37.7%; 95% CI, 34.1%–41.3%) were the most variable. After 30 days, only hemoglobin remained stable and reliable (CV, 0.8%; 95% CI, 0.4%–1.3%). Conclusions: Hemoglobin remained stable in frozen blood samples stored for up to 30 days at −70 °C and may be reliably used in research studies using short-term frozen specimens. Other CBC parameters measured in stored blood are not sufficiently reliable for research or patient care.

We investigated residual beta cell function in participants from the DCCT/EDIC study with an average of 35 (range 27, 48) years duration of T1D. Between 2015-2017 after 22-24 years of follow-up in EDIC, a 4-hour mixed meal tolerance test was administered to 944 participants and 7 timed plasma specimens were collected and assayed for C-peptide using a chemiluminescent immunoassay (Roche). We defined a significant post-stimulus response as a peak C-peptide concentration >0.03 nmol/L, based on an earlier DCCT study which demonstrated that the risk of microvascular disease progression was markedly higher among participants who entered the trial with values below this concentration. Overall, 71 (7.5%) participants were classified as “responders” with a median peak C-peptide of 0.10 nmol/L (IQR 0.06, 0.15). Among the 873 participants classified as “non-responders”, 46 had detectable peak C-peptide between 0.003 (limit of detection) and 0.029 nmol/L. Responders were slightly older than non-responders (58.5 ± 6.2 vs. 56.5 ± 6.8 y; p=0.02), yet had similar age of onset of diabetes (22.5 ± 7.0 vs. 21.4 ± 7.8 y) and duration of diabetes (35.9 ± 5.1 vs. 35.0 ± 4.9 y). Compared to non-responders, responders had lower HbA1c values (8.6 ± 1.7 vs. 8.9 ± 1.5%; p=0.01) and higher stimulated C-peptide (0.22 ± 0.14 vs. 0.11 ± 0.11 nmol/L; p In conclusion, beta cell function persists in some very long duration T1D patients and is associated with clinically meaningful reductions in the frequency of severe hypoglycemia. Disclosure R. Gubitosi-Klug: None. B. Braffett: None. S.M. Hitt: None. V. Arends: None. M. Steffes: None. A.K. Saenger: Advisory Panel; Self; Alere Inc. J. Lachin: Board Member; Self; AbbVie Inc., Celgene Corporation. J.P. Palmer: None.

OBJECTIVE Women with type 1 diabetes have increased risk of infertility compared to women without diabetes even after adjustment for irregular menses, but aetiologies are incompletely understood. Our aim was to examine the prevalence of abnormalities in ovarian markers consistent with polycystic ovary syndrome in women with type 1 diabetes and associations with irregular menses and diabetes-specific variables. DESIGN, PATIENTS AND MEASUREMENTS We conducted a secondary analysis of women in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study (DCCT/EDIC), a randomized trial and observational follow-up of intensive insulin therapy for type 1 diabetes. We included women with anti-Mullerian hormone (AMH) measurements among women not using oral contraceptives (n = 187). Initial AMH and testosterone measures were performed between EDIC years 1 and 4. History of irregular menses was assessed annually. RESULTS The median age of women was 35 (interquartile ratio 29, 40) years; 133 (35%) had elevated AMH and 62 (17%) reported irregular menses. Twelve per cent of women had relative elevations in total testosterone. In multivariable models, lower insulin dosages were associated with higher AMH concentrations (P = .0027), but not diabetes duration, glycemic control, body mass index or irregular menses. Neither irregular menses nor diabetes-specific variables were associated with testosterone concentrations. CONCLUSIONS Among women with type 1 diabetes in their thirties, abnormalities in ovarian markers are common and not associated with irregular menses and thus may partially account for decreased fecundity in women with type 1 diabetes.

Markers of ovarian reserve such as anti-Mullerian hormone (AMH) are used in the management of fertility and prediction of menopause. Although women with type 1 diabetes have a high prevalence of reproductive disorders, no studies have examined whether markers of ovarian reserve are associated with randomization to intensive insulin therapy and subsequent markers of glycemic control. Using data from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study, we found that the strongest predictor of AMH was chronologic age, and that diabetes-specific variables such as randomization to intensive therapy, insulin dose, and glycemic control were not associated with AMH concentrations.

Background: ATP-binding cassette A1 (ABCA1) and cholesteryl ester transfer protein (CETP) play important roles in the reverse cholesterol transport pathway. The associations of ABCA1 and CETP polymorphisms with lipoprotein subclasses have not been extensively studied.Methods: We genotyped 2 ABCA1 and 5 CETP polymorphisms in 999 participants of the Multi-Ethnic Study of Atherosclerosis (MESA) and studied their associations with HDL and LDL subclass particle concentrations, measured by nuclear magnetic resonance spectroscopy.Results: ABCA1 and CETP polymorphisms were associated with different and distinct changes in lipoprotein subclass concentrations. The ABCA1 1051G/A AA genotype, previously found to be associated with cardioprotective effects in this cohort, was associated with a 5.5% higher concentration of small HDL particles (P = 0.024). The CETP TaqIB B2B2, −2505C/A AA, and −629C/A AA genotypes, previously demonstrated to lack cardioprotective effects, were associated with 15.2%, 15.4%, and 11.7% higher HDL cholesterol concentrations, respectively, and 36.5%, 40.7%, and 25.4% higher large HDL particle concentrations (P < 0.0001). The minor alleles of the A373P and R451Q polymorphisms were associated with lower large HDL particle concentrations.Conclusions: Our study of the influence of ABCA1 and CETP genetic variants on lipoprotein subclasses demonstrates the importance of interpreting lipoprotein subclasses within the context of the biochemical processes involved in the alterations. In the case of HDL, the study of subclass particle numbers and sizes may not be sufficiently informative. Assays for HDL function may be needed to supplement quantification of HDL cholesterol and HDL particle numbers and sizes.

BACKGROUND— Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes. RESEARCH DESIGN AND METHODS— We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome. RESULTS— We observed association between rs17880135 in the 3′ region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64–4.18], P = 5.6 × 10−5, q = 0.06) and persistent microalbuminuria (1.82 [1.29–2.57], P = 6.4 × 10−4, q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P < 10−3) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines. CONCLUSIONS— Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

Implementation of multicenter and/or longitudinal studies requires an effective quality assurance program to identify trends, data inconsistencies and process variability of results over time. The Diabetes Control and Complications Trial (DCCT) and the follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) study represent over 30 years of data collection among a cohort of participants across 27 clinical centers. The quality assurance plan is overseen by the Data Coordinating Center and is implemented across the clinical centers and central reading units. Each central unit incorporates specific DCCT/EDIC quality monitoring activities into their routine quality assurance plan. The results are reviewed by a data quality assurance committee whose function is to identify variances in quality that may impact study results from the central units as well as within and across clinical centers, and to recommend implementation of corrective procedures when necessary. Over the 30-year period, changes to the methods, equipment, or clinical procedures have been required to keep procedures current and ensure continued collection of scientifically valid and clinically relevant results. Pilot testing to compare historic processes with contemporary alternatives is performed and comparability is validated prior to incorporation of new procedures into the study. Details of the quality assurance plan across and within the clinical and central reading units are described, and quality outcomes for core measures analyzed by the central reading units (e.g. biochemical samples, fundus photographs, ECGs) are presented.

Background: Previous studies have shown that ATP-binding cassette transporter 1 ( ABCA1) polymorphisms associated with increased ABCA1 expression result in increased small HDL (high-density lipoprotein) subclass particle concentration. This study examines the effect of treatment with fenofibrate, a drug known to bind peroxisome proliferator-activated receptor alpha (PPARα) which increases the expression of ABCA1 gene, on lipoprotein subclass profiles of individuals stratified by ABCA1 genotypes. Methods: Participants of Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) were treated with fenofibrate over a three week period. We analyzed six ABCA1 polymorphisms in 287 GOLDN participants with triglyceride concentrations ⩾150mg/dL and studied their associations with HDL subclass particle concentrations, as measured by nuclear magnetic resonance spectroscopy, before and after treatment. Results: Fenofibrate treatment did not result in significant changes in small HDL subclass particle concentration. When changes in HDL subclasses were stratified by ABCA1 polymorphism genotypes, there were no statistically significant associations between ABCA1 genotypes and small HDL subclasses before fenofibrate treatment. However, after fenofibrate treatment the KK genotype of R1587K (mean 4.40μmol/L; p =0.004) and the RK genotype of R219K (mean 1.60μmol/L; p =0.02) polymorphisms were associated with significantly increased small HDL. The R1587KKK genotype (mean 4.80μmol/L; p =0.0002) and the R219K KK genotype (mean 2.50μmol/L; p =0.02) were also associated with increased HDL particle concentrations. Conclusion: There is a synergistic effect between ABCA1 polymorphisms and fenofibrate. Thus, our study indirectly confirms the role of fenofibrate and genotype in increasing cholesterol efflux, as evidenced by increased small HDL particles.

Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes.We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome.We observed association between rs17880135 in the 3' region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64-4.18], P = 5.6 x 10(-5), q = 0.06) and persistent microalbuminuria (1.82 [1.29-2.57], P = 6.4 x 10(-4), q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P10(-3)) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines.Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

The cholesteryl ester transport protein (CETP) plays a key role in high-density lipoprotein (HDL) metabolism. Genetic variants that alter CETP activity and concentration may cause significant alterations in HDL-cholesterol (HDL-C) concentration; however, controversies remain about whether these genetic variants are associated with atherosclerosis. We genotyped the CETP R451Q, A373P, -629C/A, Taq1B, and -2505C/A polymorphisms in a cohort of Caucasian, Chinese, African-American, and Hispanic individuals within the Multi-Ethnic Study of Atherosclerosis. Genotypes were examined in relationship to HDL-C, CETP activity, CETP concentration, and three measures of subclinical cardiovascular disease (CVD): coronary artery calcium (CAC) measured by fast CT scanning, and carotid intimal-medial thickness (IMT) and carotid artery plaque, measured by ultrasonography. Carriers of the 451Q and 373P alleles have significantly higher CETP concentration (22.4% and 19.5%, respectively; p

HDL-cholesterol (HDL-C) has long been recognized as having an atheroprotective role (1). Epidemiologic studies have shown that decreased HDL-C concentrations are the most common lipid abnormality in patients with premature coronary artery disease (CAD). Thus, there has been increased recognition of the inverse relationship between HDL-C concentrations and risk for CAD (2)(3). This heightened awareness is underscored by changes in the recommendations of the Adult Treatment Panel (ATP) of the National Cholesterol Education Program. For example, ATP II, published in 1993, first introduced the concept of a low HDL-C [