Circulating metabolomic markers in association with overall burden of microvascular complications in type 1 diabetes.

INTRODUCTION: Diabetic retinopathy (DR), diabetic kidney disease (DKD) and distal symmetric polyneuropathy (DSPN) share common pathophysiology and pose an additive risk of early mortality. RESEARCH DESIGN AND METHODS: In adults with type 1 diabetes, 49 metabolites previously associated with either DR or DKD were assessed in relation to presence of DSPN. Metabolites overlapping in significance with presence of all three complications were assessed in relation to microvascular burden severity (additive number of complications-ie, presence of DKD±DR±DSPN) using linear regression models. Subsequently, the same metabolites were assessed with progression to endpoints: soft microvascular events (progression in albuminuria grade, ≥30% estimated glomerular filtration rate (eGFR) decline, or any progression in DR grade), hard microvascular events (progression to proliferative DR, chronic kidney failure, or ≥40% eGFR decline), and hard microvascular or macrovascular events (hard microvascular events, cardiovascular events (myocardial infarction, stroke, or arterial interventions), or cardiovascular mortality), using Cox models. All models were adjusted for sex, baseline age, diabetes duration, systolic blood pressure, HbA1c, body mass index, total cholesterol, smoking, and statin treatment. RESULTS: The full cohort investigated consisted of 487 participants. Mean (SD) follow-up was 4.8 (2.9, 5.7) years. Baseline biothesiometry was available in 202 participants, comprising the cross-sectional cohort. Eight metabolites were significantly associated with presence of DR, DKD, and DSPN, and six with additive microvascular burden severity. In the full cohort longitudinal analysis, higher levels of 3,4-dihydroxybutanoic acid (DHBA), 2,4-DHBA, ribonic acid, glycine, and ribitol were associated with development of events in both crude and adjusted models. Adding 3,4-DHBA, ribonic acid, and glycine to a traditional risk factor model improved the discrimination of hard microvascular events. CONCLUSIONS: While prospective studies directly assessing the predictive ability of these markers are needed, our results strengthen the role of clinical metabolomics in relation to risk assessment of diabetic complications in chronic type 1 diabetes.

Ceramides as Risk Markers for Future Cardiovascular Events and All-Cause Mortality in Long-standing Type 1 Diabetes.

Ceramides are lipid molecules involved in inflammation-related signaling. Recent studies have shown that higher amounts of specific circulating ceramides and their ratios are associated with future development of cardiovascular (CV) disease (CVD). We examined the associations between serum ceramide levels with CVD, kidney failure, and all-cause mortality in individuals with long-standing type 1 diabetes (T1D). We included 662 participants with T1D and 6-year follow-up, with a mean age of 55 years and mean diabetes duration of 33 years. Baseline serum samples were analyzed using liquid chromatography-mass spectrometry. Six predefined ceramide levels were measured, and predefined ratios were calculated. Adjusted Cox regression analyses on ceramide levels in relation to future CV events (CVE), kidney failure, and all-cause mortality were performed, with and without adjustment for age, sex, BMI, LDL, triglycerides, systolic blood pressure, HbA1c, history of CVD, smoking status, statin use, estimated glomerular filtration rate (eGFR), and urinary albumin excretion rate (UAER). The ceramide ratio cer(d18:1/18:0)/cer(d18:1/24:0) was significantly associated with risk of CVE (hazard ratio [HR] = 1.33, P = 0.01) and all-cause mortality (HR = 1.48, P = 0.01) before and after adjustments. All five investigated ceramide ratios were associated with kidney failure, before adjusting for the kidney markers eGFR and UAER. In this study, we demonstrate specific ceramides and ratios associated with 6-year cardiovascular risk and all-cause mortality in a T1D cohort. This highlights the strength of ceramide association with vascular complications and presents a new potential tool for early risk assessment if validated in other cohorts. ARTICLE HIGHLIGHTS: Improved tools for assessing risk for diabetes complication before onset will help in complication prevention. We investigated a set of six predefined ceramides and their ratios versus 6-year outcomes of cardiovascular events, kidney failure, and all-cause mortality in people with long-standing type 1 diabetes, using Cox regression with and without adjustment for potential confounders. We found that several ceramides and ceramide ratios associated with cardiovascular events and all-cause mortality. The ratio of cer(d18:1/18:0)/cer(d18:1/24:0) was an especially robust marker. These finding show that ceramides can be biomarkers of cardiovascular disease and all-cause mortality in individuals with long-standing type 1 diabetes.

Collagen turnover is associated with cardiovascular autonomic and peripheral neuropathy in type 1 diabetes: novel pathophysiological mechanism?

BACKGROUND: Diabetic cardiovascular autonomic neuropathy (CAN) and distal symmetrical polyneuropathy (DSPN) are severe diabetic complications. Collagen type VI (COL6) and III (COL3) have been associated with nerve function. We investigated if markers of COL6 formation (PRO-C6) and COL3 degradation (C3M) were associated with neuropathy in people with type 1 diabetes (T1D). METHODS: In a cross-sectional study including 300 people with T1D, serum and urine PRO-C6 and C3M were obtained. CAN was assessed by cardiovascular reflex tests: heart rate response to deep breathing (E/I ratio), to standing (30/15 ratio) and to the Valsalva maneuver (VM). Two or three pathological CARTs constituted CAN. DSPN was assessed by biothesiometry. Symmetrical vibration sensation threshold above 25 V constituted DSPN. RESULTS: Participants were (mean (SD)) 55.7 (9.3) years, 51% were males, diabetes duration was 40.0 (8.9) years, HbA1c was 63 (11 mmol/mol, (median (IQR)) serum PRO-C6 was 7.8 (6.2;11.0) ng/ml and C3M 8.3 (7.1;10.0) ng/ml. CAN and DSPN were diagnosed in 34% and 43% of participants, respectively. In models adjusted for relevant confounders a doubling of serum PRO-C6, was significantly associated with odds ratio > 2 for CAN and > 1 for DSPN, respectively. Significance was retained after additional adjustments for eGFR only for CAN. Higher serum C3M was associated with presence of CAN, but not after adjustment for eGFR. C3M was not associated with DSPN. Urine PRO-C6 analyses indicated similar associations. CONCLUSIONS: Results show previously undescribed associations between markers of collagen turnover and risk of CAN and to a lesser degree DSPN in T1D.

Non-dipping and higher nocturnal blood pressure are associated with risk of mortality and development of kidney disease in type 1 diabetes.

AIMS: People with type 1 diabetes have increased risk of cardiovascular (CV) and kidney disease. A 24-hour ambulatory blood pressure (BP) measurement (ABPM) examines diurnal variations in BP. We aimed to determine the prognostic significance of blunted decrease in nocturnal systolic BP of <10 % (non-dipping of nocturnal BP) for CV- and kidney disease and all-cause mortality in type 1 diabetes. METHODS: From 2009 to 2011, at Steno Diabetes Center Copenhagen, 654 participants with type 1 diabetes had 24-hour ABPM obtained with a tonometric wrist-watch device (BPro, HealthStats, Singapore). In 2017, outcomes (composite CV endpoint; all-cause mortality; decline in estimated glomerular filtration rate (eGFR) ≥30 %; end-stage kidney disease (ESKD); and a composite kidney endpoint including decline in eGFR ≥30 %, ESKD and all-cause mortality) were registered. Hazard Ratios (HR) were calculated using Cox regressions. RESULTS: Participants were mean ± SD 55 ± 13 years old and had median (IQR) 35 (24-44) years diabetes duration. Mean daytime and nocturnal systolic BP were 133 ± 16 and 121 ± 16 mmHg while 337 (52 %) participants demonstrated non-dipping. After CV risk factor adjustments, non-dipping was associated with all-cause mortality (HR 2.12 (1.09-4.11), p = 0.03) and the composite kidney endpoint (HR 1.92 (1.23-3.00), p = 0.004). CONCLUSIONS: Non-dipping entailed increased risk of all-cause mortality and kidney disease in type 1 diabetes.

Circulating metabolites and molecular lipid species are associated with future cardiovascular morbidity and mortality in type 1 diabetes.

BACKGROUND: Cardiovascular disease remains the leading cause of mortality in individuals with diabetes and improved understanding of its pathophysiology is needed. We investigated the association of a large panel of metabolites and molecular lipid species with future cardiovascular events in type 1 diabetes. METHODS: The study included 669 individuals with type 1 diabetes. Non-targeted serum metabolomics and lipidomics analyses were performed using mass spectrometry. Data on cardiovascular events (cardiovascular mortality, coronary artery disease, stroke, and peripheral arterial interventions) were obtained from Danish Health registries and analyzed by Cox hazards models. Metabolites and molecular lipid species were analyzed in univariate models adjusted for false discovery rate (FDR). Metabolites and molecular lipid species fulfilling a pFDR < 0.05 were subsequently analyzed in adjusted models including age, sex, hemoglobin A1c, mean arterial pressure, smoking, body mass index, low-density lipoprotein cholesterol, estimated glomerular filtration rate, urinary albumin excretion rate and previous cardiovascular disease. Analyses of molecular lipid species were further adjusted for triglycerides and statin use. RESULTS: Of the included participants, 55% were male and mean age was 55 ± 13 years. Higher 4-hydroxyphenylacetic acid (HR 1.35, CI [1.01-1.80], p = 0.04) and lower threonine (HR 0.81, CI [0.67-0.98] p = 0.03) were associated with development of cardiovascular events (n = 95). In lipidomics analysis, higher levels of three different species, diacyl-phosphatidylcholines (PC)(36:2) (HR 0.82, CI [0.70-0.98], p = 0.02), alkyl-acyl-phosphatidylcholines (PC-O)(34:2) (HR 0.76, CI [0.59-0.98], p = 0.03) and (PC-O)(34:3) (HR 0.75, CI [0.58-0.97], p = 0.03), correlated with lower risk of cardiovascular events, whereas higher sphingomyelin (SM)(34:1) (HR 1.32, CI [1.04-1.68], p = 0.02), was associated with an increased risk. CONCLUSIONS: Circulating metabolites and molecular lipid species were associated with future cardiovascular events in type 1 diabetes. While the causal effect of these biomolecules on the cardiovascular system remains unknown, our findings support that omics-based technologies, although still in an early phase, may have the potential to unravel new pathways and biomarkers in the field of cardiovascular disease in type 1 diabetes.

Cardiovascular Autonomic Neuropathy in Type 1 Diabetes Is Associated With Disturbances in TCA, Lipid, and Glucose Metabolism.

Introduction: Diabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D). Materials and Methods: Cardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders. Results: Participants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4-deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p<0.05). Upon adjustment, positive associations with the CAN diagnoses were retained for hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, citric acid, and phenols (P<0.05). Conclusion: Metabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives are associated with the CAN diagnosis in T1D. These pathway may be part of the pathogeneses leading to CAN and may be modifiable risk factors for the complication.

Treatment options for hypercalcemia after cosmetic oil injections: Lessons from human tissue cultures and a pilot intervention study.

OBJECTIVE: Granuloma formation following self-administered cosmetic oil injections can lead to severe hypercalcemia and renal calcifications due to extra-renal vitamin D activation. This translational study aims to identify Prednisolone sparing therapeutics for hypercalcemia after development of granulomatous disease secondary to paraffin oil injections. MATERIALS AND METHODS: Granuloma tissue isolated from five men were cultured ex vivo and treated with selected drugs to block generation of activated vitamin D (1,25(OH)2D3). In a retrospective study, we included data before and during different treatments of 21 men with paraffin oil induced granulomatous hypercalcemia (46 treatment courses) where serum calcium, parathyroid hormone, vitamin D metabolites, creatinine and inflammatory markers were measured. RESULTS: Addition of Ketoconazole or Ciclosporin to granuloma tissue ex vivo culture, significantly suppressed production of 1,25(OH)2D3 after 48 h (both p < 0.05). Prednisolone was the first treatment option in most men and lowered serum levels of ionized calcium after 1, 2, 3 and 6 months compared with baseline (p < 0.05). Ketoconazole or Hydroxychloroquine had no significant effect on serum calcium levels and were unable to reduce the concomitant daily Prednisolone doses (p > 0.05). Azathioprine did not reduce calcium levels. However, addition of Tacrolimus to Prednisolone treatment enabled a reduction in Prednisolone dose after 3 months (p = 0.014), but with no additional effect on calcium homeostasis. CONCLUSION: This study verifies that Prednisolone is an effective treatment and suggests that calcineurin inhibitors may be used as Prednisolone sparing treatment for paraffin oil-induced granulomatous hypercalcemia. Randomized clinical trials are needed to determine clinical efficacy.

Cardiovascular autonomic neuropathy and the impact on progression of diabetic kidney disease in type 1 diabetes.

INTRODUCTION: We investigated the association between cardiovascular autonomic neuropathy (CAN) and decline in kidney function in type 1 diabetes. RESEARCH DESIGN AND METHODS: We included 329 persons with type 1 diabetes. CAN was assessed by cardiovascular reflex tests (CARTs): heart rate response to deep breathing (E/I ratio), to standing (30/15 ratio) and to the Valsalva maneuvre. Two or more pathological CARTs defined CAN diagnosis. Outcomes were yearly change in albuminuria or yearly change in estimated glomerular filtration rate (eGFR). An endpoint of eGFR decline >30%, development of end-stage kidney disease (ESKD) or death was examined.Associations were assessed by linear and Cox regression. RESULTS: Participants were aged 55.2 (9.4) years, 52% were male, with a diabetes duration of 40.1 (8.9) years, HbA1c of 7.9% (62.5 mmol/mol), eGFR 77.9 (27.7) mL/min/1.73 m2, urinary albumin excretion rate of 14.5 (7-58) mg/24 hours, and 31% were diagnosed with CAN.CAN was associated with a 7.8% higher albuminuria increase per year (95% CI: 0.50% to 15.63%, p=0.036) versus no CAN. The endpoint of ESKD, all-cause mortality and ≥30% decline in eGFR was associated with CAN (HR=2.497, p=0.0254). CONCLUSION: CAN and sympathetic dysfunction were associated with increase in albuminuria in individuals with type 1 diabetes suggesting its role as a potential marker of diabetic kidney disease progression.

An overview of obesity mechanisms in humans: Endocrine regulation of food intake, eating behaviour and common determinants of body weight.

Obesity is one of the biggest health challenges of the 21st century, already affecting close to 700 million people worldwide, debilitating and shortening lives and costing billions of pounds in healthcare costs and loss of workability. Body weight homeostasis relies on complex biological mechanisms and the development of obesity occurs on a background of genetic susceptibility and an environment promoting increased caloric intake and reduced physical activity. The pathophysiology of common obesity links neuro-endocrine and metabolic disturbances with behavioural changes, genetics, epigenetics and cultural habits. Also, specific causes of obesity exist, including monogenetic diseases and iatrogenic causes. In this review, we provide an overview of obesity mechanisms in humans with a focus on energy homeostasis, endocrine regulation of food intake and eating behavior, as well as the most common specific causes of obesity.

Hypercalcemia After Cosmetic Oil Injections: Unraveling Etiology, Pathogenesis, and Severity.

Intramuscular injections of paraffin oil can cause foreign body granuloma formation and hypercalcemia. Macrophages with the ability to produce high levels of 1,25(OH)2 D3 may induce the mineral disturbance, but no major series of patients have been published to date. Here, medical history, physical evaluation, biochemical, and urinary analysis for calcium homeostasis were obtained from 88 males, who 6 years previously had injected paraffin or synthol oil into skeletal muscle. Moreover, granuloma tissue from three men was cultured for 48 hours ex vivo to determine 1,25(OH)2 D3 production supported by qPCR and immunohistochemistry of vitamin D metabolism and immune cell populations after treatment with 14 different drugs. The 88 men were stratified into men with hypercalcemia (34%), whereas normocalcemic men were separated into men with either normal (42%) or suppressed parathyroid hormone (PTH) (24%). All men had high calcium excretion, and nephrolithiasis was found in 48% of hypercalcemic men, 22% of normocalcemic men with normal PTH, and 47% of normocalcemic men with suppressed PTH. Risk factors for developing hypercalcemia were oil volume injected, injection of heated oil, high serum interleukin-2 receptor levels, and high urine calcium. High 1,25(OH)2 D3 /25OHD ratio, calcium excretion, and low PTH was associated with nephrolithiasis. The vitamin D activating enzyme CYP27B1 was markedly expressed in granuloma tissue, and 1,25(OH)2 D3 was released in concentrations corresponding to 40% to 50% of the production by human kidney specimens. Dexamethasone, ketoconazole, and ciclosporin significantly suppressed granulomatous production of 1,25(OH)2 D3 . In conclusion, this study shows that injection of large oil volumes alters calcium homeostasis and increases the risk of nephrolithiasis. Hypercalciuria is an early sign of disease, and high granulomatous 1,25(OH)2 D3 production is part of the cause. Prospective clinical trials are needed to determine if ciclosporin, ketoconazole, or other drugs can be used as prednisolone-sparing treatment. © 2020 American Society for Bone and Mineral Research (ASBMR).

Comparison of Natriuretic Peptides as Risk Markers for All-Cause Mortality and Cardiovascular and Renal Complications in Individuals With Type 1 Diabetes.

OBJECTIVE: Few studies have compared midregional proatrial natriuretic peptide (MR-proANP) and N-terminal probrain natriuretic peptide (NT-proBNP). We compared their value as risk markers for all-cause mortality and cardiovascular (CV) and renal complications in individuals with type 1 diabetes. RESEARCH DESIGN AND METHODS: MR-proANP and NT-proBNP were measured in 664 individuals. Hazard ratios (HRs) were assessed per doubling of NT-proBNP or MR-proANP for risk of a composite of ischemic events, heart failure (HF), a combined renal end point of end-stage kidney disease (ESKD), decline in estimated glomerular filtration rate (eGFR) ≥30%, and all-cause mortality or individual end points. Adjustments included CV risk factors and addition of MR-proANP or NT-proBNP. RESULTS: Median follow-up was 5.1-6.2 years. MR-proANP was associated with higher risk of all-cause mortality (n = 57; HR 1.7, 95% CI 1.1-2.7), combined CV end point (n = 94; 1.6, 1.1-2.2), HF (n = 27; 2.8, 1.5-5.2), combined renal end point (n = 123; 1.6, 1.2-2.1), and ESKD (n = 21; 3.1, 1.2-7.8) independent of CV risk factors (P ≤ 0.02). After addition of NT-proBNP, significance for all end points was lost. A doubling of NT-proBNP was associated with higher risk of all-cause mortality (HR 1.5, 95% CI 1.2-1.8), the combined CV end point (1.3, 1.1-1.5), HF (1.7, 1.3-2.1), and the combined renal end point (1.3, 1.1-1.4) independent of CV risk factors (model 2 [P < 0.001]) and MR-proANP (model 3 [P ≤ 0.03]). There was no association with decline in eGFR ≥30% (n = 93). CONCLUSIONS: Higher NT-proBNP was independently associated with all-cause mortality, CV disease, HF, and the combined renal end point. MR-proANP was associated with all end points but decline in eGFR, although not independent of NT-proBNP. MR-proANP may contribute to the predictive value of NT-proBNP for risk stratification in type 1 diabetes.

Copeptin and renal function decline, cardiovascular events and mortality in type 1 diabetes.

BACKGROUND: Plasma copeptin is a surrogate of arginine vasopressin (AVP) secretion and is associated with a risk of renal and cardiovascular disease. We investigated associations between copeptin and renal events, cardiovascular events and mortality in type 1 diabetes (T1D). METHODS: We conducted a prospective cohort study on 658 individuals with T1D from Steno Diabetes Center Copenhagen. Plasma copeptin concentrations and conventional risk factors were assessed at baseline. The five endpoints were traced through national registries and electronic laboratory records. RESULTS: Baseline mean age was 55 ± 13 years and estimated glomerular filtration rate (eGFR) was 81 ± 26 mL/min/1.73 m2. The median follow-up was 6.2 years (interquartile range 5.8-6.7); 123 participants reached a combined renal endpoint [decline in eGFR ≥30%, end-stage kidney disease (ESKD) or all-cause mortality], 93 had a decrease in eGFR ≥30%, 21 developed ESKD, 94 experienced a combined cardiovascular endpoint and 58 died from all causes. Higher copeptin was associated with all endpoints in unadjusted Cox regression analyses. Upon adjustment for baseline eGFR, the associations were attenuated and remained significant only for the combined renal endpoint and decrease in eGFR ≥30%. Results were similar upon further adjustment for other risk factors, after which hazard ratios for the two renal endpoints were 2.27 (95% confidence interval 1.08-4.74) and 4.49 (1.77-11.4), respectively, for the highest versus the lowest quartile of copeptin. CONCLUSIONS: Higher copeptin was an independent risk marker for a combined renal endpoint and decline in renal function. AVP may be a marker of renal damage or a factor whose contribution to renal and cardiovascular risk is partially mediated by renal damage.

Visit-to-visit variability of clinical risk markers in relation to long-term complications in type 1 diabetes.

BACKGROUND: Clinical characteristics such as HbA1c , systolic blood pressure (SBP), albuminuria and estimated glomerular filtration rate (eGFR) are important when treating type 1 diabetes. We investigated the variability in these measures as risk markers for micro- and macrovascular complications. METHODS: This prospective study included 1062 individuals with type 1 diabetes. Visit-to-visit variability of HbA1c , SBP, albuminuria and eGFR was calculated as the SD of the residuals in individual linear regression models using all available measures in a specified period of 3 years (VV). Endpoints included were as follows: cardiovascular events (CVE) defined as myocardial infarction, non-fatal stroke, or coronary or peripheral arterial intervention; end-stage kidney disease (ESKD) defined as eGFR <15 ml/min/1.73 m2 , chronic dialysis or kidney transplantation; eGFR decline ≥30%; and mortality. Adjustment included age, sex, cholesterol, HbA1c , SBP, body mass index, smoking, albuminuria, eGFR, and mean, intercept, slope of respective exposure variables and regression models. RESULTS: SBP VV was significantly associated with CVE (adjusted hazard ratio per 50% increase, (CI 95%); p: 1.21 [1.05-1.39]; p = 0.008), ESKD (1.51 [1.16-1.96]; p = 0.002) and mortality (1.25 [1.09-1.44]; p = 0.002). HbA1c VV was significantly associated with mortality (1.51 [1.30-1.75]; p < 0.001); albuminuria VV with eGFR decline (1.14 [1.08-1.20]; p = 0.024) and ESKD (1.14 [1.02-1.27]; p < 0.001), but neither CVE nor mortality. Adjusted eGFR VV was not associated with endpoints. CONCLUSION: In type 1 diabetes, higher variability of basic clinical risk markers adds important risk stratification information for the development of micro- and macrovascular complications.

Carotid-Femoral Pulse Wave Velocity as a Risk Marker for Development of Complications in Type 1 Diabetes Mellitus.

Background The value of carotid-femoral pulse wave velocity (cfPWV) as risk factor for development of complications in type 1 diabetes mellitus remains to be determined. We investigated associations between cfPWV and renal outcomes, cardiovascular events, and all-cause mortality in people with type 1 diabetes mellitus. Methods and Results cfPWV was measured with SphygmoCor in 633 people with type 1 diabetes mellitus. Median (interquartile range) follow-up was 6.2 (5.8-6.7) years. End points included progression in albuminuria group, decline in estimated glomerular filtration rate (eGFR) ≥30%, end-stage kidney disease, cardiovascular event, mortality, and a composite renal end point. Hazard ratios (HRs) were calculated per 1-SD increase in cfPWV. Adjustments included age, sex, hemoglobin A1c, mean arterial pressure, body mass index, low-density lipoprotein cholesterol, smoking, urine albumin excretion rate, and eGFR. The cohort included 45% women, mean (SD) age was 54 (13) years, mean (SD) eGFR was 83.2 (27.9) mL/min per 1.73 m2, and mean (SD) cfPWV was 10.4 (3.3) m/s. Median (interquartile range) albumin excretion rate was 17 (17-63) mg/24 h. After adjustment, higher cfPWV was associated with increased hazard of progression in albuminuria (HR, 1.59; 95% CI, 1.10-2.32); decline in eGFR ≥30% (HR, 1.38; 95% CI, 1.06-1.79); cardiovascular event (HR, 1.31; 95% CI, 1.01-1.70); mortality (HR, 1.36; 95% CI, 1.00-1.85); and the composite renal end point (HR, 1.30; 95% CI, 1.04-1.63), but not with end-stage kidney disease (HR, 1.18; 95% CI, 0.62-2.26). Higher cfPWV was associated with steeper yearly increase in albumin excretion and steeper yearly decline in eGFR after adjustment (P=0.002 and P=0.01, respectively). Conclusions cfPWV was associated with increased hazard of renal outcomes, cardiovascular event, and mortality. cfPWV may be suited for risk stratification in type 1 diabetes mellitus.

Circulating Metabolites and Lipids Are Associated to Diabetic Retinopathy in Individuals With Type 1 Diabetes.

Omics-based methods may provide new markers associated to diabetic retinopathy (DR). We investigated a wide omics panel of metabolites and lipids related to DR in type 1 diabetes. Metabolomic analyses were performed using two-dimensional gas chromatography with time-of-flight mass spectrometry and lipidomic analyses using an ultra-high-performance liquid chromatography quadruple time-of-flight mass spectrometry method in 648 individuals with type 1 diabetes. Subjects were subdivided into no DR, mild nonproliferative DR (NPDR), moderate NPDR, proliferative DR, and proliferative DR with fibrosis. End points were any progression of DR, onset of DR, and progression from mild to severe DR tracked from standard ambulatory care and investigated using Cox models. The cohort consisted of 648 participants aged a mean of 54.4 ± 12.8 years, 55.5% were men, and follow-up was 5.1-5.5 years. Cross-sectionally, 2,4-dihydroxybutyric acid (DHBA), 3,4-DHBA, ribonic acid, ribitol, and the triglycerides 50:1 and 50:2 significantly correlated (P < 0.042) to DR stage. Longitudinally, higher 3,4-DHBA was a risk marker for progression of DR (n = 133) after adjustment (P = 0.033). We demonstrated multiple metabolites being positively correlated to a higher grade of DR in type 1 diabetes and several triglycerides being negatively correlated. Furthermore, higher 3,4-DHBA was an independent risk marker for progression of DR; however, confirmation is required.

SuPAR is associated with death and adverse cardiovascular outcomes in patients with suspected coronary artery disease.

BACKGROUND: The inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) is associated with presence and severity of coronary artery disease (CAD) and incident death and myocardial infarction (MI). We sought to validate this finding in a further cohort of patients with suspected CAD. METHODS: Plasma suPAR was available in 1635 patients (73% with CAD) undergoing coronary angiography at a single regional Danish hospital between 2003 and 2005. Patients were followed for adverse cardiovascular outcomes of death, cardiac death and MI over a median follow-up of 4.2 years. RESULTS: In multivariate Cox models, adjusted for established cardiovascular risk factors, the biomarkers C-reactive protein, troponin-T and N-terminal-pro brain natriuretic peptide and the number of stenotic vessels, suPAR was independently associated with the combined endpoint of death/MI, hazard ratio (HR) 1.88; cardiovascular death, HR 2.01; and non-fatal MI, HR 1.53; (all p ≤ .037) per doubling of suPAR concentration. A plasma cutoff for suPAR ≥ 3.5 ng/mL was also significantly associated with death/MI, HR 1.51; p = .005. The C-statistic for the multivariate model predicting death/MI improved from 0.712 to 0.730 (p for difference .008) after inclusion of suPAR. However, suPAR was not associated with presence or extent of CAD (p > .05). CONCLUSION: These results validate previous findings that demonstrate suPAR to be an independent predictor of death/MI in patients with suspected or known CAD, however suPAR was not associated with presence or extent of CAD in our cohort. Probably because suPAR reflects end organ damage rather than the degree of atherosclerosis. BRIEF SUMMARY: We demonstrate that the inflammatory biomarker soluble urokinase plasminogen activator receptor is an independent predictor of death/myocardial infarction in patients with suspected or known coronary artery disease, but is not associated with the presence or severity of coronary artery disease.

Metabolomic Assessment Reveals Alteration in Polyols and Branched Chain Amino Acids Associated With Present and Future Renal Impairment in a Discovery Cohort of 637 Persons With Type 1 Diabetes.

Background: Improved understanding of the pathophysiology causing diabetic kidney disease (DKD) is imperative. The aim of this study was to uncover associations between serum metabolites and renal outcomes. Methods: Non-targeted serum metabolomics analyses were performed in samples from 637 persons with type 1 diabetes using two-dimensional gas chromatography coupled to time-of-flight mass-spectrometry. Longitudinal data at follow-up (median 5.5 years) on renal events were obtained from national Danish health registries. A composite renal endpoint (n = 123) consisted of estimated glomerular filtration rate (eGFR) decline from baseline (≥30%), progression to end-stage renal disease and all-cause mortality. Metabolites with significant associations (p < 0.05) in any of the cross-sectional analyses with eGFR and albuminuria were analyzed for specific and composite endpoints. Adjustments included traditional cardiovascular risk factors and correction for multiple testing. Results: A data-driven partial correlation analysis revealed a dense fabric of co-regulated metabolites and clinical variables dominated by eGFR. Ribonic acid and myo-inositol were inversely associated with eGFR, positively associated with macroalbuminuria (p < 0.02) and longitudinally associated with higher risk of eGFR decline ≥30% (HR 2.2-2.7, CI [1.3-4.3], p < 0.001). Ribonic acid was associated with a combined renal endpoint (HR 1.8, CI [1.3-2.3], p = 0.001). The hydroxy butyrate 3,4-dihydroxybutanoic acid was cross-sectionally associated with micro- and macroalbuminuria, urinary albumin excretion rate and inversely associated with eGFR (p < 0.04) while branched chain amino acids were associated with eGFR and lower risk of the combined renal endpoint (p < 0.02). Conclusions: Alterations in serum metabolites, particularly polyols and amino acids, were associated with renal endpoints in type 1 diabetes highlighting molecular pathways associated with progression of kidney disease. External validation is needed to further assess their roles and potentials as future therapeutic targets.

Lipidomic analysis reveals sphingomyelin and phosphatidylcholine species associated with renal impairment and all-cause mortality in type 1 diabetes.

There is an urgent need for a better molecular understanding of the pathophysiology underlying development and progression of diabetic nephropathy. The aim of the current study was to identify novel associations between serum lipidomics and diabetic nephropathy. Non-targeted serum lipidomic analyses were performed with mass spectrometry in 669 individuals with type 1 diabetes. Cross-sectional associations of lipid species with estimated glomerular filtration rate (eGFR) and urinary albumin excretion were assessed. Moreover, associations with register-based longitudinal follow-up for progression to a combined renal endpoint including ≥30% decline in eGFR, ESRD and all-cause mortality were evaluated. Median follow-up time was 5.0-6.4 years. Adjustments included traditional risk factors and multiple testing correction. In total, 106 lipid species were identified. Primarily, alkyl-acyl phosphatidylcholines, triglycerides and sphingomyelins demonstrated cross-sectional associations with eGFR and macroalbuminuria. In longitudinal analyses, thirteen lipid species were associated with the slope of eGFR or albuminuria. Of these lipids, phosphatidylcholine and sphingomyelin species, PC(O-34:2), PC(O-34:3), SM(d18:1/24:0), SM(d40:1) and SM(d41:1), were associated with lower risk of the combined renal endpoint. PC(O-34:3), SM(d40:1) and SM(d41:1) were associated with lower risk of all-cause mortality while an SM(d18:1/24:0) was associated with lower risk of albuminuria group progression. We report distinct associations between lipid species and risk of renal outcomes in type 1 diabetes, independent of traditional markers of kidney function.

Echocardiography improves prediction of major adverse cardiovascular events in a population with type 1 diabetes and without known heart disease: the Thousand & 1 Study.

AIMS/HYPOTHESIS: Cardiovascular disease is the most common comorbidity in type 1 diabetes. However, current guidelines do not include routine assessment of myocardial function. We investigated whether echocardiography provides incremental prognostic information in individuals with type 1 diabetes without known heart disease. METHODS: A prospective cohort of individuals with type 1 diabetes without known heart disease was recruited from the outpatient clinic. Follow-up was performed through Danish national registers. The association of echocardiography with major adverse cardiovascular events (MACE) and the incremental prognostic value when added to the clinical Steno T1D Risk Engine were examined. RESULTS: A total of 1093 individuals were included: median (interquartile range) age 50.2 (39.2-60.3) years and HbA1c 65 (56-74) mmol/mol; 53% men; and mean (SD) BMI 25.5 (3.9) kg/m2 and diabetes duration 25.8 (14.6) years. During 7.5 years of follow-up, 145 (13.3%) experienced MACE. Echocardiography significantly and independently predicted MACE: left ventricular ejection fraction (LVEF) <45% (n = 18) vs ≥45% (n = 1075), HR (95% CI) 3.93 (1.91, 8.08), p < 0.001; impaired global longitudinal strain (GLS), 1.65 (1.17, 2.34) (n = 263), p = 0.005; diastolic mitral early velocity (E)/early diastolic tissue Doppler velocity (e') <8 (n = 723) vs E/e' 8-12 (n = 285), 1.59 (1.04, 2.42), p = 0.031; and E/e' <8 vs E/e' ≥12 (n = 85), 2.30 (1.33, 3.97), p = 0.003. In individuals with preserved LVEF (n = 1075), estimates for impaired GLS were 1.49 (1.04, 2.15), p = 0.032; E/e' <8 vs E/e' 8-12, 1.61 (1.04, 2.49), p = 0.033; and E/e' <8 vs E/e' ≥12, 2.49 (1.41, 4.37), p = 0.001. Adding echocardiographic variables to the Steno T1D Risk Engine significantly improved risk prediction: Harrell's C statistic, 0.791 (0.757, 0.824) vs 0.780 (0.746, 0.815), p = 0.027; and net reclassification index, 52%, p < 0.001. CONCLUSIONS/INTERPRETATION: In individuals with type 1 diabetes without known heart disease, echocardiography significantly improves risk prediction over and above guideline-recommended clinical risk factors alone and could have a role in clinical care.

Targeted Clinical Metabolite Profiling Platform for the Stratification of Diabetic Patients.

Several small molecule biomarkers have been reported in the literature for prediction and diagnosis of (pre)diabetes, its co-morbidities, and complications. Here, we report the development and validation of a novel, quantitative method for the determination of a selected panel of 34 metabolite biomarkers from human plasma. We selected a panel of metabolites indicative of various clinically-relevant pathogenic stages of diabetes. We combined these candidate biomarkers into a single ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and optimized it, prioritizing simplicity of sample preparation and time needed for analysis, enabling high-throughput analysis in clinical laboratory settings. We validated the method in terms of limits of detection (LOD) and quantitation (LOQ), linearity (R2), and intra- and inter-day repeatability of each metabolite. The method's performance was demonstrated in the analysis of selected samples from a diabetes cohort study. Metabolite levels were associated with clinical measurements and kidney complications in type 1 diabetes (T1D) patients. Specifically, both amino acids and amino acid-related analytes, as well as specific bile acids, were associated with macro-albuminuria. Additionally, specific bile acids were associated with glycemic control, anti-hypertensive medication, statin medication, and clinical lipid measurements. The developed analytical method is suitable for robust determination of selected plasma metabolites in the diabetes clinic.