Plasma calprotectin and new-onset type 2 diabetes in the general population: a prospective cohort study.

BACKGROUND: Systemic inflammation plays a pivotal role in the development of type 2 diabetes (T2D). Here we hypothesized that circulating levels of calprotectin, a myeloid cell-derived biomarker of inflammation, is associated with the development of new-onset T2D in the general population. METHODS: A total of 4,815 initially non-diabetic participants of the Prevention of Renal and Vascular ENd-stage Disease (PREVEND), a prospective population-based cohort study, were assessed for plasma levels of calprotectin at baseline. Circulating levels of calprotectin were investigated for potential associations with the risk of new-onset T2D, defined as a fasting plasma glucose level ≥7.0 mmol/l, a random plasma glucose level ≥11.1 mmol/l, a self-reported physician-based diagnosis of T2D, the use of glucose-lowering drugs, or any combinations thereof. RESULTS: Median plasma calprotectin levels were 0.49 [0.35-0.69] mg/l. Plasma calprotectin levels were significantly associated with the risk of new-onset T2D (hazard ratio [HR] per doubling 1.42 [95% confidence interval: 1.22-1.66], P<0.001). The association remained independent of adjustment for age and sex (HR 1.34 [95%CI: 1.14-1.57], P<0.001), but not after further adjustment for potentially confounding factors (HR 1.11 [95% CI: 0.90-1.37], P=0.326), with adjustment for hyperlipidemia and high-sensitivity C-reactive protein explaining the loss of significance. Stratified analyses showed significant effect modification by hypertension, history of cardiovascular disease and HOMA-IR (Pinteraction≤0.001 for each), with higher HRs in individuals without hypertension, without history of cardiovascular disease and with below-median HOMA-IR. CONCLUSIONS: Elevated plasma levels of calprotectin are associated with a higher risk of developing T2D in the general population and may represent a moveable inflammatory biomarker. This association, however, does not represent a direct effect, and seems dependent on hyperlipidemia and systemic inflammation.

Fasting Plasma Ketone Bodies Are Associated with NT-proBNP: A Potential Mechanism to Provide Fuel for the Failing Heart.

Background: Heart failure (HF) features a shift in metabolism towards enhanced utilization of ketone bodies. While elevations in plasma natriuretic peptides represent a biochemical hallmark of HF, natriuretic peptides may promote lipolysis, thereby contributing to fatty acid availability for ketogenesis. Methods: We cross-sectionally tested to what extent fasting plasma total ketone bodies (measured using nuclear magnetic resonance spectroscopy) are associated with N-terminal pro-BNP (NT-proBNP; electrochemiluminescent sandwich immunoassay) in individuals with and without HF. Results: Among 6217 participants from the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study, 203 were identified with HF. NT-proBNP was four-fold and total ketone bodies were 25% higher in HF participants (each p < 0.001). In both participants with and without HF, total ketone body levels correlated with NT-proBNP (r = 0.116 and 0.185, respectively; p < 0.001). In multivariable linear regression analysis adjusted for relevant covariates, total ketone bodies remained associated with NT-proBNP in the whole cohort (std ß = 0.08, p < 0.001), without a difference in participants with and without HF (p interaction: 0.52). Conclusion: This general population-based study reveals an independent association of fasting total body ketone bodies with plasma NT-proBNP. Our findings suggest that a metabolic defense mechanism could be operative, providing the myocardium with ketone bodies to meet its energy demands.

Alcohol Consumption, High-Density Lipoprotein Particles and Subspecies, and Risk of Cardiovascular Disease: Findings from the PREVEND Prospective Study.

The associations of HDL particle (HDL-P) and subspecies concentrations with alcohol consumption are unclear. We aimed to evaluate the interplay between alcohol consumption, HDL parameters and cardiovascular disease (CVD) risk. In the PREVEND study of 5151 participants (mean age, 53 years; 47.5% males), self-reported alcohol consumption and HDL-P and subspecies (small, medium, and large) by nuclear magnetic resonance spectroscopy were assessed. Hazard ratios (HRs) with 95% CIs for first CVD events were estimated. In multivariable linear regression analyses, increasing alcohol consumption increased HDL-C, HDL-P, large and medium HDL, HDL size, and HDL subspecies (H3P, H4P, H6 and H7) in a dose-dependent manner. During a median follow-up of 8.3 years, 323 first CVD events were recorded. Compared with abstainers, the multivariable adjusted HRs (95% CIs) of CVD for occasional to light, moderate, and heavy alcohol consumers were 0.72 (0.55-0.94), 0.74 (0.54-1.02), and 0.65 (0.38-1.09), respectively. These associations remained consistent on additional adjustment for each HDL parameter. For CVD, only HDL-C was associated with a statistically significant decreased risk of CVD in a fully adjusted analysis (HR 0.84, 95% CI 0.72-0.97 per 1 SD increment). For coronary heart disease, HDL-C, HDL-P, medium HDL, HDL size, and H4P showed inverse associations, whereas HDL-C and HDL size modestly increased stroke risk. Except for H6P, alcohol consumption did not modify the associations between HDL parameters and CVD risk. The addition of HDL-C, HDL size, or H4P to a CVD risk prediction model containing established risk factors improved risk discrimination. Increasing alcohol consumption is associated with increased HDL-C, HDL-P, large and medium HDL, HDL size, and some HDL subspecies. Associations of alcohol consumption with CVD are largely independent of HDL parameters. The associations of HDL parameters with incident CVD are generally not attenuated or modified by alcohol consumption.

Urine cotinine versus self-reported smoking and the risk of chronic kidney disease.

BACKGROUND AND HYPOTHESIS: Evidence on the role of smoking in the development of chronic kidney disease (CKD) has mostly relied on self-reported smoking status. We aimed to compare the associations of smoking status as assessed by self-reports and urine cotinine with CKD risk. METHODS: Using the PREVEND prospective study, smoking status was assessed at baseline using self-reports and urine cotinine in 4333 participants (mean age, 52 years) without a history of CKD at baseline. Participants were classified as never, former, light current and heavy current smokers according to self-reports and comparable cut-offs for urine cotinine. Hazard ratios (HRs) with 95% CIs were estimated for CKD. RESULTS: The percentages of self-reported and cotinine-assessed current smokers were 27.5% and 24.0%, respectively. During a median follow-up of 7.0 years, 593 cases of CKD were recorded. In analyses adjusted for established risk factors, the HRs (95% CI) of CKD for self-reported former, light current, and heavy current smokers compared with never smokers were 1.17 (0.95-1.44), 1.48 (1.10-2.00), and 1.48 (1.14-1.93), respectively. On further adjustment for urinary albumin excretion (UAE), the HRs (95% CI) were 1.07 (0.87-1.32), 1.26 (0.93-1.70), and 1.20 (0.93-1.57), respectively. For urine cotinine-assessed smoking status, the corresponding HRs (95% CI) were 0.81 (0.52-1.25), 1.17 (0.92-1.49), and 1.32 (1.02-1.71), respectively, in analyses adjusted for established risk factors plus UAE. CONCLUSION: Self-reported current smoking is associated with increased CKD risk, but dependent on UAE. The association between urine cotinine-assessed current smoking and increased CKD risk is independent of UAE. Urine cotinine-assessed smoking status may be a more reliable risk indicator for CKD incidence than self-reported smoking status.

High-Density Lipoprotein Particles and Torque Teno Virus in Stable Outpatient Kidney Transplant Recipients.

Torque teno virus (TTV) is emerging as a potential marker for monitoring immune status. In transplant recipients who are immunosuppressed, higher TTV DNA loads are observed than in healthy individuals. TTV load measurement may aid in optimizing immunosuppressive medication dosing in solid organ transplant recipients. Additionally, there is a growing interest in the role of HDL particles in immune function; therefore, assessment of both HDL concentrations and TTV load may be of interest in transplant recipients. The objective of this study was to analyze TTV loads and HDL parameters in serum samples collected at least one year post-transplantation from 656 stable outpatient kidney transplant recipients (KTRs), enrolled in the TransplantLines Food and Nutrition Cohort (Groningen, the Netherlands). Plasma HDL particles and subfractions were measured using nuclear magnetic resonance spectroscopy. Serum TTV load was measured using a quantitative real-time polymerase chain reaction. Associations between HDL parameters and TTV load were examined using univariable and multivariable linear regression. The median age was 54.6 [IQR: 44.6 to 63.1] years, 43.3% were female, the mean eGFR was 52.5 (±20.6) mL/min/1.73 m2 and the median allograft vintage was 5.4 [IQR: 2.0 to 12.0] years. A total of 539 participants (82.2%) had a detectable TTV load with a mean TTV load of 3.04 (±1.53) log10 copies/mL, the mean total HDL particle concentration was 19.7 (±3.4) µmol/L, and the mean HDL size was 9.1 (±0.5) nm. The univariable linear regression revealed a negative association between total HDL particle concentration and TTV load (st.ß = -0.17, 95% CI st.ß: -0.26 to -0.09, p < 0.001). An effect modification of smoking behavior influencing the association between HDL particle concentration and TTV load was observed (Pinteraction = 0.024). After adjustment for age, sex, alcohol intake, hemoglobin, eGFR, donor age, allograft vintage and the use of calcineurin inhibitors, the negative association between HDL particle concentration and TTV load remained statistically significant in the non-smoking population (st.ß = -0.14, 95% CI st.ß: -0.23 to -0.04, p = 0.006). Furthermore, an association between small HDL particle concentration and TTV load was found (st.ß = -0.12, 95% CI st.ß: -0.22 to -0.02, p = 0.017). Higher HDL particle concentrations were associated with a lower TTV load in kidney transplant recipients, potentially indicative of a higher immune function. Interventional studies are needed to provide causal evidence on the effects of HDL on the immune system.

Optimal Screening for Predicting and Preventing the Risk of Heart Failure Among Adults With Diabetes Without Atherosclerotic Cardiovascular Disease: A Pooled Cohort Analysis.

BACKGROUND: The optimal approach to identify individuals with diabetes who are at a high risk for developing heart failure (HF) to inform implementation of preventive therapies is unknown, especially in those without atherosclerotic cardiovascular disease (ASCVD). METHODS: Adults with diabetes and no HF at baseline from 7 community-based cohorts were included. Participants without ASCVD who were at high risk for developing HF were identified using 1-step screening strategies: risk score (WATCH-DM [Weight, Age, Hypertension, Creatinine, HDL-C, Diabetes Control, QRS Duration, MI, and CABG] ≥12), NT-proBNP (N-terminal pro-B-type natriuretic peptide ≥125 pg/mL), hs-cTn (high-sensitivity cardiac troponin T ≥14 ng/L; hs-cTnI ≥31 ng/L), and echocardiography-based diabetic cardiomyopathy (echo-DbCM; left atrial enlargement, left ventricular hypertrophy, or diastolic dysfunction). High-risk participants were also identified using 2-step screening strategies with a second test to identify residual risk among those deemed low risk by the first test: WATCH-DM/NT-proBNP, NT-proBNP/hs-cTn, NT-proBNP/echo-DbCM. Across screening strategies, the proportion of HF events identified, 5-year number needed to treat and number needed to screen to prevent 1 HF event with an SGLT2i (sodium-glucose cotransporter 2 inhibitor) among high-risk participants, and cost of screening were estimated. RESULTS: The initial study cohort included 6293 participants (48.2% women), of whom 77.7% without prevalent ASCVD were evaluated with different HF screening strategies. At 5-year follow-up, 6.2% of participants without ASCVD developed incident HF. The 5-year number needed to treat to prevent 1 HF event with an SGLT2i among participants without ASCVD was 43 (95% CI, 29-72). In the cohort without ASCVD, high-risk participants identified using 1-step screening strategies had a low 5-year number needed to treat (22 for NT-proBNP to 37 for echo-DbCM). However, a substantial proportion of HF events occurred among participants identified as low risk using 1-step screening approaches (29% for echo-DbCM to 47% for hs-cTn). Two-step screening strategies captured most HF events (75-89%) in the high-risk subgroup with a comparable 5-year number needed to treat as the 1-step screening approaches (30-32). The 5-year number needed to screen to prevent 1 HF event was similar across 2-step screening strategies (45-61). However, the number of tests and associated costs were lowest for WATCH-DM/NT-proBNP ($1061) compared with other 2-step screening strategies (NT-proBNP/hs-cTn: $2894; NT-proBNP/echo-DbCM: $16 358). CONCLUSIONS: Selective NT-proBNP testing based on the WATCH-DM score efficiently identified a high-risk primary prevention population with diabetes expected to derive marked absolute benefits from SGLT2i to prevent HF.

Plasma Calprotectin Levels Associate With New-Onset Hypertension in the General Population: A Prospective Cohort Study.

BACKGROUND: Low-grade systemic inflammation is a relevant pathogenic mechanism underlying the development of hypertension. In this study, we hypothesized that plasma calprotectin levels, as a biomarker of neutrophil-mediated inflammation, is associated with developing new-onset hypertension in the general population. METHODS AND RESULTS: Plasma calprotectin levels were determined in 3524 participants who participated in the PREVEND (Prevention of Renal and Vascular End-Stage Disease) study, a prospective population-based cohort study. Plasma calprotectin levels were studied for associations with the risk of new-onset hypertension, defined as systolic blood pressure of at least 140 mm Hg, diastolic blood pressure of at least 90 mm Hg, or the first recorded use of antihypertensives. Participants with hypertension at baseline were excluded. Median plasma calprotectin levels were 0.48 (0.34-0.66) mg/L, and median systolic blood pressure was 117 (109-126) mm Hg. Plasma calprotectin levels were significantly associated with the risk of new-onset hypertension (hazard ratio [HR], per doubling 1.30 [95% CI, 1.21-1.41]; P<0.001), also after adjustment for age and sex (HR, 1.26 [95% CI, 1.16-1.37]; P<0.001), but not after additional adjustment for potentially confounding factors, including baseline systolic blood pressure (HR, 1.00 [95% CI, 0.90-1.11]; P=0.996). Stratified analyses showed significant effect modification by sex (Pinteraction=0.023) and urinary albumin excretion (Pinteraction=0.004), with higher HRs in men (compared with women) and in individuals with higher urinary albumin excretion (>9.3 mg per 24 hours) compared with lower urinary albumin excretion (≤9.3 mg per 24 hours). CONCLUSIONS: Higher plasma calprotectin levels are associated with an increased risk of new-onset hypertension in the general population. This association is dependent on baseline systolic blood pressure and is particularly prominent in men compared with women.

Triglyceride/HDL cholesterol ratio and lipoprotein insulin resistance Score: Associations with subclinical atherosclerosis and incident cardiovascular disease.

BACKGROUND: The triglyceride/HDL cholesterol (TG/HDL-C) ratio and the Lipoprotein Insulin Resistance (LP-IR) score are lipid markers of insulin resistance. Their associations with carotid intima media thickness (cIMT; subclinical atherosclerosis) and incident cardiovascular disease (CVD) have not been thoroughly investigated. METHODS: In a cross-sectional cohort (89 subjects without type 2 diabetes (T2D) and 81 subjects with T2D we determined cIMT (ultrasound), homeostasis model assessment of insulin resistance (HOMA-IR) and the TG/HDL-C ratio. The LP-IR score, based on 6 lipoprotein characteristics determined by nuclear magnetic resonance spectroscopy, was measured in 123 participants. A prospective study was carried out among 6232 participants (Prevention of REnal and Vascular ENd-stage Disease study). RESULTS: Cross-sectionally, the adjusted associations of HOMA-IR, the TG/HDL-C ratio and the LP-IR score with cIMT were approximately similar (standardized ß = 0.34 (95 % CI 0.19-0.48), 0.24 (95 % CI 0.09-039) and 0.41 (95 % CI 0.23--0.59), respectively). Prospectively, 507 new cases of CVD were observed after a median follow-up of 8.2 (interquartile range 7.5-8.8) years. HOMA-IR, the TG/HDL-C ratio and LP-IR were each associated with incident CVD independent of potential confounders (HR 1.12, 95 % CI 1.02-1.24;1.22, 95 % CI 1.11-1.35 and 1.15. 95 % CI 1.01-1.31, respectively). The association of the TG/HDL-C ratio with incident CVD was somewhat stronger than that of HOMA-IR. CONCLUSION: Lipoprotein-based markers of insulin resistance are at least as strongly associated with subclinical atherosclerosis and clinical atherosclerosis development as HOMA-IR, obviating the need to measure insulin to determine the impact of insulin resistance. For practical purposes, the easily obtainable TG/HDL-C ratio may suffice.

Serum peroxiredoxin-4, a biomarker of oxidative stress, is associated with the development of nephropathy in patients with type 2 diabetes (Zodiac-65).

Oxidative stress is implicated in the development and progression of type 2 diabetes (T2D). Peroxiredoxin-4 is an antioxidant protein, which may serve as biomarker of oxidative stress, and has previously been associated with new-onset T2D. In this study, we investigated associations between circulating peroxiredoxin-4 and the risk of developing new-onset microvascular complications in T2D patients. Serum peroxiredoxin-4 was measured in 536 patients with T2D with (n = 257) and without (n = 279) baseline microvascular complications who participated in a primary-care based cohort study (Zwolle Outpatient Diabetes project Integrating Available Care [ZODIAC] study). Over a median follow-up of 3.4 years, 38 (13.6%) developed nephropathy, defined as albuminuria in two consecutive urine samples. In multivariable Cox proportional hazards regression analyses, peroxiredoxin-4 was associated with new-onset nephropathy (hazard ratio [HR] per doubling 1.78 [95% CI: 1.27-2.49], P < 0.001) after adjustment for potential confounding factors, including age, sex, disease duration, HbA1c levels, macrovascular complications, systolic blood pressure, and even high-sensitive C-reactive protein. There was no interaction of peroxiredoxin-4 with hs-CRP impacting on new-onset nephropathy. No significant associations were found with new-onset retinopathy or neuropathy. In conclusion, circulating peroxiredoxin-4 associates positively with an increased risk of developing nephropathy in T2D independent and irrespective of low-grade inflammation.

Indirect Insulin Resistance Indices and Their Cut-Off Values for the Prediction of Post-Transplantation Diabetes Mellitus in Kidney Transplant Recipients.

BACKGROUND: Insulin resistance plays an important role in the development of post-transplantation diabetes mellitus (PTDM) in kidney transplant recipients (KTRs). Current methods for the direct determination of insulin resistance are complicated and invasive. Therefore, this study aimed to investigate the relevance of indirect insulin resistance indices in relation to the development of PTDM in KTRs. METHODS: We included 472 stable outpatient KTRs without diabetes at baseline from a prospective cohort study. Four indirect insulin resistance indices, namely homeostasis model assessment-insulin resistance (HOMA-IR), visceral adiposity index (VAI), lipid accumulation product (LAP), and triglycerides-glucose (TyG) index, were assessed. We analyzed each measure using the receiver operating characteristic (ROC) curve for PTDM development. The optimal cut-off value for each parameter was determined using the Youden index. RESULTS: After a median of 9.6 years (interquartile range (IQR) 6.6-10.2) of follow-up, 68 (14%) KTRs developed PTDM. In Cox regression analyses, all indirect insulin resistance indices associated with incident PTDM were independent of potential confounders. ROC curve was 0.764 (95% CI, 0.703-0.826) for HOMA-IR, 0.685 (95% CI, 0.615-0.757) for VAI, 0.743 (95% CI, 0.678-0.808) for LAP, and 0.698 (95% CI, 0.629-0.766) for TyG index, with respective optimal cut-off values of 2.47, 4.01, 87.0, and 4.94. CONCLUSIONS: Indirect insulin resistance indices can be used to predict incident PTDM in KTRs. In addition to HOMA-IR, insulin-free surrogates of insulin resistance might serve as useful methods to identify KTRs at risk of PTDM, thus obviating the necessity to measure insulin.

Novel Size-Based High-Density Lipoprotein Subspecies and Incident Vascular Events.

Background High-density lipoprotein (HDL) particle concentration likely outperforms HDL cholesterol in predicting atherosclerotic cardiovascular events. Whether size-based HDL subspecies explain the atheroprotective associations of HDL particle concentration remains unknown. Our objective was to assess whether levels of specific size-based HDL subspecies associate with atherosclerotic cardiovascular disease in a multiethnic pooled cohort and improve risk prediction beyond traditional atherosclerotic cardiovascular disease risk factors. Methods and Results Seven HDL size-based subspecies were quantified by nuclear magnetic resonance (LP4 algorithm; H1=smallest; H7=largest) among participants without prior atherosclerotic cardiovascular disease in ARIC (Atherosclerosis Risk in Communities), MESA (Multi-Ethnic Study of Atherosclerosis), PREVEND (Prevention of Renal and Vascular Endstage Disease), and DHS (Dallas Heart Study) cohorts (n=15 371 people). Multivariable Cox proportional hazards models were used to evaluate the association between HDL subspecies and incident myocardial infarction (MI) or ischemic stroke at follow-up (average 8-10 years) adjusting for HDL cholesterol and risk factors. Improvement in risk prediction was assessed via discrimination and reclassification analysis. Within the pooled cohort (median age 57 years; female 54%; Black 22%) higher H1 (small) and H4 (medium) concentrations were inversely associated with incident MI (hazard ratio [HR]/SD, H1 0.88 [95% CI, 0.81-0.94]; H4 0.89 [95% CI, 0.82-0.97]). H4 but not H1 improved risk prediction indices for incident MI. Increasing H2 and H4 were inversely associated with improved risk prediction indices for composite end point of stroke, MI, and cardiovascular death (HR/SD, H2 0.94 [95% CI, 0.88-0.99]; H4 0.91 [95% CI, 0.85-0.98]). Levels of the large subspecies (H6 and H7) were not associated with any vascular end point. Conclusions Two of 7 HDL size-based subspecies modestly improved risk prediction for MI and composite vascular end points in a large multiethnic pooled cohort. These findings support assessment of precise HDL subspecies for future studies regarding clinical utility.

Plasma Citrate Levels Are Associated with an Increased Risk of Cardiovascular Mortality in Patients with Type 2 Diabetes (Zodiac-64).

Circulating citrate may represent a proxy of mitochondrial dysfunction which plays a role in the development of vascular complications in type 2 diabetes (T2D). Here, we determined the associations between plasma citrate levels and cardiovascular (CV) mortality in T2D patients. In this prospective cohort study, 601 patients were included who participated in the Zwolle Outpatient Diabetes project Integrating Available Care (ZODIAC). Plasma citrate levels were measured by nuclear magnetic resonance spectroscopy. Cox proportional hazards regression models were used to evaluate the associations between plasma citrate and the risk of CV mortality. Over a median follow-up of 11.4 years, 119 (19.8%) of the 601 patients died from a CV cause. In multivariable Cox proportional hazards regression models, adjusting for conventional risk factors, plasma citrate was associated with an increased risk of CV mortality (the hazard ratio (HR) per 1-SD increment was 1.19 (95%CI: 1.00-1.40), p = 0.048). This association was prominent in males (n = 49 with CV mortality) (HR 1.52 (95%CI: 1.14-2.03), p = 0.005), but not in females (n = 70 with CV mortality) (HR 1.11 (95%CI: 0.90-1.37), p = 0.319) (age-adjusted Pinteraction = 0.044). In conclusion, higher plasma citrate levels are associated with an increased risk of CV mortality in patients with established T2D. Future studies are warranted to unravel the potential role of citrate-related pathways in the pathogenesis of T2D-related vascular complications.

Antibody against apolipoprotein-A1, non-alcoholic fatty liver disease and cardiovascular risk: a translational study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common liver disease increasing cardiovascular disease (CVD) morbidity and mortality. Autoantibodies against apolipoprotein A-1 (AAA-1) are a possible novel CVD risk factor promoting inflammation and disrupting cellular lipid homeostasis, two prominent pathogenic features of NAFLD. We explored the role of AAA-1 in NAFLD and their association with CVD risk. METHODS: HepaRG cells and liver sections from ApoE-/- mice exposed to AAA-1 were used for lipid quantification and conditional protein expression. Randomly selected sera from 312 subjects of the Prevention of Renal and Vascular End-stage Disease (PREVEND) general population cohort were used to measure AAA-1. A Fatty Liver Index (FLI) ≥ 60 and a 10-year Framingham Risk Score (FRS) ≥ 20% were used as proxy of NAFLD and high CVD risk, respectively. RESULTS: In-vitro and mouse models showed that AAA-1 increased triglyceride synthesis leading to steatosis, and promoted inflammation and hepatocyte injury. In the 112 PREVEND participants with FLI ≥ 60, AAA-1 were associated with higher FRS, alkaline phosphatase levels, lower HDL cholesterol and tended to display higher FLI values. Univariate linear and logistic regression analyses (LRA) confirmed significant associations between AAA-1, FLI and FRS ≥ 20%, while in adjusted LRA, FLI was the sole independent predictor of FRS ≥ 20% (OR: 1.05, 95%CI 1.01-1.09, P = 0.003). AAA-1 was not an independent FLI predictor. CONCLUSIONS: AAA-1 induce a NAFLD-compatible phenotype in vitro and in mice. Intricate associations exist between AAA-1, CVD risk and FLI in the general population. Further work is required to refine the role of AAA-1 in NAFLD and to determine if the AAA-1 association with CVD is affected by hepatic steatosis.

Circulating Citrate Is Associated with Liver Fibrosis in Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is associated with mitochondrial damage. Circulating mitochondrial metabolites may be elevated in NAFLD but their associations with liver damage is not known. This study aimed to assess the association of key mitochondrial metabolites with the degree of liver fibrosis in the context of NAFLD and nonalcoholic steatohepatitis (NASH). Cross-sectional analyses were performed on two cohorts of biopsy-proven NAFLD and/or NASH subjects. The association of circulating mitochondrial metabolite concentrations with liver fibrosis was assessed using linear regression analysis. In the single-center cohort of NAFLD subjects (n = 187), the mean age was 54.9 ±13.0 years, 40.1% were female and 86.1% were White. Type 2 diabetes (51.3%), hypertension (43.9%) and obesity (72.2%) were prevalent. Those with high citrate had a higher proportion of moderate/significant liver fibrosis (stage F ≥ 2) (68.4 vs. 39.6%, p = 0.001) and advanced fibrosis (stage F ≥ 3) (31.6 vs. 13.6%, p = 0.01). Citrate was associated with liver fibrosis independent of age, sex, NAFLD activity score and metabolic syndrome (per 1 SD increase: ß = 0.19, 95% CI: 0.03-0.35, p = 0.02). This association was also observed in a cohort of NASH subjects (n = 176) (ß = 0.21, 95% CI: 0.07-0.36, p = 0.005). The association of citrate with liver fibrosis was observed in males (p = 0.005) but not females (p = 0.41). In conclusion, circulating citrate is elevated and associated with liver fibrosis, particularly in male subjects with NAFLD and NASH. Mitochondrial function may be a target to consider for reducing the progression of liver fibrosis and NASH.

The optimal healthy ranges of thyroid function defined by the risk of cardiovascular disease and mortality: systematic review and individual participant data meta-analysis.

BACKGROUND: Reference intervals of thyroid-stimulating hormone (TSH) and free thyroxine (FT4) are statistically defined by the 2·5-97·5th percentiles, without accounting for potential risk of clinical outcomes. We aimed to define the optimal healthy ranges of TSH and FT4 based on the risk of cardiovascular disease and mortality. METHODS: This systematic review and individual participant data (IPD) meta-analysis identified eligible prospective cohorts through the Thyroid Studies Collaboration, supplemented with a systematic search via Embase, MEDLINE (Ovid), Web of science, the Cochrane Central Register of Controlled Trials, and Google Scholar from Jan 1, 2011, to Feb 12, 2017 with an updated search to Oct 13, 2022 (cohorts found in the second search were not included in the IPD). We included cohorts that collected TSH or FT4, and cardiovascular outcomes or mortality for adults (aged ≥18 years). We excluded cohorts that included solely pregnant women, individuals with overt thyroid diseases, and individuals with cardiovascular disease. We contacted the study investigators of eligible cohorts to provide IPD on demographics, TSH, FT4, thyroid peroxidase antibodies, history of cardiovascular disease and risk factors, medication use, cardiovascular disease events, cardiovascular disease mortality, and all-cause mortality. The primary outcome was a composite outcome including cardiovascular disease events (coronary heart disease, stroke, and heart failure) and all-cause mortality. Secondary outcomes were the separate assessment of cardiovascular disease events, all-cause mortality, and cardiovascular disease mortality. We performed one-step (cohort-stratified Cox models) and two-step (random-effects models) meta-analyses adjusting for age, sex, smoking, systolic blood pressure, diabetes, and total cholesterol. The study was registered with PROSPERO, CRD42017057576. FINDINGS: We identified 3935 studies, of which 53 cohorts fulfilled the inclusion criteria and 26 cohorts agreed to participate. We included IPD on 134 346 participants with a median age of 59 years (range 18-106) at baseline. There was a J-shaped association of FT4 with the composite outcome and secondary outcomes, with the 20th (median 13·5 pmol/L [IQR 11·2-13·9]) to 40th percentiles (median 14·8 pmol/L [12·3-15·0]) conveying the lowest risk. Compared with the 20-40th percentiles, the age-adjusted and sex-adjusted hazard ratio (HR) for FT4 in the 80-100th percentiles was 1·20 (95% CI 1·11-1·31) for the composite outcome, 1·34 (1·20-1·49) for all-cause mortality, 1·57 (1·31-1·89) for cardiovascular disease mortality, and 1·22 (1·11-1·33) for cardiovascular disease events. In individuals aged 70 years and older, the 10-year absolute risk of composite outcome increased over 5% for women with FT4 greater than the 85th percentile (median 17·6 pmol/L [IQR 15·0-18·3]), and men with FT4 greater than the 75th percentile (16·7 pmol/L [14·0-17·4]). Non-linear associations were identified for TSH, with the 60th (median 1·90 mIU/L [IQR 1·68-2·25]) to 80th percentiles (2·90 mIU/L [2·41-3·32]) associated with the lowest risk of cardiovascular disease and mortality. Compared with the 60-80th percentiles, the age-adjusted and sex-adjusted HR of TSH in the 0-20th percentiles was 1·07 (95% CI 1·02-1·12) for the composite outcome, 1·09 (1·05-1·14) for all-cause mortality, and 1·07 (0·99-1·16) for cardiovascular disease mortality. INTERPRETATION: There was a J-shaped association of FT4 with cardiovascular disease and mortality. Low concentrations of TSH were associated with a higher risk of all-cause mortality and cardiovascular disease mortality. The 20-40th percentiles of FT4 and the 60-80th percentiles of TSH could represent the optimal healthy ranges of thyroid function based on the risk of cardiovascular disease and mortality, with more than 5% increase of 10-year composite risk identified for FT4 greater than the 85th percentile in women and men older than 70 years. We propose a feasible approach to establish the optimal healthy ranges of thyroid function, allowing for better identification of individuals with a higher risk of thyroid-related outcomes. FUNDING: None.

Creatine and creatinine quantified using nuclear magnetic resonance: A method validation study and clinical associations between circulating creatine and fatigue in kidney transplant recipients.

BACKGROUND: A potential contributor to fatigue in kidney transplant recipients (KTR) may be impaired creatine homeostasis. We developed and validated a high-throughput NMR assay allowing for simultaneous measurement of circulating creatine and creatinine, and determined plasma creatine and estimated intramuscular creatine concentrations in KTRs, delineated their determinants and explored their associations with self-reported fatigue. METHODS: An NMR assay was developed and validated for measurement of circulating creatinine and creatine concentrations. Plasma creatine and creatinine concentrations were measured in 618 KTR. Fatigue was assessed using the checklist individual strength. Associations of creatine parameters with fatigue was assessed using linear mixed effect models. RESULTS: The NMR-based assay had good sensitivity, precision and demonstrated linearity across a large range of values. Among KTR, the mean age was 56 ± 13 years, 62% were men and eGFR was 54 ± 18 ml/min/1.73 m2. Plasma creatine concentration was 27 [19-39] µmol/L. Estimated intramuscular creatine concentration was 27 ± 7 mmol/kg. Higher plasma creatine concentration and higher estimated intramuscular creatine concentration were independently associated with a lower total fatigue score and less motivation problems. CONCLUSION: An NMR method for measurement of circulating creatine and creatinine which offers the potential for accurate and efficient quantification was developed. The found associations suggest that improving creatine status may play a beneficial role in mitigating fatigue.

Serum Level of Cytokeratin 18 (M65) as a Prognostic Marker of High Cardiovascular Disease Risk in Individuals with Non-Alcoholic Fatty Liver Disease.

Alterations in apoptosis, as reflected by circulating Cytokeratin 18 (CK18), are involved in the progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis and atherogenesis. We aimed to explore the discriminant accuracy of Cytokeratin 18 (CK18, including M65 and M30 forms) for an elevated fatty liver index (FLI) as a validated proxy of NAFLD, and cardiovascular disease (CVD) risk in the general population. Both serum CK18 forms were measured using a commercial immunoassay in randomly selected samples from 312 participants of the PREVEND general population cohort. FLI ≥ 60 was used to indicate NAFLD. Framingham Risk Score (FRS) and the SCORE2 were used to estimate the 10-year risk of CVD. The Receiver Operating Characteristic (ROC) curve, linear/logistic regression models, and Spearman's correlations were used. Intricate associations were found between CK18, FLI, and CVD risk scores. While M30 was the only independent predictor of FLI ≥ 60, M65 best discriminated NAFLD individuals at very-high 10-year CVD risk according to SCORE2 (AUC: 0.71; p = 0.001). Values above the predefined manufacturer cutoff (400 U/L) were associated with an independent 5-fold increased risk (adjusted odds ratio: 5.44, p = 0.01), with a negative predictive value of 93%. Confirming that NAFLD is associated with an increased CVD risk, our results in a European general population-based cohort suggest that CK18 M65 may represent a candidate biomarker to identify NAFLD individuals at low CVD risk.

Both LDL and HDL particle concentrations associate positively with an increased risk of developing microvascular complications in patients with type 2 diabetes: lost protection by HDL (Zodiac-63).

BACKGROUND: Triglyceride-rich lipoproteins (TRL) and low-density lipoproteins (LDL) are associated positively whereas high-density lipoproteins (HDL) are associated inversely with the development of new-onset type 2 diabetes (T2D). Here we studied potential associations between these lipoprotein particle concentrations and the risk of developing microvascular complications in patients with established T2D. METHODS: Lipoprotein particle concentrations (TRLP, LDLP, and HDLP) were determined in 278 patients with T2D participating in a primary care-based longitudinal cohort study (Zwolle Outpatient Diabetes project Integrating Available Care [ZODIAC] study) leveraging the Vantera nuclear magnetic resonance (NMR) platform using the LP4 algorithm. Associations between lipoprotein particles and incident microvascular complications (nephropathy, neuropathy, and retinopathy) were assessed using Cox proportional hazards regression models. RESULTS: In total, 136 patients had microvascular complications at baseline. During a median follow-up of 3.2 years, 49 (34.5%) of 142 patients without microvascular complications at baseline developed new-onset microvascular complications. In multivariable Cox proportional hazards regression analyses, both total LDLP and HDLP concentrations, but not total TRLP concentrations, were positively associated with an increased risk of developing any microvascular complications after adjustment for potential confounding factors, including age, sex, disease duration, HbA1c levels, history of macrovascular complications, and statin use (adjusted hazard ratio [HR] per 1 SD increment: 1.70 [95% CI 1.24-2.34], P < 0.001 and 1.63 [95% CI 1.19-2.23], P = 0.002, respectively). When analyzing each microvascular complication individually, total LDLP concentrations were positively associated with retinopathy (adjusted HR 3.35, 95% CI 1.35-8.30, P = 0.009) and nephropathy (adjusted HR 2.13, 95% CI 1.27-3.35, P = 0.004), and total HDLP concentrations with neuropathy (adjusted HR 1.77, 95% CI 1.15-2.70, P = 0.009). No significant associations were observed for lipoprotein particle subfractions. CONCLUSIONS: Total lipoprotein particle concentrations of both LDL and HDL associate positively with an increased risk of developing microvascular complications in T2D. We propose that the protective role of HDL on the development of microvascular complications may be lost in established T2D.

Fasting Ketone Bodies and Incident Type 2 Diabetes in the General Population.

With rising incidence and prevalence of type 2 diabetes, prevention including identification of prospective biomarkers becomes increasingly relevant. Although ketone bodies recently received a renewed interest as potential biomarkers, data linking these metabolites to diabetes risk are scarce. Therefore, the present prospective study investigated a potential association between fasting ketone bodies and incident type 2 diabetes in the general population. This study from the PREVEND cohort included 3,307 participants from the general population initially free of diabetes or impaired fasting glucose. Baseline fasting ketone body concentrations were measured by nuclear magnetic resonance spectroscopy. One hundred twenty-six participants (3.8%) developed type 2 diabetes during a median (interquartile range) follow-up of 7.3 (6.3-7.6) years. In Kaplan-Meier analysis, sex-stratified ketone body levels strongly positively associated with incident type 2 diabetes, which was confirmed in Cox regression analyses adjusted for several potential confounders. There was no significant interaction by sex. Both 3-ß-hydroxybutyrate and acetoacetate+acetone individually associated with incident type 2 diabetes. In conclusion, fasting plasma ketone body levels are strongly positively associated with incident type 2 diabetes in the general population independent of several other recognized risk factors. These results may have important implications for diabetes prevention including dietary strategies. ARTICLE HIGHLIGHTS: The identification of biomarkers that predict type 2 diabetes is increasingly relevant for personalized medicine strategies. Data regarding ketone bodies and incident type 2 diabetes are scarce. This study shows that ketone bodies, either combined or as individual subspecies, are strongly associated with incident type 2 diabetes in the general population, independent of potential confounders. These results may have important implications for diabetes prevention including dietary strategies.

Branched Chain Amino Acids Are Associated with Physical Performance in Patients with End-Stage Liver Disease.

Decreased circulating branched chain amino acids (BCAA) represent a prominent change in amino acid profiles in patients with end-stage liver disease (ESLD). These alterations are considered to contribute to sarcopenia and hepatic encephalopathy and may relate to poor prognosis. Here, we cross-sectionally analyzed the association between plasma BCAA levels and the severity of ESLD and muscle function in participants of the liver transplant subgroup of TransplantLines, enrolled between January 2017 and January 2020. Plasma BCAA levels were measured by nuclear magnetic resonance spectroscopy. Physical performance was analyzed with a hand grip strength test, 4 m walking test, sit-to-stand test, timed up and go test, standing balance test and clinical frailty scale. We included 92 patients (65% men). The Child Pugh Turcotte classification was significantly higher in the lowest sex-stratified BCAA tertile compared to the highest tertile (p = 0.015). The times for the sit-to-stand (r = -0.352, p < 0.05) and timed up and go tests (r = -0.472, p < 0.01) were inversely correlated with total BCAA levels. In conclusion, lower circulating BCAA are associated with the severity of liver disease and impaired muscle function. This suggests that BCAA may represent a useful prognostic marker in the staging of liver disease severity.