High Betaine, a Trimethylamine N-Oxide Related Metabolite, Is Prospectively Associated with Low Future Risk of Type 2 Diabetes Mellitus in the PREVEND Study

Background: Gut microbiota-related metabolites, trimethylamine-N-oxide (TMAO), choline, and betaine, have been shown to be associated with cardiovascular disease (CVD) risk. Moreover, lower plasma betaine concentrations have been reported in subjects with type 2 diabetes mellitus (T2DM). However, few studies have explored the association of betaine with incident T2DM, especially in the general population. The goals of this study were to evaluate the performance of a newly developed betaine assay and to prospectively explore the potential clinical associations of betaine and future risk of T2DM in a large population-based cohort. Methods: We developed a high-throughput, nuclear magnetic resonance (NMR) spectroscopy procedure for acquiring spectra that allow for the accurate quantification of plasma/serum betaine and TMAO. Assay performance for betaine quantification was assessed and Cox proportional hazards regression was employed to evaluate the association of betaine with incident T2DM in 4336 participants in the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study. Results: Betaine assay results were linear (y = 1.02X - 3.75) over a wide range of concentrations (26.0-1135 mu M). The limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ) were 6.4, 8.9, and 13.2 mu M, respectively. Coefficients of variation for intra- and inter-assay precision ranged from 1.5-4.3% and 2.5-5.5%, respectively. Deming regression analysis of results produced by NMR and liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS) revealed an R-2 value of 0.94 (Y = 1.08x - 1.89) and a small bias for higher values by NMR. The reference interval, in a cohort of apparently healthy adult participants (n = 501), was determined to be 23.8 to 74.7 mu M (mean of 42.9 +/- 12.6 mu M). In the PREVEND study (n = 4336, excluding subjects with T2DM at baseline), higher betaine was associated with older age and lower body mass index, total cholesterol, triglycerides, and hsCRP. During a median follow-up of 7.3 (interquartile range (IQR), 5.9-7.7) years, 224 new T2DM cases were ascertained. Cox proportional hazards regression models revealed that the highest tertile of betaine was associated with a lower incidence of T2DM. Hazard ratio (HR) for the crude model was 0.61 (95% CI: 0.44-0.85, p = 0.004). The association remained significant even after adjusting for multiple clinical covariates and T2DM risk factors, including fasting glucose. HR for the fully-adjusted model was 0.50 (95% CI: 0.32-0.80, p = 0.003). Conclusions: The newly developed NMR-based betaine assay exhibits performance characteristics that are consistent with usage in the clinical laboratory. Betaine levels may be useful for assessing the risk of future T2DM

TMAO is Associated with Mortality:Impact of Modestly Impaired Renal Function

Abstract Trimethylamine-N-Oxide (TMAO) is a microbiome-related metabolite that is cleared by the kidney and linked to renal function. We explored the relationship between TMAO and all-cause mortality, and determined whether this association was modified by renal function. A prospective study was performed among PREVEND participants to examine associations of plasma TMAO with all-cause mortality. After median follow-up of 8.3 years in 5,469 participants, 322 subjects died. TMAO was positively associated with age, body mass index, type 2 diabetes mellitus and inversely with estimated glomerular filtration rate (eGFRcreatcysC)(all P < 0.001). Subjects in the highest versus lowest TMAO quartile had a crude 1.86-fold higher mortality risk (Ptrend < 0.001). After adjustment for several risk factors, TMAO remained associated with all-cause mortality [HR:1.36 (95% CI, 0.97–1.91),Ptrend = 0.016]. This association was lost after further adjustment for urinary albumin excretion and eGFR [HR:1.15 (95% CI, 0.81–1.64),Ptrend = 0.22]. The association of TMAO with mortality was modified by eGFR in crude and age- and sex-adjusted analyses (interaction P = 0.002). When participants were stratified by renal function (eGFR < vs. ≥90 mL/min/1.73 m2), TMAO was associated with all-cause mortality only in subjects with eGFR <90 mL/min/1.73 m2 [adjusted HR:1.18 (95% CI, 1.02–1.36),P = 0.023]. In conclusion, TMAO is associated with all-cause mortality, particularly in subjects with eGFR <90 mL/min/1.73 m2

Characterization of LP-Z Lipoprotein Particles and Quantification in Subjects with Liver Disease Using a Newly Developed NMR-Based Assay

Background: Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. Methods: Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. Results: The assay showed good linearity and precision (2.5-6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. Conclusions: LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease

Genome- and CD4\u3csup\u3e+\u3c/sup\u3e T-Cell Methylome-Wide Association Study of Circulating Trimethylamine-N-Oxide in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)

Background: Trimethylamine-N-oxide (TMAO), an atherogenic metabolite species, has emerged as a possible new risk factor for cardiovascular disease. Animal studies have shown that circulating TMAO levels are regulated by genetic and environmental factors. However, large-scale human studies have failed to replicate the observed genetic associations, and epigenetic factors such as DNA methylation have never been examined in relation to TMAO levels. Methods and results: We used data from the family-based Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) to investigate the heritable determinants of plasma TMAO in humans. TMAO was not associated with other plasma markers of cardiovascular disease, e.g. lipids or inflammatory cytokines. We first estimated TMAO heritability at 27%, indicating a moderate genetic influence. We used 1000 Genomes imputed data (n = 626) to estimate genome-wide associations with TMAO levels, adjusting for age, sex, family relationships, and study site. The genome-wide study yielded one significant hit at the genome-wide level, located in an intergenic region on chromosome 4. We subsequently quantified epigenome-wide DNA methylation using the Illumina Infinium array on CD4þ Tcells. We tested for association of methylation loci with circulating TMAO (n = 847), adjusting for age, sex, family relationships, and study site as the genome-wide study plus principal components capturing CD4þ T-cell purity. Upon adjusting for multiple testing, none of the epigenetic findings were statistically significant. Conclusions: Our findings contribute to the growing body of evidence suggesting that neither genetic nor epigenetic factors play a critical role in establishing circulating TMAO levels in humans

Characterization of LP-Z Lipoprotein Particles and Quantification in Subjects with Liver Disease Using a Newly Developed NMR-Based Assay

Background: Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. Methods: Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. Results: The assay showed good linearity and precision (2.5-6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. Conclusions: LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease