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Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl − system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl residues serves as a mechanism for producing 2-AAA and LysCN in vivo . These studies further support involvement of MPO-catalyzed oxidative processes in both the development of atherosclerosis and diabetes risk. [ABSTRACT FROM AUTHOR]

Summary Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e −/− mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases. [ABSTRACT FROM AUTHOR]

Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary l-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of l-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma l-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary l-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk. [ABSTRACT FROM AUTHOR]

Context: Formation of nitric oxide–derived oxidants may serve as a mechanism linking inflammation to development of atherosclerosis. Nitrotyrosine, a specific marker for protein modification by nitric oxide–derived oxidants, is enriched in human atherosclerotic lesions and low-density lipoprotein (LDL) recovered from human atheroma. Objectives: To determine whether systemic levels of nitrotyrosine are associated with the prevalence of coronary artery disease (CAD) and are modulated by hydroxymethylglutaryl coenzyme-A reductase inhibitor (statin) therapy. Design, Setting, and Patients: A case-control and interventional study at 2 urban tertiary-care referral centers; recruitment for each was from June 1, 2001, until January 1, 2002. For the case-control study, 100 case-patients with established CAD and 108 patients with no clinically evident CAD were recruited consecutively. In the interventional study, participants aged 21 years or older with hypercholesterolemia (LDL cholesterol ≥130 mg/dL [≥3.5 mmol/L]) underwent nutrition and exercise counseling. Those whose levels did not decrease with 6 to 8 weeks were enrolled in the study (n = 35). For 12 weeks, they received 10 mg/d of oral atorvastatin therapy. Main Outcome Measures: In the case-control study, the association between systemic levels of protein-bound nitrotyrosine, CAD risk, and presence of CAD. In the interventional study, the change in nitrotyrosine, lipoprotein, and C-reactive protein (CRP) levels. Results: Nitrotyrosine levels were significantly higher among patients with CAD (median 9.1 µmol/mol [interquartile range, 4.8-13.8 µmol/mol] tyrosine vs 5.2 µmol/mol [interquartile range, 2.2-8.4 µmol/mol]; P<.001). Patients in the upper quartile of nitrotyrosine (29%; P<.001) had a higher odds of CAD compared with those in the lowest quartile (unadjusted odds ratio, 6.1; 95% confidence interval, 2.6-14.0; P<.001). In multivariate models adjusting for... [ABSTRACT FROM AUTHOR]