Protocol to assess the efficacy of carnosine supplementation in mitigating the adverse cardiovascular responses to particulate matter (PM) exposure: the Nucleophilic Defense Against PM Toxicity (NEAT) trial.

INTRODUCTION: Exposure to airborne particulate matter (PM) is associated with cardiovascular disease. These outcomes are believed to originate from pulmonary oxidative stress and the systemic delivery of oxidised biomolecules (eg, aldehydes) generated in the lungs. Carnosine is an endogenous di-peptide (ß-alanine-L-histidine) which promotes physiological homeostasis in part by conjugating to and neutralising toxic aldehydes. We hypothesise that an increase of endogenous carnosine by dietary supplementation would mitigate the adverse cardiovascular outcomes associated with PM exposure in humans. METHODS AND ANALYSIS: To test this, we designed the Nucleophilic Defense Against PM Toxicity trial. This trial will enroll 240 participants over 2 years and determine if carnosine supplementation mitigates the adverse effects of PM inhalation. The participants will have low levels of endogenous carnosine to facilitate identification of supplementation-specific outcomes. At enrollment, we will measure several indices of inflammation, preclinical cardiovascular disease and physical function. Participants will be randomly allocated to carnosine or placebo groups and instructed to take their oral supplement for 12 weeks with two return clinical visits and repeated assessments during times of peak PM exposure (June-September) in Louisville, Kentucky, USA. Statistical modelling approaches will be used to assess the efficacy of carnosine supplementation in mitigating adverse outcomes. ETHICS AND DISSEMINATION: This study protocol has been approved by the Institutional Review Board at the University of Louisville. Results from this study will be disseminated at scientific conferences and in peer-reviewed publications.Trial registration: NCT03314987; Pre-results.

Carnosine Supplementation Enhances Post Ischemic Hind Limb Revascularization.

High (millimolar) concentrations of the histidine containing dipeptide - carnosine (ß-alanine-L-histidine) are present in the skeletal muscle. The dipeptide has been shown to buffer intracellular pH, chelate transition metals, and scavenge lipid peroxidation products; however, its role in protecting against tissue injury remains unclear. In this study, we tested the hypothesis that carnosine protects against post ischemia by augmenting HIF-1α angiogenic signaling by Fe2+ chelation. We found that wild type (WT) C57BL/6 mice, subjected to hind limb ischemia (HLI) and supplemented with carnosine (1g/L) in drinking water, had improved blood flow recovery and limb function, enhanced revascularization and regeneration of myocytes compared with HLI mice placed on water alone. Carnosine supplementation enhanced the bioavailability of carnosine in the ischemic limb, which was accompanied by increased expression of proton-coupled oligopeptide transporters. Consistent with our hypothesis, carnosine supplementation augmented HIF-1α and VEGF expression in the ischemic limb and the mobilization of proangiogenic Flk-1+/Sca-1+ cells into circulation. Pretreatment of murine myoblast (C2C12) cells with octyl-D-carnosine or carnosine enhanced HIF-1α protein expression, VEGF mRNA levels and VEGF release under hypoxic conditions. Similarly pretreatment of WT C57/Bl6 mice with carnosine showed enhanced blood flow in the ischemic limb following HLI surgery. In contrast, pretreatment of hypoxic C2C12 cells with methylcarcinine, a carnosine analog, lacking Fe2+ chelating capacity, had no effect on HIF-1α levels and VEGF release. Collectively, these data suggest that carnosine promotes post ischemic revascularization via augmentation of pro-angiogenic HIF-1α/VEGF signaling, possibly by Fe2+ chelation.

Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by ß-alanine transamination.

KEY POINTS: Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that ß-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of ß-alanine, which is in turn controlled by degradation of ß-alanine in liver and kidney. Chronic oral ß-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the ß-alanine supplementation protocol is so inefficient, by demonstrating that exogenous ß-alanine can be effectively routed toward oxidation. ABSTRACT: The metabolic fate of orally ingested ß-alanine is largely unknown. Chronic ß-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (ß-alanyl-l-histidine) in muscle. However, only a small fraction (3-6%) of the ingested ß-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two ß-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate ß-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on ß-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating ß-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating ß-alanine levels, which are suppressed by hepatic and renal ß-alanine transamination upon oral ß-alanine intake.

Dietary carnosine prevents early atherosclerotic lesion formation in apolipoprotein E-null mice.

OBJECTIVE: Atherosclerotic lesions are associated with the accumulation of reactive aldehydes derived from oxidized lipids. Although inhibition of aldehyde metabolism has been shown to exacerbate atherosclerosis and enhance the accumulation of aldehyde-modified proteins in atherosclerotic plaques, no therapeutic interventions have been devised to prevent aldehyde accumulation in atherosclerotic lesions. APPROACH AND RESULTS: We examined the efficacy of carnosine, a naturally occurring ß-alanyl-histidine dipeptide, in preventing aldehyde toxicity and atherogenesis in apolipoprotein E-null mice. In vitro, carnosine reacted rapidly with lipid peroxidation-derived unsaturated aldehydes. Gas chromatography mass-spectrometry analysis showed that carnosine inhibits the formation of free aldehydes 4-hydroxynonenal and malonaldialdehyde in Cu(2+)-oxidized low-density lipoprotein. Preloading bone marrow-derived macrophages with cell-permeable carnosine analogs reduced 4-hydroxynonenal-induced apoptosis. Oral supplementation with octyl-D-carnosine decreased atherosclerotic lesion formation in aortic valves of apolipoprotein E-null mice and attenuated the accumulation of protein-acrolein, protein-4-hydroxyhexenal, and protein-4-hydroxynonenal adducts in atherosclerotic lesions, whereas urinary excretion of aldehydes as carnosine conjugates was increased. CONCLUSIONS: The results of this study suggest that carnosine inhibits atherogenesis by facilitating aldehyde removal from atherosclerotic lesions. Endogenous levels of carnosine may be important determinants of atherosclerotic lesion formation, and treatment with carnosine or related peptides could be a useful therapy for the prevention or the treatment of atherosclerosis.

Purification and characterization of kininogens from sheep plasma.

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep (Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. Ki values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. Ki values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.