Evaluation of supplementary carnosine accumulation and distribution: an initial analysis of participants in the Nucleophilic Defense Against PM Toxicity (NEAT) clinical trial.

Carnosine is an endogenous dipeptide that buffers intracellular pH and quenches toxic products of lipid peroxidation. Used as a dietary supplement, it also supports exercise endurance. However, the accumulation and distribution of carnosine after supplementation has not been rigorously evaluated. To do this, we randomized a cohort to receive daily supplements of either placebo or carnosine (2 g/day). Blood and urine samples were collected twice over the subsequent 12 week supplementation period and we measured levels of red blood cell (RBC) carnosine, urinary carnosine, and urinary carnosine-propanol and carnosine-propanal conjugates by LC/MS-MS. We found that, when compared with placebo, supplementation with carnosine for 6 or 12 weeks led to an approximate twofold increase in RBC carnosine, while levels of urinary carnosine increased nearly sevenfold. Although there were no changes in the urinary levels of carnosine propanol, carnosine propanal increased nearly twofold. RBC carnosine levels were positively associated with urinary carnosine and carnosine propanal levels. No adverse reactions were reported by those in the carnosine or placebo arms, nor did carnosine supplementation have any effect on kidney, liver, and cardiac function or blood electrolytes. In conclusion, irrespective of age, sex, or BMI, oral carnosine supplementation in humans leads to its increase in RBC and urine, as well as an increase in urinary carnosine-propanal. RBC carnosine may be a readily accessible pool to estimate carnosine levels. Clinical trial registration: This study is registered with ClinicalTrials.gov (Nucleophilic Defense Against PM Toxicity (NEAT Trial)-Full Text View-ClinicalTrials.gov), under the registration: NCT03314987.

Molecular investigation of vitamin D receptor (VDR) genetic variants and their impact on VDR mRNA and serum vitamin D levels in allergic rhinitis in an Indian population: A case-control study.

Vitamin D deficiency is widespread and poses a significant health concern, as emerging research links it to allergic diseases owing to its immunomodulatory functions. The optimal functioning of vitamin D and its activation depend on its nuclear receptor, vitamin D receptor (VDR). Genetic variants of VDR have been explored as potential factors in autoimmune and allergic diseases, with limited studies on their association with allergic rhinitis (AR). The present investigation aims to analyse the role of three VDR genetic variants - TaqI, FokI and BsmI - in AR susceptibility and their impact on VDR mRNA and serum vitamin D levels. A total of 550 subjects, consisting of 250 AR cases and 300 age- and gender-matched controls, underwent genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). VDR mRNA and vitamin D levels were determined by quantitative real-time PCR and chemiluminescence, respectively. Although TaqI did not exhibit significant differences, FokI demonstrated a noteworthy association with AR, particularly with the CC genotype (odds ratio [OR]: 3.34; confidence interval [CI]: 1.79-6.23). Similarly, BsmI revealed an increased risk for AR, with the GA + AA genotypes showing a 2.2-fold elevated risk (OR: 2.20; CI: 1.53-3.16). VDR mRNA expression was threefold lower in AR patients (p < .0001), accompanied by reduced serum vitamin D levels (p < .0001). In addition, CC (p = .01) and AA (p = .02) genotypes of FokI and BsmI were associated with reduced VDR mRNA levels, whereas TaqI showed no such association. Similarly, heterozygous genotypes of TaqI and FokI, as well as homozygous AA of BsmI, correlated with lower serum vitamin D levels (p < .001). This study emphasizes the intricate relationship among VDR genetic variations, altered VDR activity, immune modulation and vitamin D metabolism in AR. Further research involving diverse populations is crucial for confirming and generalizing these findings, paving the way for personalized therapeutic interventions in vitamin D-related disorders.

Carnosine as a potential therapeutic for the management of peripheral vascular disease.

AIMS: To evaluate the potential role of carnosine in the management of peripheral vascular disease. DATA SYNTHESIS: Peripheral vascular disease is growing in its burden and impact; however it is currently under researched, and there are a lack of strong, non-invasive therapeutic options for the clinicians. Carnosine is a dipeptide stored particularly in muscle and brain tissue, which exhibits a wide range of physiological activities, which may be beneficial as an adjunct treatment for peripheral vascular disease. Carnosine's strong anti-inflammatory, antioxidant and antiglycating actions may aid in the prevention of plaque formation, through protective actions on the vascular endothelium, and the inhibition of foam cells. Carnosine may also improve angiogenesis, exercise performance and vasodilatory response, while protecting from ischemic tissue injury. CONCLUSIONS: Carnosine may have a role as an adjunct treatment for peripheral vascular disease alongside typical exercise and surgical interventions, and may be used in high risk individuals to aid in the prevention of atherogenesis. CLINICAL RECOMMENDATION: This review identifies a beneficial role for carnosine supplementation in the management of patients with peripheral vascular disease, in conjunction with exercise and revascularization. Carnosine as a supplement is safe, and associated with a host of beneficial effects in peripheral vascular disease and its key risk factors.

Implications of risk conferred by 5p15.33 loci genetic variants; human telomerase reverse transcriptase rs2736098 and rs2736100 in predisposition of bladder cancer.

Background: The polymorphic variations of human telomerase reverse transcriptase (hTERT) gene play an important role in predisposition to carcinogenesis. The current study aimed to elucidate the genetic predisposition to bladder cancer in two important variants, rs2736098 and rs2736100 of hTERT gene. Materials and methods: Confirmed 130 patients of bladder cancer and 200 healthy controls were genotyped by PCR-RFLP to determine different variants of hTERT rs2736098 and rs2736100. Results: hTERT rs2736098 homozygous variant AA genotype frequency was observed to significantly differ 2-fold between cases and controls (26.15% vs. 13.5%) (p = 0.02). In addition, rare 'A' allele significantly differed among two groups (cases: 47% versus controls: 39%: p = 0.03). hTERT rs2736098 was observed to be presented significantly more in high stage tumors (p = 0.02). hTERT rs2736100 genotype AA or variant allele A showed no significant difference between cases and controls. Haplotype CA displayed significantly different pattern of frequency as 0.5 in cases as compared to 0.16 in controls (p < 0.0001). Combination of variant A/G haplotype frequency implicated more in cases than in controls (0.34 vs. 0.14, p = 0.001). Conclusions: It is concluded that hTERT rs2736098 polymorphic variant has a vital role to confer a strong risk to bladder cancer in our population. Further, hTERT haplotypes CA and AG inhTERT could prove to be a promising tool to screen the risk for bladder cancer.

Carnosine quenches the reactive carbonyl acrolein in the central nervous system and attenuates autoimmune neuroinflammation.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease driven by sustained inflammation in the central nervous system. One of the pathological hallmarks of MS is extensive free radical production. However, the subsequent generation, potential pathological role, and detoxification of different lipid peroxidation-derived reactive carbonyl species during neuroinflammation are unclear, as are the therapeutic benefits of carbonyl quenchers. Here, we investigated the reactive carbonyl acrolein and (the therapeutic effect of) acrolein quenching by carnosine during neuroinflammation. METHODS: The abundance and localization of acrolein was investigated in inflammatory lesions of MS patients and experimental autoimmune encephalomyelitis (EAE) mice. In addition, we analysed carnosine levels and acrolein quenching by endogenous and exogenous carnosine in EAE. Finally, the therapeutic effect of exogenous carnosine was assessed in vivo (EAE) and in vitro (primary mouse microglia, macrophages, astrocytes). RESULTS: Acrolein was substantially increased in inflammatory lesions of MS patients and EAE mice. Levels of the dipeptide carnosine (ß-alanyl-L-histidine), an endogenous carbonyl quencher particularly reactive towards acrolein, and the carnosine-acrolein adduct (carnosine-propanal) were ~ twofold lower within EAE spinal cord tissue. Oral carnosine treatment augmented spinal cord carnosine levels (up to > tenfold), increased carnosine-acrolein quenching, reduced acrolein-protein adduct formation, suppressed inflammatory activity, and alleviated clinical disease severity in EAE. In vivo and in vitro studies indicate that pro-inflammatory microglia/macrophages generate acrolein, which can be efficiently quenched by increasing carnosine availability, resulting in suppressed inflammatory activity. Other properties of carnosine (antioxidant, nitric oxide scavenging) may also contribute to the therapeutic effects. CONCLUSIONS: Our results identify carbonyl (particularly acrolein) quenching by carnosine as a therapeutic strategy to counter inflammation and macromolecular damage in MS.

Urinary Levels of the Acrolein Conjugates of Carnosine Are Associated with Cardiovascular Disease Risk.

Carnosine is a naturally occurring dipeptide (ß-alanine-L-histidine) which supports physiological homeostasis by buffering intracellular pH, chelating metals, and conjugating with and neutralizing toxic aldehydes such as acrolein. However, it is not clear if carnosine can support cardiovascular function or modify cardiovascular disease (CVD) risk. To examine this, we measured urinary levels of nonconjugated carnosine and its acrolein conjugates (carnosine-propanal and carnosine-propanol) in participants of the Louisville Healthy Heart Study and examined associations with indices of CVD risk. We found that nonconjugated carnosine was significantly associated with hypertension (p = 0.011), heart failure (p = 0.015), those categorized with high CVD risk (p < 0.001), body mass index (BMI; p = 0.007), high sensitivity C-reactive protein (hsCRP; p = 0.026), high-density lipoprotein (HDL; p = 0.007) and certain medication uses. Levels of carnosine-propanal and carnosine-propanol demonstrated significant associations with BMI, blood glucose, HDL and diagnosis of diabetes. Carnosine-propanal was also associated with heart failure (p = 0.045) and hyperlipidemia (p = 0.002), but no associations with myocardial infarction or stroke were identified. We found that the positive associations of carnosine conjugates with diabetes and HDL remain statistically significant (p < 0.05) in an adjusted, linear regression model. These findings suggest that urinary levels of nonconjugated carnosine, carnosine-propanal and carnosine-propanol may be informative biomarkers for the assessment of CVD risk-and particularly reflective of skeletal muscle injury and carnosine depletion in diabetes.

Carnosinase-1 overexpression, but not aerobic exercise training, affects the development of diabetic nephropathy in BTBR ob/ob mice.

Manipulation of circulating histidine-containing dipeptides (HCD) has been shown to affect the development of diabetes and early-stage diabetic nephropathy (DN). The aim of the present study was to investigate whether such interventions, which potentially alter levels of circulating HCD, also affect the development of advanced-stage DN. Two interventions, aerobic exercise training and overexpression of the human carnosinase-1 (hCN1) enzyme, were tested. BTBR ob/ob mice were either subjected to aerobic exercise training (20 wk) or genetically manipulated to overexpress hCN1, and different diabetes- and DN-related markers were compared with control ob/ob and healthy (wild-type) mice. An acute exercise study was performed to elucidate the effect of obesity, acute running, and hCN1 overexpression on plasma HCD levels. Chronic aerobic exercise training did not affect the development of diabetes or DN, but hCN1 overexpression accelerated hyperlipidemia and aggravated the development of albuminuria, mesangial matrix expansion, and glomerular hypertrophy of ob/ob mice. In line, plasma, kidney, and muscle HCD were markedly lower in ob/ob versus wild-type mice, and plasma and kidney HCD in particular were lower in ob/ob hCN1 versus ob/ob mice but were unaffected by aerobic exercise. In conclusion, advanced glomerular damage is accelerated in mice overexpressing the hCN1 enzyme but not protected by chronic exercise training. Interestingly, we showed, for the first time, that the development of DN is closely linked to renal HCD availability. Further research will have to elucidate whether the stimulation of renal HCD levels can be a therapeutic strategy to reduce the risk for developing DN.

Association of strong risk of hTERT gene polymorphic variants to malignant glioma and its prognostic implications with respect to different histological types and survival of glioma cases.

BACKGROUND: Germline genetic variants of human telomerase reverse transcriptase (hTERT) are known to predispose for various malignancies, including glioma. The present study investigated genetic variation of hTERT T/G (rs2736100) and hTERT G/A (rs2736098) with respect to glioma risk. METHODS: Confirmed cases (n = 106) were tested against 210 cancer-free healthy controls by the polymerase chain reaction-restriction fragment length polymorphism technique for genotyping. RESULTS: Homozygous variant 'GG' genotype of rs2736100 frequency was > 4-fold significantly different in cases versus controls (39.6% 17.2%; p < 0.0001). Furthermore, variant 'G' allele was found to be significantly associated with cases (0.5 versus 0.2 in controls; p < 0.0001). Homozygous variant rs2736098 'AA' genotype (35.8% versus 23.8%) and allele 'A' (0.49 versus 0.34) showed a marked significant difference in cases and controls, respectively (p < 0.05). In hTERT rs2736100, the GG genotype significantly presented more in higher grades and GBM (p < 0.0001). Furthermore, the GG variant of hTERT rs2736100 had a poor probability with respect to the overall survival of patients compared to TG and TT genotypes (log rank p = 0.03). Interestingly, two haplotypes of hTERT rs2736100/rs2736098 were identified as GG and GA that conferred a > 3- and 5-fold risk to glioma patients respectively, where variant G/A haplotype was observed to have the highest impact with respect to glioma risk (p < 0.0001). CONCLUSIONS: The results of the present study indicate that hTERT rs2736098 and rs2736100 variants play an important role in conferring a strong risk of developing glioma. Furthermore, hTERT rs2736100 GG variant appears to play a role in the bad prognosis of glioma patients. Haplotypes GG and GA could prove to be vital tools for monitoring risk in glioma patients.

Urinary levels of the acrolein conjugates of carnosine are associated with inhaled toxicants.

OBJECTIVE: The inhalation of air-borne toxicants is associated with adverse health outcomes which can be somewhat mitigated by enhancing endogenous anti-oxidant capacity. Carnosine is a naturally occurring dipeptide (ß-alanine-L-histidine), present in high abundance in skeletal and cardiac muscle. This multi-functional dipeptide has anti-oxidant properties, can buffer intracellular pH, chelate metals, and sequester aldehydes such as acrolein. Due to these chemical properties, carnosine may be protective against inhaled pollutants which can contain metals and aldehydes and can stimulate the generation of electrophiles in exposed tissues. Thus, assessment of carnosine levels, or levels of its acrolein conjugates (carnosine-propanal and carnosine-propanol) may inform on level of exposure and risk assessment. METHODS: We used established mass spectroscopy methods to measure levels of urinary carnosine (n = 605) and its conjugates with acrolein (n = 561) in a subset of participants in the Louisville Healthy Heart Study (mean age = 51 ± 10; 52% male). We then determined associations between these measures and air pollution exposure and smoking behavior using statistical modeling approaches. RESULTS: We found that higher levels of non-conjugated carnosine, carnosine-propanal, and carnosine-propanol were significantly associated with males (p < 0.02) and those of Caucasian ethnicity (p < 0.02). Levels of carnosine-propanol were significantly higher in never-smokers (p = 0.001) but lower in current smokers (p = 0.037). This conjugate also demonstrated a negative association with mean-daily particulate air pollution (PM2.5) levels (p = 0.01). CONCLUSIONS: These findings suggest that urinary levels of carnosine-propanol may inform as to risk from inhaled pollutants.

Correction to: An update on carnosine and anserine research.

The original version of this article unfortunately contained a mistake. The author "Shahid Baba" would like to include the middle name "P" in the online published article.

Carnosine protects cardiac myocytes against lipid peroxidation products.

Endogenous histidyl dipeptides such as carnosine (ß-alanine-L-histidine) form conjugates with lipid peroxidation products such as 4-hydroxy-trans-2-nonenal (HNE and acrolein), chelate metals, and protect against myocardial ischemic injury. Nevertheless, it is unclear whether these peptides protect against cardiac injury by directly reacting with lipid peroxidation products. Hence, to examine whether changes in the structure of carnosine could affect its aldehyde reactivity and metal chelating ability, we synthesized methylated analogs of carnosine, balenine (ß-alanine-Nτ-methylhistidine) and dimethyl balenine (DMB), and measured their aldehyde reactivity and metal chelating properties. We found that methylation of Nτ residue of imidazole ring (balenine) or trimethylation of carnosine backbone at Nτ residue of imidazole ring and terminal amine group dimethyl balenine (DMB) abolishes the ability of these peptides to react with HNE. Incubation of balenine with acrolein resulted in the formation of single product (m/z 297), whereas DMB did not react with acrolein. In comparison with carnosine, balenine exhibited moderate acrolein quenching capacity. The Fe2+ chelating ability of balenine was higher than that of carnosine, whereas DMB lacked chelating capacity. Pretreatment of cardiac myocytes with carnosine increased the mean lifetime of myocytes superfused with HNE or acrolein compared with balenine or DMB. Collectively, these results suggest that carnosine protects cardiac myocytes against HNE and acrolein toxicity by directly reacting with these aldehydes. This reaction involves both the amino group of ß-alanyl residue and the imidazole residue of L-histidine. Methylation of these sites prevents or abolishes the aldehyde reactivity of carnosine, alters its metal-chelating property, and diminishes its ability to prevent electrophilic injury.

Deficiency of aldose reductase exacerbates early pressure overload-induced cardiac dysfunction and autophagy in mice.

Pathological cardiac hypertrophy is associated with the accumulation of lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein in the heart. These aldehydes are metabolized via several pathways, of which aldose reductase (AR) represents a broad-specificity route for their elimination. We tested the hypothesis that by preventing aldehyde removal, AR deficiency accentuates the pathological effects of transverse aortic constriction (TAC). We found that the levels of AR in the heart were increased in mice subjected to TAC for 2 weeks. In comparison with wild-type (WT), AR-null mice showed lower ejection fraction, which was exacerbated 2 weeks after TAC. Levels of atrial natriuretic peptide and myosin heavy chain were higher in AR-null than in WT TAC hearts. Deficiency of AR decreased urinary levels of the acrolein metabolite, 3-hydroxypropylmercapturic acid. Deletion of AR did not affect the levels of the other aldehyde-metabolizing enzyme - aldehyde dehydrogenase 2 in the heart, or its urinary product - (N-Acetyl-S-(2-carboxyethyl)-l-cystiene). AR-null hearts subjected to TAC showed increased accumulation of HNE- and acrolein-modified proteins, as well as increased AMPK phosphorylation and autophagy. Superfusion with HNE led to a greater increase in p62, LC3II formation, and GFP-LC3-II punctae formation in AR-null than WT cardiac myocytes. Pharmacological inactivation of JNK decreased HNE-induced autophagy in AR-null cardiac myocytes. Collectively, these results suggest that during hypertrophy the accumulation of lipid peroxidation derived aldehydes promotes pathological remodeling via excessive autophagy, and that metabolic detoxification of these aldehydes by AR may be essential for maintaining cardiac function during early stages of pressure overload.

Genetic Deficiency of Glutathione S-Transferase P Increases Myocardial Sensitivity to Ischemia-Reperfusion Injury.

RATIONALE: Myocardial ischemia-reperfusion (I/R) results in the generation of oxygen-derived free radicals and the accumulation of lipid peroxidation-derived unsaturated aldehydes. However, the contribution of aldehydes to myocardial I/R injury has not been assessed. OBJECTIVE: We tested the hypothesis that removal of aldehydes by glutathione S-transferase P (GSTP) diminishes I/R injury. METHODS AND RESULTS: In adult male C57BL/6 mouse hearts, Gstp1/2 was the most abundant GST transcript followed by Gsta4 and Gstm4.1, and GSTP activity was a significant fraction of the total GST activity. mGstp1/2 deletion reduced total GST activity, but no compensatory increase in GSTA and GSTM or major antioxidant enzymes was observed. Genetic deficiency of GSTP did not alter cardiac function, but in comparison with hearts from wild-type mice, the hearts isolated from GSTP-null mice were more sensitive to I/R injury. Disruption of the GSTP gene also increased infarct size after coronary occlusion in situ. Ischemia significantly increased acrolein in hearts, and GSTP deficiency induced significant deficits in the metabolism of the unsaturated aldehyde, acrolein, but not in the metabolism of 4-hydroxy-trans-2-nonenal or trans-2-hexanal; on ischemia, the GSTP-null hearts accumulated more acrolein-modified proteins than wild-type hearts. GSTP deficiency did not affect I/R-induced free radical generation, c-Jun N-terminal kinase activation, or depletion of reduced glutathione. Acrolein exposure induced a hyperpolarizing shift in INa, and acrolein-induced cell death was delayed by SN-6, a Na(+)/Ca(++) exchange inhibitor. Cardiomyocytes isolated from GSTP-null hearts were more sensitive than wild-type myocytes to acrolein-induced protein crosslinking and cell death. CONCLUSIONS: GSTP protects the heart from I/R injury by facilitating the detoxification of cytotoxic aldehydes, such as acrolein.

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.

Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice.

High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100-300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK · MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein-acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1-5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10-20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity.

Role of aldose reductase in the metabolism and detoxification of carnosine-acrolein conjugates.

Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.

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.

Metabolic pathways for removing reactive aldehydes are diminished in the skeletal muscle during heart failure.

Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14 weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in the Gastrocnemius and soleus muscles were analyzed by LC/MS-MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius muscle were decreased after 14 weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5-2fold in the gastrocnemius muscle after 14 weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7 nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4 nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting.

Comparative evaluation of different modalities for measuring in vivo carnosine levels.

Carnosine is an endogenous di-peptide (ß-alanine -L- histidine) involved in maintaining tissue homeostasis. It is most abundant in skeletal muscle where its concentration has been determined in biopsy samples using tandem mass spectrometry (MS-MS). Carnosine levels can also be assessed in intact leg muscles by proton magnetic resonance spectroscopy (1H-MRS) or in blood and urine samples using mass spectrometry. Nevertheless, it remains uncertain how carnosine levels from these distinct compartments are correlated with each other when measured in the same individual. Furthermore, it is unclear which measurement modality might be most suitable for large-scale clinical studies. Hence, in 31 healthy volunteers, we assessed carnosine levels in skeletal muscle, via 1H-MRS, and in erythrocytes and urine by MS-MS. While muscle carnosine levels were higher in males (C2 peak, p = 0.010; C4 peak, p = 0.018), there was no sex-associated difference in urinary (p = 0.433) or erythrocyte (p = 0.858) levels. In a linear regression model adjusted for age, sex, race, and diet, there was a positive association between erythrocyte and urinary carnosine. However, no association was observed between 1H-MRS and erythrocytes or urinary measures. In the relationship between muscle versus urinary and erythrocyte measures, females had a positive association, while males did not show any association. We also found that 1H-MRS measures were highly sensitive to location of measurement. Thus, it is uncertain whether 1H-MRS can accurately and reliably predict endogenous carnosine levels. In contrast, urinary and erythrocyte carnosine measures may be stable and in greater synchrony, and given financial and logistical concerns, may be a feasible alternative for large-scale clinical studies.