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Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18–24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Histidine is a dietary essential amino acid because it cannot be synthesized in humans. The WHO/FAO requirement for adults for histidine is 10 mg · kg body weight(−1) · d(−1). Histidine is required for synthesis of proteins. It plays particularly important roles in the active site of enzymes, such as serine proteases (e.g., trypsin) where it is a member of the catalytic triad. Excess histidine may be converted to trans-urocanate by histidine ammonia lyase (histidase) in liver and skin. UV light in skin converts the trans form to cis-urocanate which plays an important protective role in skin. Liver is capable of complete catabolism of histidine by a pathway which requires folic acid for the last step, in which glutamate formiminotransferase converts the intermediate N-formiminoglutamate to glutamate, 5,10 methenyl-tetrahydrofolate, and ammonia. Inborn errors have been recognized in all of the catabolic enzymes of histidine. Histidine is required as a precursor of carnosine in human muscle and parts of the brain where carnosine appears to play an important role as a buffer and antioxidant. It is synthesized in the tissue by carnosine synthase from histidine and β-alanine, at the expense of ATP hydrolysis. Histidine can be decarboxylated to histamine by histidine decarboxylase. This reaction occurs in the enterochromaffin-like cells of the stomach, in the mast cells of the immune system, and in various regions of the brain where histamine may serve as a neurotransmitter.

Korat chicken (KRC) is a slow-growing chicken bred in Thailand, whose meat exhibits a unique toughness. A previous study produced KRC breast meat containing high carnosine content through dietary supplementation with β-alanine or L-histidine, however, the KRC that were fed an L-histidine-supplemented diet produced meat that was significantly more tender. Herein, we performed RNA-Seq to identify candidate genes involved in the regulation of carnosine content and meat toughness. Total RNA was isolated from five female KRC breast muscles in each treatment group that KRC fed diets without supplementation, supplemented with β-alanine or L-histidine. Compared to the non-supplemented group, we identified 118 and 198 differentially expressed genes (DEGs) in the β-alanine or L-histidine supplementation groups, respectively. Genes potentially related to meat tenderness—i.e., those regulating myosin, collagen, intramuscular fat, and calpain—were upregulated (LOC107051274, ACSBG1, and CAPNS2) and downregulated (MYO7B, MYBPH, SERPINH1, and PGAM1). However, carnosine synthase gene was not identified. Functional enrichment analysis identified pathways affected by dietary supplementation, including the insulin signaling pathway (β-alanine supplementation) and the insulin resistance and adipocytokine signaling pathways (L-histidine supplementation). The FoxO signaling pathway was identified as a regulatory network for both supplementation groups. The identified genes can be used as molecular markers of meat tenderness in slow-growing chickens.

Histidyl dipeptides, are synthesized in the heartviaenzyme carnosine synthase (Carns), which facilitates glycolysis and glucose oxidation by proton buffering and attenuate ischemia and reperfusion injury. However, a composite understanding of the histidyl dipeptide mediated responses in the heart are lacking. We performed multilayer omics in the cardio specificCarnsoverexpressing mice, showing higher myocardial levels of histidyl dipeptides lead to extensive changes in microRNAs that could target the expression of contractile proteins and enzymes involved inβ-fatty acid oxidation and citric acid cycle (TCA). Similarly, global proteomics showed contractile function, fatty acid degradation and TCA cycle, pathways were enriched in the CarnsTg heart. Parallel with these changes, free fatty acids, and TCA intermediate-succinic acid were lower under aerobic and significantly attenuated under anaerobic conditions in the CarnsTg heart. Integration of multiomics data showsβ-fatty acid oxidation and TCA cycle exhibit correlative changes at all three levels in CarnsTg heart, suggesting histidyl dipeptides are critical regulators of myocardial structure, function and energetics.

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H+ buffering, regulation of Ca2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1-/-) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1-/- resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1-/- resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca2+ peaks and slowed Ca2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca2+ handling and excitation-contraction coupling.

The current study investigated the effect of dietary l-histidine and beta-alanine supplementation on growth performance, meat quality, carnosine content, and gene expression of carnosine-related enzymes in broilers. A two-factor design was adopted in this study. A total of 640 1-day-old male broilers were assigned to eight treatments with factorial arrangement containing four levels of l-histidine (0, 650, 1300, or 1950 mg/kg) and two levels of beta-alanine (0 or 1200 mg/kg) supplementation, 0 mg/kg histidine and/or 0 mg/kg were treated as control groups. Each treatment including eight replicates with 10 birds each and the feeding trial lasted for 42 days. Dietary supplementation with l-histidine and beta-alanine did not affect average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) of broilers during the grower (22–42 days) and the entire phase (1–42 days), compared with the control group (p &gt, 0.05). The only exception was a significantly reduced ADG in the 1950 mg/kg l-histidine group in the starter period (1–21 days, p &lt, 0.05). l-Histidine at 1950 mg/kg significantly decreased redness (a*) and yellowness (b*) values of the meat at 45 min postmortem (p &lt, 0.05), whereas it increased b* value and pH in breast muscle at 24 h postmortem. Moreover, dietary supplementation with beta-alanine alone or combination with l-histidine significantly increased ΔpH in breast muscle (p &lt, 0.01). Dietary l-histidine markedly increased total superoxide dismutase activity and total antioxidant capacity (T-AOC) both in breast muscle (p &lt, 0.01) and in plasma (p &lt, 0.01), and it decreased malondialdehyde (MDA) concentration in breast muscle (p &lt, 0.01). Dietary addition of beta-alanine, alone or combination, significantly increased T-AOC in breast muscle (p &lt, 0.01) and markedly decreased MDA content both in breast muscle and in plasma (p &lt, 0.01). Addition of l-histidine and beta-alanine significantly increased muscle peptide (carnosine and anserine) content (p &lt, 0.05) and upregulated the expression of carnosine synthase, transporter of carnosine/ l-histidine, and l-histidine decarboxylase genes (p &lt, 0.05), with greater change occurring in the combination group of 1300 mg/kg l-histidine and 1200 mg/kg beta-alanine. Overall, dietary l-histidine and beta-alanine could improve meat quality and antioxidant capacity, enhance the carnosine and anserine content, and upregulate the gene expression of carnosine synthesis-related enzymes in broilers.

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC–electrospray ionization–tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine–containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H(2)O(2) exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H(2)O(2) cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

Carnosine and anserine are dipeptides synthesized from histidine and β-alanine by carnosine synthase (ATPGD1). These dipeptides, present in high concentration in the skeletal muscle, form conjugates with lipid peroxidation products such as 4-hydroxy trans-2-nonenal (HNE). Although skeletal muscle levels of these dipeptides could be elevated by feeding β-alanine, it is unclear how these dipeptides and their conjugates are affected by exercise training with or without β-alanine supplementation. We recruited 20 physically active men, who were allocated to either β-alanine or placebo-feeding group matched for peak oxygen consumption, lactate threshold, and maximal power. Participants completed 2 wk of a conditioning phase followed by 1 wk of exercise training, a single session of high-intensity interval training (HIIT), followed by 6 wk of HIIT. Analysis of muscle biopsies showed that the levels of carnosine and ATPGD1 expression were increased after CPET and decreased following a single session and 6 wk of HIIT. Expression of ATPGD1 and levels of carnosine were increased upon β-alanine-feeding after CPET, whereas ATPGD1 expression decreased following a single session of HIIT. The expression of fiber type markers myosin heavy chain I and IIa remained unchanged after CPET. Levels of carnosine, anserine, carnosine-HNE, carnosine-propanal, and carnosine-propanol were further increased after 9 wk of β-alanine supplementation and exercise training but remained unchanged in the placebo-fed group. These results suggest that carnosine levels and ATPGD1 expression fluctuates with different phases of training. Enhancing carnosine levels by β-alanine feeding could facilitate the detoxification of lipid peroxidation products in the human skeletal muscle.NEW & NOTEWORTHY Carnosine synthase expression and carnosine levels are altered in the human skeletal muscle during different phases of training. During high-intensity interval training, β-alanine feeding promotes detoxification of lipid peroxidation products and increases anserine levels in the skeletal muscle.

BACKGROUND Myocardial ischemia reperfusion (I/R) injury is associated with complex pathophysiological changes characterized by pH imbalance, the accumulation of lipid peroxidation products acrolein and 4‐hydroxy trans ‐2‐nonenal, and the depletion of ATP levels. Cardioprotective interventions, designed to address individual mediators of I/R injury, have shown limited efficacy. The recently identified enzyme ATPGD1 (Carnosine Synthase), which synthesizes histidyl dipeptides such as carnosine, has the potential to counteract multiple effectors of I/R injury by buffering intracellular pH and quenching lipid peroxidation products and may protect against I/R injury . METHODS AND RESULTS We report here that β‐alanine and carnosine feeding enhanced myocardial carnosine levels and protected the heart against I/R injury. Cardiospecific overexpression of ATPGD 1 increased myocardial histidyl dipeptides levels and protected the heart from I/R injury. Isolated cardiac myocytes from ATPGD 1‐transgenic hearts were protected against hypoxia reoxygenation injury. The overexpression of ATPGD 1 prevented the accumulation of acrolein and 4‐hydroxy trans ‐2‐nonenal–protein adducts in ischemic hearts and delayed acrolein or 4‐hydroxy trans ‐2‐nonenal–induced hypercontracture in isolated cardiac myocytes. Changes in the levels of ATP , high‐energy phosphates, intracellular pH, and glycolysis during low‐flow ischemia in the wild‐type mice hearts were attenuated in the ATPGD 1‐transgenic hearts. Two natural dipeptide analogs (anserine and balenine) that can either quench aldehydes or buffer intracellular pH , but not both, failed to protect against I/R injury. CONCLUSIONS Either exogenous administration or enhanced endogenous formation of histidyl dipeptides prevents I/R injury by attenuating changes in intracellular pH and preventing the accumulation of lipid peroxidation derived aldehydes.

The effects of dietary carnosine were evaluated on the growth performance, meat quality, antioxidant capacity and muscle fiber characteristics in thigh muscle of 256 one-day-old male broilers assigned to four diets - basal diets supplemented with 0, 100, 200 or 400 mg kg-1 carnosine respectively - during a 42 day experiment.; Results: Carnosine concentration and carnosine synthase expression in thigh muscle were linearly increased (P < 0.05) and the feed/gain ratio was decreased (P < 0.05) in the starter period by carnosine addition. Dietary supplementation with carnosine resulted in linear increases in pH45min , redness and cohesiveness and decreases in drip loss, cooking loss, shear force and hardness (P < 0.05). Carnosine addition elevated the activities of antioxidant enzymes and reduced contents of malondialdehyde and carbonyl compounds (P < 0.05). Dietary carnosine linearly decreased diameters and increased densities of muscle fibers (P < 0.01). The ratios of myosin heavy chain (MyHC) I and IIa were increased while that of MyHC IIb was decreased (P < 0.01). The mRNA expressions of genes related to fiber type transformation were linearly up-regulated (P < 0.05).; Conclusion: These findings indicated that carnosine supplementation was beneficial to improve the growth performance, meat quality, antioxidant capacity and muscle fiber characteristics of broilers. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Carnosine (β-alanyl-L-histidine) and its methylated derivatives: anserine (β-alanyl-Nπ- methyl-L-histidine) and balenine (β-alanyl-Nτ-methyl-L-histidine) are abundant constituents of excitable tissues of vertebrates. While carnosine and anserine are present at high concentrations and in variable proportions in skeletal muscle and brain of most vertebrates, balenine appears to be rather more abundant in marine mammals and certain reptilian species. Since the discovery of these compounds at the beginning of 20th century, numerous studies have been devoted to identification of the biochemical and physiological properties of carnosine and related dipeptides. These led to the discovery of the pHbuffering, metal-chelation and antioxidant, capabilities of carnosine and anserine, although no definitive ideas concerning their physiological role has yet been formulated. Only recently the molecular identities of the enzymes catalyzing synthesis of carnosine (carnosine synthase, EC 6.3.2.11) and anserine (carnosine N-methyltransferase, EC 2.1.1.22) have been elucidated, which has given a new insight into their metabolism in vertebrates. These findings have opened new research areas and provide authentic opportunities for understanding the biological function of these "enigmatic" dipeptides. This review aims to summarize recent advances in our knowledge concerning enzymes responsible for the biosynthesis of carnosine and related dipeptides and to evaluate their importance in vertebrate physiology.

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈ 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.

I congratulate Prokopieva et al. for their review “Use of Carnosine for Oxidative Stress Reduction in Different Pathologies” [1]. The authors overview properties and biological effects of the antioxidant carnosine (Cn). Data on the use of Cn in various conditions are discussed. Special attention is given to the use of carnosine in neurologic, mental diseases and alcoholism. I have read the paper with a great interest. However, a comment seems to be necessary to formulate a praxis-relevant and evidence-based conclusion. Cn or β-alanyl-L-histidine, an endogenously synthesized dipeptide, was reported to possess antioxidant properties [1, 2]. Cn enters blood being absorbed in the small intestine upon ingestion [2]. Cn is also synthesized in the human body; carnosine synthase is present in skeletal and heart muscle and certain brain regions and in other tissues. The synthesis rate of Cn appears to be predominantly rate-limited by the availability of β-alanine. Another precursor, L-histidine, is normally present in blood in sufficient concentrations [3]. The supply of β-alanine is dependent both on the hepatic synthesis from uracil and on the diet [2]. Cn penetrates through blood-brain barrier and is generally characterized by high bioavailability [1]. To decide whether supplementation of a substance is indicated, the question should be answered whether there can be a deficiency of that substance and if yes, whether it can be compensated for by a diet modification. Such deficiency appears to be improbable for Cn, which is abundant in muscles and can be synthesized in the body. Tissue Cn concentrations are influenced by the diet and are lower in vegetarians than in the general population [2, 4]. In vegetarians, muscle Cn content is limited by hepatic synthesis of β-alanine, whereas in omnivores there is additional dietary supply [5]. Cn was reported to be of importance for the antioxidant lens protection and preservation of its transparency, for prevention of certain neurologic and mental diseases. These potencies have been ascribed to the antioxidant properties of Cn [1, 2, 6, 7]. Among other potential applications, Cn eye drops have been recommended for cataracts [1, 6, 7]. However, if Cn concentration in body fluids is of importance, the incidence of corresponding conditions in vegetarians or population groups consuming less meat would be higher than average, which, to the best of our knowledge, has never been reported. Antioxidants affecting reactive oxygen species may have both harmful and beneficial effects [8]. The same is true particularly for Cn [9]. Generation of reactive oxygen species is a normal phenomenon in the course of aerobic metabolism [8]. Free radicals are not invariably toxic; some of them are necessary for the proper physiological functioning [10]. The redox status is maintained in equilibrium under the influence of many factors [11, 12]. The artificial support of the antioxidant status is considered to be not necessarily beneficial [11]. The topic of antioxidants is further complicated by conflicts of interest. Some antioxidants are propagated as dietary supplements or inexpensive substitutes for evidence-based medications. There are many examples of marketed substances without satisfactorily proven effectiveness beyond the placebo effect [13–17]. Publications of questionable reliability are sometimes used for advertising of drugs and dietary supplements, for their official registration, and for obtaining permissions for practical use. As a result, substances with unproven effects can be offered to the patients misinformed not only by advertising but also by some publications supposed to be scientific. The Cn eye drops sold in Russia are relatively expensive; they are prescribed to aged patients. In theory, Cn eye drops could be replaced by adequately prepared meat extract. Analogous suggestions have been made, for example, for osteoarthritis, to recommend for patients with low incomes to replace glycosaminoglycan-containing chondroprotectors with natural glycosaminoglycans that are abundant, for example, in animal cartilages and chicken wings [17]. To support the placebo effect, patients may be advised that the natural products can supply their organism with Cn similarly to drugs or dietary supplements. However, considering uncertainties about practical usefulness of antioxidants, discussed above, we would rather abstain from such recommendations. Even more precarious, because of complication risks [18], are recommendations of peribulbar injections of carcinine (an analog of Cn) as an antioxidant for prophylactic purposes [19]. Until recently, peribulbar injections of amino acid taurine (one of the most abundant amino acids in mammalian tissues [20, 21]) were used in the former SU as a preparation named Taufon in elderly patients for prophylactic purposes as well as for the treatment of macular dystrophy associated with atherosclerosis [22], while hematomas were observed as complications. Taufon has also been used as eye drops [23]. In conclusion, a stereotype that can be observed in some papers on normal metabolites such as Cn or taurine is as follows: metabolic importance of a substance is pointed out, which is true for many normal metabolites. In the second part of the paper, practical applications are discussed, although it remains unproven whether a deficiency of the substance ever occurs, and if it does, whether it can be compensated for by a diet, or a supplementation by drugs is really necessary. Note that drugs or dietary supplements might be more expensive for patients than a diet modification, for example, consumption of more meat products as a natural source of Cn or taurine. In any case, considering reported efficiency of Cn in chronic discirculatory encephalopathy, dementia, alcoholism, some mental disorders, and diabetes [1, 24, 25], an increase in consumption of meat products should be recommended for inhabitants of homes for the aged and psychiatric facilities. This seems to be the most important conclusion that can be derived from the article [1]. Benefits from the intake of Cn, other peptides, or amino acids in such patients may be seen as circumstantial evidence of malnutrition.

In the historical city center of Ghent in Belgium, an international group of scientists gathered for a 3-day meeting dedicated to carnosine research. The meeting took place in July 2011, which is more than 10 years following the previous congress of this kind, organized by Alexander Boldyrev in Moscow in the year 2000. The Moscow meeting was held on the occasion of the centenary of Gulewitch and Amiradzhibi’s discovery of the dipeptide carnosine in meat extract. A special issue with contributions from the invited participants was published in Biochemistry (Moscow) in 2000, entitled: ‘‘Biological role of carnosine in the functioning of excitable tissues’’ (Guest editor: A. A. Boldyrev; Original Russian text). The Ghent meeting was therefore considered the Second International Meeting on Carnosine. The souvenir medals that were created to mark the occasion of both meetings are displayed in Fig. 1. A thorough understanding of the biological role of carnosine and its methylated analogues anserine and ophidine/ balenine, is still hampered by the relatively low frequency of scientific discoveries on this topic. However, in the 11-year period separating both meetings there has been an exponential increase in the number of publications and citations on carnosine (see Fig. 2). Indeed, significant progress was made in the fields of both biochemistry and medicine, as well as in sport science. The genes for carnosine synthase and carnosinase, the principal enzymes responsible for carnosine synthesis and degradation, were molecularly identified and cloned in 2003 and 2010, respectively. A polymorphism in the gene for the carnosine-degrading enzyme carnosinase was defined as an important genetic risk factor for diabetic nephropathy. The evidence for a therapeutic role of carnosine in aging and neurologic diseases has expanded and new target diseases, such as diabetes/metabolic syndrome, liver and kidney disease and cancer, have been successfully explored in animal experiments. Carnosinase-resistant analogues of carnosine are developed in the pharmaceutical industry for increased therapeutic potential and stability. Finally, the content of muscle carnosine appears to influence athletic performance and can be augmented by oral beta-alanine supplementation, making beta-alanine increasingly popular in the athletic community. Thus, a new meeting was timely and needed. The current special issue in Amino Acids provides a selection of contributions presented at the Ghent meeting, containing original papers, review articles and a new perspective. Carnosine was originally discovered in skeletal muscle, where it has a far greater concentration compared to all other tissues. The regulation of the carnosine content in muscle is still incompletely understood, but the current knowledge is elegantly summarized in this special issue by Harris et al. (2012, this issue). A recent development of a non-invasive magnetic resonance spectroscopy (MRS) based method to quantify muscle carnosine in humans has further facilitated the research on this topic. Accordingly, a study based on over 260 male and female healthy humans aged 9–83 years (Baguet et al. 2012, this issue) indicates This is the introduction paper to the Special Issue of Amino Acids, entitled ‘Carnosine in Exercise and Disease’ and edited by Craig Sale and Wim Derave.

Carnosine is an abundant dipeptide in human skeletal muscle with proton buffering capacity. There is controversy as to whether training can increase muscle carnosine and thereby provide a mechanism for increased buffering capacity. This study investigated the effects of 5 weeks sprint training combined with a vegetarian or mixed diet on muscle carnosine, carnosine synthase mRNA expression and muscle buffering capacity. Twenty omnivorous subjects participated in a 5 week sprint training intervention (2-3 times per week). They were randomized into a vegetarian and mixed diet group. Measurements (before and after the intervention period) included carnosine content in soleus, gastrocnemius lateralis and tibialis anterior by proton magnetic resonance spectroscopy ((1)H-MRS), true-cut biopsy of the gastrocnemius lateralis to determine in vitro non-bicarbonate muscle buffering capacity, carnosine content (HPLC method) and carnosine synthase (CARNS) mRNA expression and 6 × 6 s repeated sprint ability (RSA) test. There was a significant diet × training interaction in soleus carnosine content, which was non-significantly increased (+11%) with mixed diet and non-significantly decreased (-9%) with vegetarian diet. Carnosine content in other muscles and gastrocnemius buffer capacity were not influenced by training. CARNS mRNA expression was independent of training, but decreased significantly in the vegetarian group. The performance during the RSA test improved by training, without difference between groups. We found a positive correlation (r = 0.517; p = 0.002) between an invasive and non-invasive method for muscle carnosine quantification. In conclusion, this study shows that 5 weeks sprint training has no effect on the muscle carnosine content and carnosine synthase mRNA.

Anserine (β-alanyl-N(Pi)-methyl-l-histidine), a methylated derivative of carnosine (β-alanyl-l-histidine), is an abundant constituent of vertebrate skeletal muscles. Although it has been suggested to serve as a proton buffer and radical scavenger, its physiological function remains mysterious. The formation of anserine is catalyzed by carnosine N-methyltransferase, recently identified in chicken as histamine N-methyltransferase-like (HNMT-like) protein. Although the HNMT-like gene is absent in mammalian genomes, the activity of carnosine N-methyltransferase was reported in most mammalian species. In the present investigation, we purified carnosine N-methyltransferase from rat muscles about 2600-fold. Three polypeptides of ∼45, 50, and 70 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of UPF0586 protein C9orf41 homolog as the only meaningful candidate. Rat UPF0586 and its yeast, chicken, and human orthologs were expressed in COS-7 cells and purified to homogeneity. Although all recombinant proteins catalyzed the formation of anserine, as confirmed by chromatographic and mass spectrometry analysis, rat UPF0586 was more active on carnosine than other orthologs. Confocal microscopy of HeLa cells expressing recombinant UPF5086 proteins revealed their presence in both cytosol and nucleus. Carnosine and Gly-His were the best substrates for all UPF0586 orthologs studied, although the enzymes also methylated other l-histidine-containing di- and tripeptides. Finally, cotransfection of COS-7 cells with rat or human UPF0586 and carnosine synthase transformed the cells into efficient anserine producers. We conclude that UPF0586 is mammalian carnosine N-methyltransferase and hypothesize that it may also serve as a peptide or protein methyltransferase in eukaryotes.

Beta-alanine in blood-plasma when administered as A) histidine dipeptides (equivalent to 40 mg . kg(-1) bwt of beta-alanine) in chicken broth, or B) 10, C) 20 and D) 40 mg . kg(-1) bwt beta-alanine (CarnoSyn, NAI, USA), peaked at 428 +/- SE 66, 47 +/- 13, 374 +/- 68 and 833 +/- 43 microM. Concentrations regained baseline at 2 h. Carnosine was not detected in plasma with A) although traces of this and anserine were found in urine. Loss of beta-alanine in urine with B) to D) was5%. Plasma taurine was increased by beta-alanine ingestion but this did not result in any increased loss via urine. Pharmacodynamics were further investigated with 3 x B) per day given for 15 d. Dietary supplementation with I) 3.2 and II) 6.4 g . d(-1) beta-alanine (as multiple doses of 400 or 800 mg) or III) L-carnosine (isomolar to II) for 4 w resulted in significant increases in muscle carnosine estimated at 42.1, 64.2 and 65.8%.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).

Even though myocardial ischemia/reperfusion (I/R) remains the leading cause of death, the underlying mechanisms remain incompletely understood. Increased formation of reactive carbonyl has been shown to be a common biochemical feature of I/R injury. These carbonyls are generated from the oxidation of proteins and membrane lipids. Reactive carbonyls such as methylglyoxal are generated from increased glycolytic activity during ischemia. Previous work in our lab has shown that the endogenous dipeptide carnosine (β-alanine-histidine) quenches both protein and lipid derived carbonyls. It can also buffer changes in intracellular pH and chelate metals that catalyze ROS production. In the heart, carnosine is synthesized by the ATP grasp enzyme (ATPGD1). Hence, we examined whether overexpression of ATPGD1 could increase carnosine synthesis in the heart and attenuate I/R injury. To overexpress ATPGD1, we generated mice in which the expression of the transgene was driven by cardiospecific α-MHC promoter. Two different ATPGD1Tg mouse lines were generated, which showed 10-15 fold higher abundance of ATPGD1 protein in the heart compared with their wild-type (WT) littermates. Cardiac levels of the histidyl dipeptides anserine and carnosine were approximately 100 fold higher in the ATPGD1Tg than WT mice hearts (WT: anserine 1.8±0.3 pmoles/mg protein, carnosine 6±1 pmoles/mg protein; ATPGD1-Tg: anserine 114±15 pmoles/mg protein, carnosine 615±44 pmoles/mg protein). No changes in the levels of these dipeptides were observed in other tissues of the ATPGD1Tg mice. Echocardiographic analysis showed that ATPGD1 overexpression did not affect cardiac function. When subjected to 30 min of coronary occlusion followed by 24 h of reperfusion, the infarct size was significantly lower in ATPGD1Tg than WT mice. Infarct size as the area of risk of left ventricle was 59±3.02% in WT mice and 38±2.73% in the ATPGD1-Tg mice (p

It is shown that histidine-containing dipeptide carnosine ( β -alanyl- l -histidine), which is present in skeletal muscles in millimolar concentrations, decreases the rate of Ca 2+ accumulation by the heavy fraction of sarcoplasmic reticulum from rabbit skeletal muscles. This effect results from the ability of carnosine to induce a rapid Ca 2+ release from the heavy sarcoplasmic reticulum vesicles via activation of the ruthenium red-sensitive Ca-channels. The effect of carnosine is dose-dependent that indicates the presence of saturable site(s) for carnosine in the molecules of Ca-channels. The C 0.5 value for carnosine (the concentration that induces the half-maximal Ca 2+ release) is 8.7 mM. The 1 N -methylated derivative of carnosine, i.e., anserine, also induces a rapid Ca 2+ release with the half-maximal effect at 2.7 mM. Conversely, neither histidine nor β -alanine (both separately and in the mixture) cause Ca 2+ release. In addition, carnosine increases the sensitivity of Ca-channels to their well-known activators (caffeine, AMP, and Ca 2+ ) and decreases inhibitory effect of low concentrations of Mg 2+ . It is concluded that carnosine as a component of skeletal muscles can be an endogenous regulator of the sarcoplasmic reticulum Ca-channel activity.

Carnosine (β-alanyl-l-histidine) was discovered in 1900 as an abundant non-protein nitrogen-containing compound of meat. The dipeptide is not only found in skeletal muscle, but also in other excitable tissues. Most animals, except humans, also possess a methylated variant of carnosine, either anserine or ophidine/balenine, collectively called the histidine-containing dipeptides. This review aims to decipher the physiological roles of carnosine, based on its biochemical properties. The latter include pH-buffering, metal-ion chelation, and antioxidant capacity as well as the capacity to protect against formation of advanced glycation and lipoxidation end-products. For these reasons, the therapeutic potential of carnosine supplementation has been tested in numerous diseases in which ischemic or oxidative stress are involved. For several pathologies, such as diabetes and its complications, ocular disease, aging, and neurological disorders, promising preclinical and clinical results have been obtained. Also the pathophysiological relevance of serum carnosinase, the enzyme actively degrading carnosine into l-histidine and β-alanine, is discussed. The carnosine system has evolved as a pluripotent solution to a number of homeostatic challenges. l-Histidine, and more specifically its imidazole moiety, appears to be the prime bioactive component, whereas β-alanine is mainly regulating the synthesis of the dipeptide. This paper summarizes a century of scientific exploration on the (patho)physiological role of carnosine and related compounds. However, far more experiments in the fields of physiology and related disciplines (biology, pharmacology, genetics, molecular biology, etc.) are required to gain a full understanding of the function and applications of this intriguing molecule.

Beta-alanine (BA) is a popular ergogenic supplement because it can induce muscle carnosine loading. We hypothesize that, by analogy with creatine supplementation, 1) an inverse relationship between urinary excretion and muscle loading is present, and 2) the latter is stimulated by carbohydrate- and protein-induced insulin action.In study A, the effect of a 5-wk slow-release BA (SRBA) supplementation (4.8 g · d(-1)) on whole body BA retention was determined in seven men. We further determined whether the coingestion of carbohydrates and proteins with SRBA would improve retention. In study B (34 subjects), we explored the effect of meal timing on muscle carnosine loading (3.2 g · d(-1) during 6-7 wk). One group received pure BA (PBA) in between the meals; the other received PBA at the start of the meals, to explore the effect of meal-induced insulin release. Further, we compared with a third group receiving SRBA at the start of the meals.Orally ingested SRBA has a very high whole body retention (97%-98%) that is not declining throughout the 5-wk supplementation period, nor is it influenced by the coingestion of macronutrients. Thus, a very small portion (1%-2%) is lost through urinary excretion, and equally only a small portion is incorporated into muscle carnosine (≈ 3%), indicating that most ingested BA is metabolized (possibly through oxidation). Second, in soleus muscles, the efficiency of carnosine loading is significantly higher when PBA is coingested with a meal (+64%) compared with in between the meals (+41%), suggesting that insulin stimulates muscle carnosine loading. Finally, the chronic supplementation of SRBA versus PBA seems equally effective.

Social isolation has been widely described to induce a compulsive aggressive behavior. The aggressiveness due to isolation in mice has often been used as a means for the better understanding of disturbed behavior in human beings. In the course of a study of the behavioral effects, we have noticed that fighting injuries, usually observed among male ICR mice, tend to decrease in mice irradiated with low-dose X-rays. We, therefore, quantitatively examined the effects of low-dose X-irradiation on aggressive behavior using a resident-intruder paradigm in which a resident mouse attacks an intruder that entered its territory. Male ICR white Swiss mice became gradually calm, and showed remarkably quiet behavior 7-10 days after whole-head 5 or 15 cGy X-irradiation. Only exposure of the anterior part of the head (olfactory system including orbits) also induced the remarkable suppression of the aggressive behavior. The olfactory system has direct access to the limbic system, a central part of the brain concerned with emotion. The calm behavior induced by low-dose X-irradiation might be related to the changes in the olfactory function. We also obtained data on brain biochemistry giving further support for the above low-dose effects on mouse behavior. The carnosine content and its synthetase activity in the olfactory bulbs decreased significantly after only the anterior part of the head had been exposed. Higher doses (25-35 cGy), however, did not induce such effects. The results suggest that the depression of aggressive behavior is limited to animals irradiated with the smaller doses.

Chronic oral beta-alanine supplementation can elevate muscle carnosine (beta-alanyl-L-histidine) content and improve high-intensity exercise performance. However, the regulation of muscle carnosine levels is poorly understood. The uptake of the rate-limiting precursor beta-alanine and the enzyme catalyzing the dipeptide synthesis are thought to be key steps. The aims of this study were to investigate the expression of possible carnosine-related enzymes and transporters in both human and mouse skeletal muscle in response to carnosine-altering stimuli. Human gastrocnemius lateralis and mouse tibialis anterior muscle samples were subjected to HPLC and qPCR analysis. Mice were subjected to chronic oral supplementation of beta-alanine and carnosine or to orchidectomy (7 and 30 days, with or without testosterone replacement), stimuli known to, respectively, increase and decrease muscle carnosine and anserine. The following carnosine-related enzymes and transporters were expressed in human and/or mouse muscles: carnosine synthase (CARNS), carnosinase-2 (CNDP2), the carnosine/histidine transporters PHT1 and PHT2, the beta-alanine transporters TauT and PAT1, beta-alanine transaminase (ABAT) and histidine decarboxylase (HDC). Six of these genes showed altered expression in the investigated interventions. Orchidectomy led to decreased muscle carnosine content, which was paralleled with decreased TauT expression, whereas CARNS expression was surprisingly increased. Beta-alanine supplementation increased both muscle carnosine content and TauT, CARNS and ABAT expression, suggesting that muscles increase beta-alanine utilization through both dipeptide synthesis (CARNS) and deamination (ABAT) and further oxidation, in conditions of excess availability. Collectively, these data show that muscle carnosine homeostasis is regulated by nutritional and hormonal stimuli in a complex interplay between related transporters and enzymes.

Glycolysis involves the oxidation of two neutral hydroxyl groups on each glycosyl (or glucosyl) unit metabolised, yielding two carboxylic acid groups. During low-intensity exercise these, along with the remainder of the carbon skeleton, are further oxidised to CO(2) and water. But during high-intensity exercise a major portion (and where blood flow is impaired, then most) is accumulated as lactate anions and H(+). The accumulation of H(+) has deleterious effects on muscle function, ultimately impairing force production and contributing to fatigue. Regulation of intracellular pH is achieved over time by export of H(+) out of the muscle, although physicochemical buffers in the muscle provide the first line of defence against H(+) accumulation. In order to be effective during high-intensity exercise, buffers need to be present in high concentrations in muscle and have pK(a)s within the intracellular exercise pH transit range. Carnosine (β-alanyl-L-histidine) is ideal for this role given that it occurs in millimolar concentrations within the skeletal muscle and has a pK(a) of 6.83. Carnosine is a cytoplasmic dipeptide formed by bonding histidine and β-alanine in a reaction catalysed by carnosine synthase, although it is the availability of β-alanine, obtained in small amounts from hepatic synthesis and potentially in greater amounts from the diet that is limiting to synthesis. Increasing muscle carnosine through increased dietary intake of β-alanine will increase the intracellular buffering capacity, which in turn might be expected to increase high-intensity exercise capacity and performance where this is pH limited. In this study we review the role of muscle carnosine as an H(+) buffer, the regulation of muscle carnosine by β-alanine, and the available evidence relating to the effects of β-alanine supplementation on muscle carnosine synthesis and the subsequent effects of this on high-intensity exercise capacity and performance.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on beta-alanine than on gamma-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Carnosine is a dipeptide with a high concentration in mammalian skeletal muscle. It is synthesized by carnosine synthase from the amino acids L-histidine and beta-alanine, of which the latter is the rate-limiting precursor, and degraded by carnosinase. Recent studies have shown that the chronic oral ingestion of beta-alanine can substantially elevate (up to 80%) the carnosine content of human skeletal muscle. Interestingly, muscle carnosine loading leads to improved performance in high-intensity exercise in both untrained and trained individuals. Although carnosine is not involved in the classic adenosine triphosphate-generating metabolic pathways, this suggests an important role of the dipeptide in the homeostasis of contracting muscle cells, especially during high rates of anaerobic energy delivery. Carnosine may attenuate acidosis by acting as a pH buffer, but improved contractile performance may also be obtained by improved excitation-contraction coupling and defence against reactive oxygen species. High carnosine concentrations are found in individuals with a high proportion of fast-twitch fibres, because these fibres are enriched with the dipeptide. Muscle carnosine content is lower in women, declines with age and is probably lower in vegetarians, whose diets are deprived of beta-alanine. Sprint-trained athletes display markedly high muscular carnosine, but the acute effect of several weeks of training on muscle carnosine is limited. High carnosine levels in elite sprinters are therefore either an important genetically determined talent selection criterion or a result of slow adaptation to years of training. beta-Alanine is rapidly developing as a popular ergogenic nutritional supplement for athletes worldwide, and the currently available scientific literature suggests that its use is evidence based. However, many aspects of the supplement, such as the potential side effects and the mechanism of action, require additional and thorough investigation by the sports science community.

The content of anserine and carnosine in the lateral portion of the quadriceps femoris muscle of 50 healthy, human subjects has been studied. Anserine was undetectable in all muscle samples examined. Muscle carnosine values for the group conformed to a normal distribution with a mean (SD) value of 20.0 (4.7) mmol.kg-1 of dry muscle mass. The concentration of carnosine was significantly higher in the muscle of male subjects (21.3, 4.2 mmol.kg-1 dry mass) than in females of a similar age and training status (17.5, 4.8 mmol.kg-1 dry mass) (P less than 0.005). The test-retest reliability of measures was determined on a subgroup of 17 subjects. No significant difference in mean carnosine concentration was found between the two trials [21.5 (4.0) and 22.0 (5.2) mmol.kg-1 dry muscle mass; P greater than 0.05]. The importance of carnosine as a physicochemical buffer within human muscle was examined by calculating its buffering ability over the physiological pH range. From the range of carnosine concentrations observed (7.2-30.7 mmol.kg-1 dry muscle mass), it was estimated that the dipeptide could buffer between 2.4 and 10.1 mmol H+.kg-1 dry mass over the physiological pH range 7.1-6.5, contributing, on average, approximately 7% to the total muscle buffering. This suggests that in humans, in contrast to many other species, carnosine is of only limited importance in preventing the reduction in pH observed during high intensity exercise.

High-intensity exercise results in reduced substrate levels and accumulation of metabolites in the skeletal muscle. The accumulation of these metabolites (e.g. ADP, Pi and H(+)) can have deleterious effects on skeletal muscle function and force generation, thus contributing to fatigue. Clearly this is a challenge to sport and exercise performance and, as such, any intervention capable of reducing the negative impact of these metabolites would be of use. Carnosine (beta-alanyl-L-histidine) is a cytoplasmic dipeptide found in high concentrations in the skeletal muscle of both vertebrates and non-vertebrates and is formed by bonding histidine and beta-alanine in a reaction catalysed by carnosine synthase. Due to the pKa of its imidazole ring (6.83) and its location within skeletal muscle, carnosine has a key role to play in intracellular pH buffering over the physiological pH range, although other physiological roles for carnosine have also been suggested. The concentration of histidine in muscle and plasma is high relative to its K (m) with muscle carnosine synthase, whereas beta-alanine exists in low concentration in muscle and has a higher K (m) with muscle carnosine synthase, which indicates that it is the availability of beta-alanine that is limiting to the synthesis of carnosine in skeletal muscle. Thus, the elevation of muscle carnosine concentrations through the dietary intake of carnosine, or chemically related dipeptides that release beta-alanine on absorption, or supplementation with beta-alanine directly could provide a method of increasing intracellular buffering capacity during exercise, which could provide a means of increasing high-intensity exercise capacity and performance. This paper reviews the available evidence relating to the effects of beta-alanine supplementation on muscle carnosine synthesis and the subsequent effects on exercise performance. In addition, the effects of training, with or without beta-alanine supplementation, on muscle carnosine concentrations are also reviewed.

Muscle carnosine synthesis is limited by the availability of beta-alanine. Thirteen male subjects were supplemented with beta-alanine (CarnoSyn) for 4 wks, 8 of these for 10 wks. A biopsy of the vastus lateralis was obtained from 6 of the 8 at 0, 4 and 10 wks. Subjects undertook a cycle capacity test to determine total work done (TWD) at 110% (CCT(110%)) of their maximum power (Wmax). Twelve matched subjects received a placebo. Eleven of these completed the CCT(110%) at 0 and 4 wks, and 8, 10 wks. Muscle biopsies were obtained from 5 of the 8 and one additional subject. Muscle carnosine was significantly increased by +58.8% and +80.1% after 4 and 10 wks beta-alanine supplementation. Carnosine, initially 1.71 times higher in type IIa fibres, increased equally in both type I and IIa fibres. No increase was seen in control subjects. Taurine was unchanged by 10 wks of supplementation. 4 wks beta-alanine supplementation resulted in a significant increase in TWD (+13.0%); with a further +3.2% increase at 10 wks. TWD was unchanged at 4 and 10 wks in the control subjects. The increase in TWD with supplementation followed the increase in muscle carnosine.

1. Muscle buffering capacity (beta m) and dipeptide content were measured in locomotory muscles of the Thoroughbred horse, Greyhound dog and Man. 2. Beta m and carnosine contents were highest in the horse. Anserine was only found in dog muscle. 3. The higher beta m in horse and dog muscle, compared with man, appears to be predominantly due to higher muscle contents of histidine containing dipeptides in these species.

The in vitro deproteinized vastus lateralis muscle buffer capacity, carnosine, and histidine levels were examined in 20 men from 4 distinct populations (5 sprinters, 800-m runners; 5 rowers; 5 marathoners; 5 untrained). Needle biopsies were obtained at rest from the vastus lateralis muscle. The buffer capacity was determined in deproteinized homogenates by repeatedly titrating supernatant extracts over the pH range of 7.0–6.0 with 0.01 N HCl. Carnosine and histidine levels were determined on an amino acid AutoAnalyzer. Fast-twitch fiber percentage was determined by staining intensity of myosin adenosinetriphosphatase. High-intensity running performance was assessed on an inclined treadmill run to fatigue (20% incline; 3.5 m X s-1). Significantly (P less than 0.01) elevated buffer capacities, carnosine levels, and high-intensity running performances were demonstrated by the sprinters and rowers, but no significant differences existed between these variables for the marathoners vs. untrained subjects. Low but significant (P less than 0.05) interrelationships were demonstrated between buffer capacity, carnosine levels, and fast-twitch fiber composition. These findings indicate that the sprinters and rowers possess elevated buffering capabilities and carnosine levels compared with marathon runners and untrained subjects.

1. 1. The occurrence of carnosine, anserine, ophidine, β-alanine, γ-aminobutyric acid, histidine, 1-methylhistidine and 3-methylhistidine in the muscle tissues of twenty-six vetebrate species and the skins of two reptiles was studied using paper chromatography. 2. 2. One or two of the dipeptides was found in each tissue. Some tissues also contained β-alanine and histidine, but γ-aminobutyric acid, 1-methylhistidine and 3-methylhistidine were not detected. 3. 3. Relationships appear to exist between the zoological classification of a species and the identities and relative amounts of its dipeptides. 4. 4. The reptile skins contained the same dipeptides and other substances as their corresponding muscle tissues. 5. 5. Biochemical and biological information about the dipeptides is discussed.

Carnosine synthetase was purified about 500-fold from mouse olfactory bulb to a specific activity of approx 25 nmol/min/mg. This is an increase of 800-fold over that previously reported for this enzyme from rat brain and 11 times higher than the most highly purified enzyme from chicken pectoral muscle. ATP was essential for activity and could not be replaced by ADP. NAD had no effect on the synthesis of carnosine. Of the β-alanine analogues tested, the purified mouse enzyme incorporated only γ-aminobutyric acid and β-amino-n-butyric acid into peptide linkage with histidine. Synthesis of carnosine by the mouse olfactory bulb enzyme was competitively inhibited by the histidine analogues, 1-methyl histidine and 3-methyl histidine, with Ki values which were at least 40 times the Km value for histidine (16 μM). Ornithine and lysine were more efficient β-alanine acceptors than 1-methyl histidine for the mouse enzyme. Enzyme from olfactory epithelium and leg skeletal muscle of mice also showed higher Ki values for 1–methyl histidine than the Km value for histidine. In contrast, carnosine-anserine synthetase from chicken pectoral muscle gave Km values for histidine, 1-methyl histidine and 3-methyl histidine, which were all in the range of 4–12 μM. The differences in substrate specificity between the enzyme from mouse and chicken implies alternate routes of anserine synthesis in these species and predicts the occurrence of certain novel peptides in mouse brain.

Background Carnosine is a dipeptide that improves exercise performance. The carnosine synthesis mechanism through carnosine and ß-alanine ingestion remains unclear. Therefore, we investigated the tissue distribution of carnosine synthase, ATP-grasp domain-containing protein-1 (ATPGD1) mRNA, and ATPGD1 and carnosine specific dipeptidase (CN1) gene expression profiles in mice that were given carnosine or ß-alanine orally. Methods ddY mice (7-week-old) were randomly divided into three groups (n = 6 to 8 animals per group) and were orally given 2 g/kg body weight of carnosine, ß-alanine, or water. After 15, 30, 60, 120, 180, or 360 min of treatment, the tissues (brain, blood, liver, kidneys, olfactory bulbs, hindleg muscles) were collected. The obtained tissues measured the expression of ATPGD1 and CN1 genes using quantitative PCR methods. Results The ATPGD1 gene was expressed in muscle and to a lesser extent in brain. The expression of ATPGD1 in the vastus lateralis muscle increased significantly at 180 min (P = 0.023) after carnosine ingestion and 60 (P = 0.023) and 180 min (P = 0.025) after ß-alanine ingestion. Moreover, the carnosine group showed a significantly increased renal expression of the CN1 gene 60 min after ingestion (P = 0.0015). Conclusions The ATPGD1 gene showed high expression levels in brain and muscle. The ß-alanine or carnosine administration significantly increased ATPGD1 and CN1 expression in mice.