The use of carnosine in medical practice. Priorities: past and future]

The history of the discovery of curative effects of carnosine and its perspective applications in the clinical practice are reviewed. The molecular mechanisms of carnosine interactions with free oxygen radicals (hypochlorite anion, in particular) are considered.     

Copper(II) complexes of carnosine, glycylglycine, and glycylglycine-imidazole mixtures

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Spectroscopic studies on the copper(II) complexes of carnosine

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Formation of carnosine-Cu(II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors

To further analyze the action of copper on brain synaptic mechanisms, the brain dipeptide carnosine (beta-alanyl-L-histidine) was tested in Xenopus laevis oocytes expressing the rat P2X4 or P2X7 receptors. Ten micromolar copper halved the currents evoked by ATP in both receptors; co-application of carnosine plus copper prevented the metal induced-inhibition with a median effective concentration of 12.1 +/- 3.9 and 12.0 +/- 5.5 microm for P2X4 and P2X7, respectively. Zinc potentiated only the P2X4 ATP-evoked currents; carnosine had no effect over this metal. The relative potency and selectivity of classical metal chelators to prevent the copper inhibition was compared between carnosine and penicillamine (PA), bathophenanthroline (BPh) or L-histidine (His). Their rank order of potency in P2X4 and P2X7 receptors was carnosine = PA = His > BPh > Glycine (Gly) and carnosine = BPh = His > PA > Gly, respectively. The potency to prevent the zinc-induced potentiation in the P2X4 receptor was BPh > PA > His; carnosine, Gly and beta-alanine were inactive. Whereas 1-100 microm carnosine or His alone did not modify the ATP-evoked currents, 10-100 microm PA augmented and 100 microm BPh decreased the ATP-evoked currents. Carnosine was able to revert the copper-induced inhibition restoring the maximal ATP gated current in a concentration-dependent manner. Electronic spectroscopy confirm the formation of carnosine-Cu(II) complexes, mechanism that can account for the prevention and reversal of the copper inhibition, revealing its potential in copper intoxication treatment.     

Reverse structure of carnosine-induced sedative and hypnotic effects in the chick under acute stress

In the central nervous system, beta-alanine is thought to act as an inhibitory neurotransmitter, but the role or precise mechanism of beta-alanine in the brain has not been clearly defined. beta-Alanine is found in high levels in the chicken brain as a component of the dipeptides carnosine (beta-alanyl-L-histidine) and anserine, or as a free amino acid. We focused on the position of beta-alanine, i.e., at the carboxyl terminus. In Experiment 1, the central effects of glycyl-beta-alanine, L-histidyl-beta-alanine and L-valyl-beta-alanine were compared with a saline control in chicks. L-Histidyl-beta-alanine significantly induced sedative and hypnotic effects. In Experiment 2, the effects of carnosine, its reverse (L-histidyl-beta-alanine), and their combination were investigated. Central carnosine-induced hyperactivity while reverse carnosine-induced hypoactivity, and the behaviors were intermediate following the combination of the two peptides. Finally, the central effect of reverse carnosine was compared with beta-alanine alone and L-seryl-beta-alanine in Experiment 3. Reverse carnosine showed similar effects to beta-alanine. In conclusion, L-histidyl-beta-alanine not only has the reverse structure of carnosine, but also reverse function. Thus, we propose to name reverse carnosine (L-histidyl-beta-alanine) rev-carnosine.     

Comparison of the carnosine and taurine contents of vastus lateralis of elderly Korean males, with impaired glucose tolerance, and young elite Korean swimmers

The carnosine and taurine contents of the vastus lateralis of two diverse groups of Korean male subjects (elderly and impaired glucose-tolerant (IGT) subjects and young elite swimmers at a national sport university) having a similar national diet, were examined. Despite marked differences in age, fitness and clinical status the two groups showed almost identical muscle carnosine and taurine contents. In the case of carnosine, the results suggest a similar contribution to intracellular buffering capacity in the two groups of subjects, with no evidence of a reduction of this in elderly IGT subjects. In addition, both groups showed the same inverse relationship between the muscle carnosine and taurine contents; the spread of values between subjects, within-groups, most likely reflect variations in the type I (low carnosine, high taurine) or type II (high carnosine, low taurine) composition of the vastus lateralis. The relationship is consistent with a role of taurine in osmoregulation, compensating for variations between fibre types in the carnosine content.     

Regulation of peroxo-bridge formation between cobalt(II)-carnosine complexes by histidine: Possible involvement in polycythemia

The mechanisms by which histidine stabilizes the cobalt(II)-carnosine complex from oxidation to cobalt(III) in aqueous solution are investigated with ¹H-nmr, laser Raman, and Fourier transform-infrared spectroscopy. Histidine has at least three effects on the cobalt(II)-carnosine complex. First, over the concentration range of at least 5 to 250 mM, histidine stabilizes the cobalt(II)-carnosine complex from oxidation by excluding solvent molecules from the equatorial coordination positions. Second, at the upper end of this concentration range, histidine reduces the strained nonplanarity of the equatorial coordination positions around the cobalt(II) ion that results from tridentate chelation by carnosine. Bidentate ligation by histidine causes the carnosine to bind as a bidentate ligand also. Third, bidentate ligation of two carnosine molecules to the equatorial coordination positions of Co(II) ion places the β-alanyl residues inthe vicinity of the two axial coordination positions and thereby inhibits the binding of molecular oxygen. Substitution of a molecule of histidine for one of these two carnosine molecules makes an axial coordination position available for binding oxygen. The first two effects are expected to stabilize the cobalt(II) ion from rapid oxidation, whereas the third effect is expected to give long-term stability of the peroxo-bridged complex. Since bidentate ligation of histidine is favored over monodentate ligation only when the concentration of Co(II) ion is not limiting and is inhibited by high concentrations of carnosine in the same solution, the results presented provide a possible explanation for the observation that the stability of the Co(II) complexes toward oxidation and their ability to bind molecular oxygen depend on both the relative and absolute concentrations of Co(II) ion, carnosine, and histidine in solution. Furthermore, these results provide additional support to the suggestion that the high activity of carnosinase in kidney is involved in part in regulation of the oxygen sensor in this organ.     

New glycoside derivatives of carnosine and analogs resistant to carnosinase hydrolysis: synthesis and characterization of their copper(II) complexes

Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide widely and abundantly distributed in muscle and nervous tissues of several animal species. Many functions have been proposed for this compound, such as antioxidant and metal ion-chelator properties. However, the main limitation on therapeutic use of carnosine on pathologies related to increased oxidative stress and/or metal ion dishomeostasis is associated with the hydrolysis by the specific dipeptidase carnosinase. Several attempts have been made to overcome this limitation. On this basis, we functionalized carnosine and its amide derivative with small sugars such as glucose and lactose. The resistance of these derivatives to the carnosinase hydrolysis was tested and compared with that of carnosine. We found that the glycoconjugation protects the dipeptide moiety from carnosinase hydrolysis, thus potentially improving the availability of carnosine. The copper(II) binding properties of all the new synthesized compounds were investigated by spectroscopic (UV-Visible and circular dichroism) and ESI-MS studies. Particularly, the new family of amide derivatives that are not significantly hydrolyzed by carnosinase is a very promising class of carnosine derivatives. The sugar moiety can act as a recognition element. These new derivatives are potentially able to act as chelating agents in the development of clinical approaches for the regulation of metal homeostasis in the field of medicinal inorganic chemistry.     

Biochemical and Physiological Evidence that Carnosine Is an Endogenous Neuroprotector Against Free Radicals

1. Carnosine, anserine, and homocarnosine are endogenous dipeptides concentrated in brain and muscle whose biological functions remain in doubt.2. We have tested the hypothesis that these compounds function as endogenous protective substances against molecular and cellular damage from free radicals, using two isolated enzyme systems and two models of ischemic brain injury. Carnosine and homocarnosine are both effective in activating brain Na, K-ATPase measured under optimal conditions and in reducing the loss of its activity caused by incubation with hydrogen peroxide.3. In contrast, all three endogenous dipeptides cause a reduction in the activity of brain tyrosine hydroxylase, an enzyme activated by free radicals. In hippocampal brain slices subjected to ischemia, carnosine increased the time to loss of excitability.4. In in vivo experiments on rats under experimental hypobaric hypoxia, carnosine increased the time to loss of ability to stand and breath and decreased the time to recovery.5. These actions are explicable by effects of carnosine and related compounds which neutralize free radicals, particularly hydroxyl radicals. In all experiments the effective concentration of carnosine was comparable to or lower than those found in brain. These observations provide further support for the conclusion that protection against free radical damage is a major role of carnosine, anserine, and homocarnosine.     

Effects of carnosine on contractile apparatus Ca²⁺ sensitivity and sarcoplasmic reticulum Ca²⁺ release in human skeletal muscle fibers

There is considerable interest in potential ergogenic and therapeutic effects of increasing skeletal muscle carnosine content, although its effects on excitation-contraction (EC) coupling in human muscle have not been defined. Consequently, we sought to characterize what effects carnosine, at levels attained by supplementation, has on human muscle fiber function, using a preparation with all key EC coupling proteins in their in situ positions. Fiber segments, obtained from vastus lateralis muscle of human subjects by needle biopsy, were mechanically skinned, and their Ca(2+) release and contractile apparatus properties were characterized. Ca(2+) sensitivity of the contractile apparatus was significantly increased by 8 and 16 mM carnosine (increase in pCa(50) of 0.073 ± 0.007 and 0.116 ± 0.006 pCa units, respectively, in six type I fibers, and 0.063 ± 0.018 and 0.103 ± 0.013 pCa units, respectively, in five type II fibers). Caffeine-induced force responses were potentiated by 8 mM carnosine in both type I and II fibers, with the potentiation in type II fibers being entirely explicable by the increase in Ca(2+) sensitivity of the contractile apparatus caused by carnosine. However, the potentiation of caffeine-induced responses caused by carnosine in type I fibers was beyond that expected from the associated increase in Ca(2+) sensitivity of the contractile apparatus and suggestive of increased Ca(2+)-induced Ca(2+) release. Thus increasing muscle carnosine content likely confers benefits to muscle performance in both fiber types by increasing the Ca(2+) sensitivity of the contractile apparatus and possibly also by aiding Ca(2+) release in type I fibers, helping to lessen or slow the decline in muscle performance during fatiguing stimulation.     

Biological role of histidine-containing dipeptides

The biological role of histidine-containing dipeptides is reviewed. The role of carnosine and anserine in muscle function is discussed from the evolutionary viewpoint. Evidence on the antioxidative effect of carnosine and its protection of biological membranes against lipid peroxidation-induced damages is presented. The effects of presently known natural antioxidative agents and carnosine on lipid peroxidation are compared. Carnosine has been shown to be a more universal protector of membranes as compared to free radical scavengers.     

Problems and perspectives in studying the biological role of carnosine

In describing carnosine among the constituents of muscle tissue in 1900, V. Gulevitsch opened the question of its real biological role. Investigation of carnosine-related phenomena occurred simultaneously with the study of its metabolic transformation within the cell. It has now been demonstrated that carnosine has the ability to protect cells against oxidative stress as well as to increase their resistance toward functional exhaustion and accumulation of senile features. Mechanisms of such protection are explained in terms of proton buffering, heavy metal chelating, as well as free radical and active sugar molecule scavenging, preventing modification of biomacromolecules and keeping their native functional activity under oxidative stress. Several carnosine derivatives are characterized by different rates of splitting by tissue carnosinase and by different biological efficiencies, thus the biological significance of enzymatic modification of carnosine during its tissue metabolism may be increased resistance of cells operating under unfavorable conditions.     

Evidence that carnosine and anserine may participate in Wilson's disease

Carnosine, anserine and copper(II) ion all bind to specific sites on bovine serum albumin, and, in addition, both dipeptides chelate copper(II) ion in the absence of serum albumin. Thus a solution of dipeptide, copper(II) ion and serum albumin exhibits several complexes that arise from the competing binding reactions. Since a change in this complex equilibrium might occur in Wilson's disease, we have investigated the reactions between the various complexes with NMR and ESR spectroscopy. Serum albumin simultaneously binds the copper(II) ion and carnosine to separate sites rather than forming a mixed chelate, but carnosine still is capable of competing with serum albumin for subsaturating amounts of copper.     

Multiple forms of the cobalt(II)-carnosine complex.

Cobalt(II) ion and L-carnosine produce two different complexes when mixed in aqueous solution at pH 7.2. One complex has coordination of N-3 of the imidazole ring to the cobalt(II) and is produced when the concentration of peptide exceeds that of cobalt(II). The second complex has chelation of three nitrogen atoms of a single carnosine. This second complex produces a reversible oxygen carrier by making stable mixed chelates with additional carnosine, histidine or cysteine. These results indicate that cobalt complexes with mixed ligands should be of more importance $\text{in}\text{vivo}$ than those with carnosine as the only ligand. They provide an explanation for the high activity and substrate specificity of carnosinase in kidney.     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.     

Direct measurement of the interaction of carnosine and its analogs with free radicals]

An ESR study of interactions of carnosine and its derivatives with free radicals has been carried out. In model systems the spin trap OH. radical adduct generation has been shown to decrease significantly in the presence of carnosine in a pronounced concentration-dependent manner. A comparative study of effects of some other histidine-containing dipeptides on this process has revealed a similarity in anserine, homocarnosine, and acetylcarnosine actions.     

Neuroprotective effect of carnosine on primary culture of rat cerebellar cells under oxidative stress

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatographymass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.     

Mechanism of antioxidant action of carnosine

The comparative study of the antiradical activity of carnosine and vitamin C was carried out by the means of the evaluation of quenching of ESR signals of 2,2-diphenyl-1-picrylhydrazyl (DFPH) and semiquinone radical of alpha-tocopherol. It was shown that carnosine is not able to quench the ESR signals of the stable radical of DFPH and semiquinone radical of alpha-tocopherol. It permits to conclude that: a) carnosine does not interact directly with highly active free radicals; b) carnosine is unable to regenerate the radical of alpha-tocopherol to form the antiradical synergistic couple. The data obtained are consistent with the idea that there is a difference between on the antioxidant mechanism action of vitamin C and carnosine due to the difference in the antiradical activity of these compounds.     

Evidence for rapid inter- and intramolecular chlorine transfer reactions of histamine and carnosine chloramines: implications for the prevention of hypochlorous-acid-mediated damage

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and Cl(-) by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that is implicated in a wide range of human diseases (e.g., atherosclerosis). Histamine and carnosine have been proposed as protective agents against such damage. However, as recent studies have shown that histidine-containing compounds readily form imidazole chloramines that can rapidly chlorinate other targets, it was hypothesized that similar reactions may occur with histamine and carnosine, leading to propagation, rather than prevention, of HOCl-mediated damage. In this study, the reactions of HOCl with histamine, histidine, carnosine, and other compounds containing imidazole and free amine sites were examined. In all cases, rapid formation (k, 1.6 x 10(5) M(-)(1) s(-)(1)) of imidazole chloramines was observed, followed by chlorine transfer to yield more stable, primary chloramines (R-NHCl). The rates of most of these secondary reactions are dependent upon substrate concentrations, consistent with intermolecular mechanisms (k, 10(3)-10(4) M(-)(1) s(-)(1)). However, for carnosine, the imidazole chloramine transfer rates are independent of the concentration, indicative of intramolecular processes (k, 0.6 s(-)(1)). High-performance liquid chromatography studies show that in all cases the resultant R-NHCl species can slowly chlorinate N-alpha-acetyl-Tyr. Thus, the current data indicate that the chloramines formed on the imidazole and free amine groups of these compounds can oxidize other target molecules but with limited efficiency, suggesting that histamine and particularly carnosine may be able to limit HOCl-mediated oxidation in vivo.     

Characterization of carnosine uptake and its physiological function in human intestinal epithelial Caco-2 cells

Carnosine (beta-Ala-L-His) is known to have the physiological functions of an antioxidant. Although dietary carnosine is thought to be absorbed across intestinal epithelial cells, the mechanism for this absorption is not yet well understood and its function in the intestinal tract is also obscure. The intestinal transport of carnosine was characterized in the present study by using human intestinal Caco-2 cells, and its physiological function in these cells was further examined. The carnosine uptake was proton-dependent, being activated by lowering the apical pH value. Its uptake was significantly inhibited by other dipeptides, whereas it was not inhibited by other amino acids. These characteristics of the carnosine uptake strongly suggest its transport into the cells via peptide transporter 1 (PepT1). Since carnosine has antioxidative activity, we studied its effect on the H2O2-induced secretion of inflammatory cytokines in Caco-2 cells. The H2O2 induced increase in IL-8 secretion was inhibited by a pretreatment with carnosine for 3 h, this inhibition being presented in a dose-dependent manner. These results suggest that carnosine had a protective effect against oxidative stress in intestinal epithelial cells.