The interaction of taurine and beta-alanyl-L-histidine (carnosine) with hypochlorite anion (ClO-)]

Dipeptide carnosine and amino acid taurine have been found to actively interact with hypochlorite anion. Chloramine complexes obtained during this reaction were more stable in case of taurine. It is suggested that therapeutic effect of new Russian eye drops taufon and sevitin is due to neutralization of the reaction product hypochlorite anion catalyzed by myeloperoxidase.     

Mechanism of action of biogenic chloramines and hypochlorite on initial aggregation of blood platelets

The antiaggregant action of two reactive oxidants N,N-dichlorotaurine (chloramine of biogenic type) and sodium hypochlorite on the initial ADP-induced aggregation of rabbit blood platelets has been studied. Platelet aggregation in the reconstructed platelet-rich plasma (PRP) was measured by the nephelometric method, and the aggregation index was an increase in the intensity of small-angle light scattering. The introduction of chloramine at comparatively small concentrations (no greater than 1 mM active chlorine) directly into the reconstructed platelet-rich plasma induces the suppression of the initial aggregation (formation of small aggregates) several times stronger than in the case of its preliminary incubation with plasma alone. This suggests that N,N-dichlorotaurine exerts its antiaggeregant action on the platelet-rich plasma by direct interaction with cells. The effects of the inhibition of platelet aggregation in two variants of introduction of high concentrations of N,N-dichlorotaurine do not significantly differ. In this case a great amount of residual chloramine remains in the plasma, which just induces the suppression of platelet aggregation during subsequent reconstruction of the platelet-rich plasma. Similar data have been obtained in the study of the antiaggregant action of hypochlorite. N,N-Dichlorotaurine and hypochlorite at final concentrations of 0.2-0.3 and 0.15 mM, respectively, inhibit strongly the initial aggregation of isolated platelets (approximately 2 x 10(8) cells in 1 ml) preliminarily activated for 1.5 min by the addition of 100-500 nM ADP. However, the antiaggregants show a more profound suppression of aggregation of unstimulated platelets. The antiaggregant effects of N,N-dichlorotaurine and hypochlorite are probably due to the oxidative modification of sulfur-containing groups in platelet plasmatic membrane.     

Effects of Calpain on Antioxidant Enzyme Activities

The activities of superoxide dismutase, catalase and glutathione reductase were not affected by in vitro incubation with the intracellular proteinase calpain, suggesting that these enzymes are not in vivo substrates of calpain. In contrast, the activity of another important antioxidant enzyme, glutathione peroxidase, is stimulated in vitro by calpain. This may explain the correlation between elevations in glutathione peroxidase activity and calpain activity which occur in aging, exercised and dystrophic muscle. Calpain treatment in vitro caused a large decrease in the activity of carnosine synthetase which is involved in the synthesis of the putative antioxidant carnosine. This may be the reason for the in vivo correlation between elevated calpain and diminished carnosine levels in aging, hypertensive, denervated and dystrophic muscles.     

Hypochlorite-Modified Adenine Nucleotides: Structure, Spin-Trapping, and Formation by Activated Guinea Pig Polymorphonuclear Leukocytes

Adenosine and its nucleotides react with hypochlorite to form unstable products that have been identified as the N⁶ chloramine derivatives. These chloramines spontaneously oligomerize. form stable adducts with proteins and nucleic acids, and are converted with loss of chlorine to the original nucleoside or nucleotide by reducing agents. The chloramines are associated with a free radical. and the spin-trapping of adenosine chloramine with 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) yielded a mixture of unstable nitroxyl adducts that corresponded to nitrogen-centered radicals from the parent nucleoside. When activated guinea pig polymorphonuclear leukocytes were stimulated with phorbol myristate acetate to produce hypochlorite, they actively incorporated [¹⁴C] adenosine into acid-insoluble products by a process that was dependent on oxygen and inhibited by azide and thiols. These findings suggest that adenine nucleotide chloramines are generated by activated phagocytic cells and form ligands with proteins and nucleic acids as observed in model systems. The results imply that nucleotide chloramines are among the cytotoxic and possibly mutagenic factors that are associated with the icflammatory process.     

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.     

Studies on reaction of glutathionylcobalamin with hypochlorite. Evidence of protective action of glutathionyl-ligand against corrin modification by hypochlorite

Glutathionylcobalamin (GSCbl), a tight complex of glutathione (GSH) with cobalamin(III), is readily oxidized to aquacobalamin by hypochlorite. Corrin macrocycle remains unmodified in the presence of threefold excess of hypochlorite, whereas aqua- and cyanocobalamins are partially transformed to chlorinated species under the same conditions. The suggested mechanism of reaction between GSCbl and hypochlorite involves subsequent oxidation of thiol and amino groups and dissociation of oxidized glutathione from Co(III)-ion.     

Hypochlorite-Modified Adenine Nucleotides: Structure,Spin-Trapping,and Formation by Activated Guinea Pig Polymorphonuclear Leukocytes

Adenosine and its nucleotides react with hypochlorite to form unstable products that have been identified as the N⁶ chloramine derivatives. These chloramines spontaneously oligomerize. form stable adducts with proteins and nucleic acids, and are converted with loss of chlorine to the original nucleoside or nucleotide by reducing agents. The chloramines are associated with a free radical. and the spin-trapping of adenosine chloramine with 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) yielded a mixture of unstable nitroxyl adducts that corresponded to nitrogen-centered radicals from the parent nucleoside. When activated guinea pig polymorphonuclear leukocytes were stimulated with phorbol myristate acetate to produce hypochlorite, they actively incorporated [¹⁴C] adenosine into acid-insoluble products by a process that was dependent on oxygen and inhibited by azide and thiols. These findings suggest that adenine nucleotide chloramines are generated by activated phagocytic cells and form ligands with proteins and nucleic acids as observed in model systems. The results imply that nucleotide chloramines are among the cytotoxic and possibly mutagenic factors that are associated with the icflammatory process.     

Carnosine as a potential scavenger of oxidants generated by stimulated neutrophils

Luminol-dependent chemiluminescence of PMA-stimulated human neutrophils decrease more than by 50% in the presence of physiological concentrations of carnosine (20 mM). This inhibition is the result of carnosine ability to scavenge hypochlorite (OCl-), since carnosine exerts a similar effect on chemiluminescence produced by myeloperoxidase-H2O2-Cl- and OCl(-)-H2O2 systems. The previously undocumented property of this dipeptide to scavenge active oxygen species requires further experiments.     

薄层色谱和高效液相色谱联用测定暗纹东方鲀肌肉中肌肽和谷胱甘肽的含量

通过薄层色谱(TLC)与高效液相色谱(HPLC)联用技术鉴定了暗纹东方鲀(Takifugu obscurus)肌肉中存在肌肽和谷胱甘肽,同时利用高效液相色谱法测定了肌肽和谷胱甘肽(GSH)的含量。薄层层析采用的展开剂为正丁醇:乙酸:水(4∶2∶1),层析板为硅胶板。高效液相色谱利用Kromasil C18反相柱分析,流动相为10%的纯乙腈和90%含有0.05%三氟乙酸的超纯水。结果表明:通过薄层色谱和高效液相色谱鉴定了暗纹东方鲀肌肉中肌肽和谷胱甘肽,其中暗纹东方鲀肌肉中肌肽含量约213μg/g(鲜重),还原性谷胱甘肽含量约211μg/g(鲜重)。本法样品无需衍生,操作简便,适合于暗纹东方鲀肌肉中肌肽和谷胱甘肽的测定。本文为生物体内肌肽和谷胱甘肽的研究提供借鉴意义。     

Mechanism of the action of biogenic chloramines and hypochlorite on the initial aggregation of platelets

The antiaggregant effect of two reactive oxidants—N,N-dichlorotaurine (a biogenic chloramine) and sodium hypochlorite—on the initial ADP-induced aggregation of rabbit blood platelets was studied. Platelet aggregation in reconstituted platelet-rich plasma was measured nephelometrically; an increase in the intensity of small-angle light scattering served as an index of aggregation. Addition of chloramine at relatively small concentrations (no greater than 1 mM available chlorine) directly to the reconstituted platelet-rich plasma suppressed the initial aggregation (formation of small aggregates) several times more strongly than preincubation of native plasma with chloramine. This suggests that N,N-dichlorotaurine realizes its antiaggregant effect on the platelet-rich plasma by directly interacting with cells. The effects of the inhibition of platelet aggregation in two variants of addition of high concentrations of N,N-dichlorotaurine did not differ significantly. In this case, a large amount of residual unreacted chloramine remained in the plasma, which caused the suppression of platelet aggregation during subsequent reconstitution of the platelet-rich plasma. Similar data were obtained in studying the antiaggregant effect of hypochlorite. N,N-Dichlorotaurine and hypochlorite at concentrations of 0.2–0.3 and 0.15 mM, respectively, strongly inhibited the initial aggregation of isolated platelets (approximately 2·10⁸ cells/ml) preliminarily activated for 1.5 min by addition of 0.1–0.5 μM ADP. However, the antiaggregants had a more profound suppressive effect on the aggregation of unstimulated platelets. The antiaggregant effects of N,N-dichlorotaurine and hypochlorite probably stem from the oxidative modification of the sulfur-containing groups in platelet plasma membrane.     

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.     

Use of carnosine as a natural anti-senescence drug for human beings

Carnosine is an endogenous free-radical scavenger. The latest research has indicated that apart from the function of protecting cells from oxidation-induced stress damage, carnosine appears to be able to extend the lifespan of cultured cells, rejuvenate senescent cells, inhibit the toxic effects of amyloid peptide (A beta), malondialdehyde, and hypochlorite to cells, inhibit glycosylation of proteins and protein-DNA and protein-protein cross-linking, and maintain cellular homeostasis. Also, carnosine seems to delay the impairment of eyesight with aging, effectively preventing and treating senile cataract and other age-related diseases. Therefore, carnosine may be applied to human being as a drug against aging.     

A re-evaluation of the antioxidant activity of purified carnosine

The antioxidant activity of carnosine has been re-evaluated due to the presence of contaminating hydrazine in commercial carnosine preparations. Purified carnosine is capable of scavenging peroxyl radicals. Inhibition of the oxidation of phosphatidylcholine liposomes by purified carnosine is greater in the presence of copper than iron, a phenomenon likely to be due to the copper chelating properties of carnosine. Purified carnosine is capable of forming adducts with aldehydic lipid oxidation products. Adduct formation is greatest for alpha,beta-monounsaturated followed by polyunsaturated and saturated aldehydes. While the ability of carnosine to form adducts with aldehydic lipid oxidation products is lower than other compounds such as glutathione, the higher concentrations of carnosine in skeletal muscle are likely to make it the most important molecule that forms aldehyde adducts. Monitoring changes in carnosine concentrations in oxidizing skeletal muscle shows that carnosine oxidation does not occur until the later stages of oxidation suggesting that carnosine may not be as effective free radical scavenger in vivo as other antioxidants like alpha-tocopherol.     

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.     

Oxidation of ceruloplasmin by hypochlorite. The loss of blue color and preservation of oxidase activity

Ceruloplasmin oxidation by hypochlorite results in the bleaching of the protein solution; the oxidase activity remains, however, unchanged. Hypochlorite exerts a complex effect on the protein activity. After a short-term (5 min) incubation with hypochlorite the enzyme activity increases with a further progressive decrease. It is supposed that the oxidase activity of ceruloplasmin is not coupled with copper ions of the first type responsible for the blue staining of the protein solution. The low susceptibility of functional properties of ceruloplasmin to hypochlorite raises its potency as an antioxidative agent.     

In vitro demonstration of histamine biosynthesis from carnosine by kidneys of pregnant mice

Kidneys of pregnant mice synthesize histamine when incubated in the presence of carnosine, manganese, and pyridoxal phosphate. Intensity of biosynthesis increases linearly with the amount of enzyme and the incubation time. The reaction can only be catalysed by two enzymes that are located in kidneys and act in succession: carnosinase, which hydrolyzes carnosine into its two moieties, and histidine decarboxylase, which transforms histidine, a product of carnosine degradation, into histamine. The biosynthesis of histamine from carnosine seems to increase with the progress of pregnancy. In nonpregnant mice, kidneys do not effect this biosynthesis. The above results directly demonstrate that carnosine may be used for histamine synthesis when the activity of histidine decarboxylase is high, as in pregnant mouse kidney. Vertebrate carnosine, its role still enigmatic, might thus be mainly a potential histidine reservoir that would be mobilized any time there is a significant requirement for histidine, such as for histamine biosynthesis.     

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.     

Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain‐sensitive Na/K‐ATPase

It is known that ouabain, a selective inhibitor of Na/K‐ATPase, not only can cause the activation of signal cascades, which regulate the cell viability, but also can cause the accumulation of free radicals, which can evoke the oxidative stress. We have shown that the nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals, but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover, the long‐term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain‐mediated apoptosis and necrosis were also abolished by carnosine. Copyright © 2012 John Wiley & Sons, Ltd.     

Carnosine protection of Ca2+ transport against damage induced by lipid peroxidation

The authors studied the protective action of carnosine on sarcoplasmic reticulum (SR) membranes from frog skeletal muscles destroyed by ascorbic acid-dependent lipid peroxidation (LPO). It was demonstrated that addition of carnosine to the incubation medium at a concentration of 25 mM sharply decelerated inactivation of Ca-ATPase of SR membranes, maintaining at the same time the coupling of hydrolysing and transport functions of the Ca-pump. When given at the same concentration carnosine inhibited the accumulation of LPO products reacting with 2-thiobarbituric acid. This effect of carnosine was followed by its utilization.     

The effect of dopamine on in vitro methemoglobin formation in erythrocytes of patients with Parkinson’s disease under oxidative stress

Using electron paramagnetic resonance, the dose-dependence effect of dopamine on methemoglobin formation in erythrocytes of patients with Parkinson’s disease under the activation of oxidative stress induced by acrolein and the possibilities for the correction of this pathological process using carnosine in vitro experiments have been examined. It was shown that incubation of erythrocytes with 1.5 mM dopamine did not change the methemoglobin content, while incubation with 15 mM dopamine caused a two fold increase in the methemoglobin content compared to its initial level; 10 μM acrolein increased methemoglobin formation threefold. Administration of 15 mM dopamine and, after 1 h, 10 μM acrolein to the incubation system increased methemoglobin formation tenfold compared to its initial level. Preincubation of erythrocytes with 5 mM carnosine followed by acrolein addition prevented the increase in the methemoglobin content, while carnosine had no effect on methemoglobin formation induced by dopamine.