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.     

Retrospectives and perspectives on the biological activity of histidine-containing dipeptides

1. An evaluation of the biological function of histidine-containing dipeptides, is presented. 2. These have been found to be potent pH-buffering compounds, strong antioxidants and membrane stabilizers. 3. Analysis of the biological and biomedical properties of carnosine and other related dipeptides suggests their use as universal membrane repairing compounds.     

Muscle Histidine-Containing Dipeptides Are Elevated by Glucose Intolerance in Both Rodents and Men

Objective

Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.

Design and Methods

Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured.

Results

Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects.

Conclusion

Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.     

Adsorption of histidine-containing dipeptides on copper(II) immobilized chelating resin from saline solution

Adsorption of histidine-containing dipeptides such as carnosine (Car) was investigated using copper(II) immobilized cation exchange resins. Adsorption of Car was enhanced using Cu(II) immobilized resins, on the basis of metal affinity interactions. In particular, iminodiacetic acid chelating resin with immobilized Cu(II) (Cu-IDA) can adsorb Car from saline water. Car was adsorbed on Cu-IDA even in the presence of 1000 mM of NaCl. Adsorption of various amino acids on Cu-IDA was compared under same conditions. Histidine and the histidine-containing dipeptides were selectively adsorbed on Cu-IDA over other amino acids, both in the absence and in the presence of NaCl. Therefore, immobilized metal affinity adsorption is an efficient method for recovering histidine-containing dipeptides from saline water.     

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.     

Anserine and carnosine in chicks (Gallus gallus) rat pups (Rattus rattus) and ducklings (Anas platyrhynchos): comparative ontogenetic observations.

1. Muscle and brain from developing chick embryos, as well as from day-old chicks, rats, and ducks were analyzed for the histidine-containing dipeptides, anserine and carnosine. 2. Anserine was found in the brain of all species studied, whereas in muscle, anserine was found only in chicks. 3. At 15 days, the muscle of developing chick embryo contained 41 +/- 9 mumoles/100 g anserine while carnosine was present at a level of less than 3 mumoles/100 g. 4. In day-old chicks the anserine level in muscle was 100 +/- 35 mumoles/100 g while the carnosine level was 22.5 +/- 1 mumoles/100 g. 5. These findings cast doubt on earlier hypotheses relating anserine and carnosine to muscle activity.     

Some properties of a homocarnosine-carnosine synthetase isolated from rat brain

—An enzyme from rat brain catalysing the synthesis of the histidine-containing dipeptides carnosine and homocarnosine (l .-histidine: β-alanine ligase (AMP) [EC 6.3.2.11]) was purified about 30-40-fold from a 100,000 g supernatant. Assays were conducted by measuring the incorporation of L-[14C]histidine into carnosine and homocarnosine isolated by paper electrophoresis from the incubation mixture. The ratios of specific activities for the formation of carnosine and homocarnosine were not significantly different for the various purification steps. This was taken as evidence of one enzyme synthesizing both dipeptides. In studying the properties of this enzyme, a pH optimum of 7.4 was shown for carnosine synthesis. The concentrations of amino acid substrates giving maximal synthesis of both dipeptides were in the physiological range found for rat brain. An apparent requirement for ATP, Mg²⁺, and DPN was seen for dipeptide synthesis. A substrate dependent, enzymecatalysed ³²PPi-ATP exchange reaction was observed, suggesting the formation of an aminoacyl-AMP intermediate. Certain other nucleoside triphosphates could substitute for the ATP; this effect showed a specificity toward the dipeptide being synthesized. The apparent requirement for DPN was quite specific, with a number of related compounds having no effect. The stoichiometry of enzyme-catalysed carnosine synthesis was studied. A one to one relationship between carnosine formed and ATP hydrolysed was demonstrated. However, the ratio between carnosine synthesized and DPN hydrolysed was about 6 to 1, indicating a catalytic role for the DPN. The breakdown of DPN did not occur with enzyme alone but was dependent on the presence of substrate.     

Superoxide-scavenging activity of carnosine in the presence of copper and zinc ions

In the presence of Cu2+ and Zn2+ carnosine (beta-alanyl-L-histidine) possesses a superoxide-scavenging activity. The efficiency of scavenging as measured by the inhibition of tetrazolium nitroblue reduction in superoxide anion generation systems (phenazine methasulfate/NADH and xanthine/xanthine oxidase) is concentration-dependent and shows a maximum in the presence of millimolar concentrations of carnosine and equimolar concentrations of Cu2+ and Zn2+. In the presence of Cu2+ and Zn2+ histidine also exhibits a superoxide-scavenging activity. The feasible role of the superoxide-scavenging activity of histidine-containing dipeptide complexes with bivalent metal ions in the realization of physiological function of these dipeptides in skeletal muscles is discussed.     

Antioxidative properties of histidine-containing dipeptides from skeletal muscles of vertebrates

1. Ascorbate-dependent peroxidation of lipid components of biological membranes is inhibited by the natural histidine-containing dipeptides, carnosine and anserine, used at physiological concentrations. 2. Carnosine and anserine exhibit an equal antioxidative activity, whereas the preventing effect of homocarnosine is manifested only at low concentrations of oxidized lipid material. 3. The inhibiting effect of the dipeptides is enhanced either by the rise in the dipeptide concentration or by the decrease in the level of membrane components. 4. Addition of the dipeptides results in a marked decrease in the level of primary molecular products of lipid peroxidation. 5. In this case the optical spectrum of primary molecular products of polyunsaturated fatty acids changes significantly.     

A comparative study of synthetic carnosine analogs as antioxidants

1. The antioxidative activity of carnosine and 16 related compounds, both synthetic and natural, was determined. 2. The antioxidative effect was estimated by the ability of the dipeptides to prevent MDA accumulation in the course of LPO induced in rabbit sarcoplasmic reticulum membranes by the Fe2+ ascorbate system. 3. It was found that the antioxidative effect comparable to that of carnosine was exerted by water-soluble (cyclo-L-histidyl-L-proline) and alcohol-soluble (cyclo-L-histidyl-L-phenilalanine) dipeptides as well as by the histidine-free cyclodipeptides (cyclo-L-tyrosyl-L-proline). 4. However, in contrast to its synthetic analogs, carnosine not only inhibited the LPO, but also diminished the level of products accumulated during membrane lipid peroxidation.     

Role of Aldose Reductase in the Metabolism and Detoxification of Carnosine-Acrolein Conjugates

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

Discovery of carnosine and anserine. Some of their properties]

The history of discovery of carnosine and anserine is reviewed with special reference to the structure and distribution of the dipeptides in various tissues during ontogenesis. The state of the dipeptides in muscle cells, their metabolism and role in muscle activity are considered. The properties of carnosine and anserine phosphoric esters are described, and their putative role in mitochondrial oxidative phosphorylation is discussed. The membranotropic activity of carnosine and anserine is demonstrated.     

Carnosine: biological role and prospects for use in medicine]

The biological role of the histidine-containing dipeptide carnosine (beta-alanyl-L-histidine) has been reviewed. The properties and putative biological role of the dipeptide in vertebrate tissues are considered. The antioxidative activity of carnosine and related compounds is described. The author's conception of the membranoprotective effect of carnosine on cells, tissues, and whole organism has been formulated. The properties of carnosine as an antistressory radioprotective agent are discussed. The data presented suggest that carnosine is a perspective immunomodulating tool which has many applications in medicine.     

N-Acetylhistidine, Glutamate, and β-Alanine Are Concentrated in a Receptor Cell Layer of the Trout Inner Ear

An epithelial sheet isolated from the trout saccular macula, highly enriched in acousticolateralis receptor cells (hair cells), has been analyzed for primary amine-containing compounds. The hair cell preparation, compared to the saccular nerve, was found to contain elevated levels of the presumptive receptoneural transmitter, glutamate, as well as beta-alanine, and components eluting in the positions of the standards phosphoserine and phosphoethanolamine on cation-exchange HPLC. Saccular nerve contained a different spectrum of primary amines and was elevated specifically in carnosine/homocarnosine. Acid hydrolysis of perchlorate extracts of both hair cell and nerve fractions yielded large amounts of histidine. For the saccular nerve fraction, production of histidine by acid hydrolysis was matched by production of beta-alanine and gamma-aminobutyric acid (GABA) and disappearance of carnosine/homocarnosine. The dipeptides carnosine and homocarnosine have been chromatographically resolved by expanded HPLC and found to be present in saccular nerve in a ratio of approximately 10:1, respectively. Production of histidine in the hair cell extract was not coupled with production of beta-alanine and GABA. The hair cell histidine-containing unknown, present in millimolar concentration, has been identified as N-acetylhistidine by the hydrolysis and rechromatography of fractions from cation-exchange HPLC. The large and specific presence of N-acetylhistidine in the hair cell preparation, together with electrophysiological evidence for its facilitatory action on afferent fibers in the frog semicircular canal, is suggestive of a role for this molecule as well as glutamate in acousticolateralis receptoneural transmission.     

Muscle dipeptides--natural inhibitors of lipid peroxidation

The effects of carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) on ascorbate-dependent lipid peroxidation in frog skeletal muscle sarcoplasmic reticulum were studied. It was found that the dipeptides (10-50 mM) cause a 25-90% inhibition of ascorbate-dependent lipid peroxidation and decrease the reaction rate and the amount of end products. The nature of lipid peroxidation primary products in the presence of the dipeptides changes which can be evidenced from changes in their spectral properties. Unlike other known natural antioxidants, skeletal muscle dipeptides do not only inhibit lipid peroxidation but also decrease the level of accumulated lipid peroxidation products. Histidine and beta-alanine, similar to imidazole, glycyl-glycine, arginyl-phenyl alanine and alpha-alanyl-D-histidine do not inhibit lipid peroxidation. At the same time, the carnosine stereoisomer D-carnosine which does not exist in nature exhibits a far greater inhibiting effect as compared to its natural counterpart. It is assumed that the skeletal muscle dipeptides carnosine and anserine are highly effective as natural antioxidants.     

Carnosine and related dipeptides as quenchers of reactive carbonyl species: from structural studies to therapeutic perspectives

Carnosine (beta-alanyl-L-histidine) and related peptides such as homocarnosine (gamma-amino-butyryl-histidine), balenine beta-alanyl-L-3-methylhistidine) and anserine beta-alanyl-L-1-methylhistidine) are histidine-containing dipeptides (HD) particularly abundant in excitable tissues such as nervous system and skeletal muscle. Although their biochemical role is still unknown, several evidences indicate that these endogenous compounds act as quenchers of reactive and cytotoxic carbonyl species. In this presentation we will review the structural evidences and ex vivo data supporting this hypothesis. We first elucidated the reaction mechanism of carnosine as quencher of alpha, beta-unsaturated aldehydes such as 4-hydroxy-trans-2,3-nonenal (HNE) and acrolein (ACR) and then demonstrated the efficacy of carnosine and related peptides as detoxifying agents of HNE in spontaneously oxidized rat skeletal muscle, by detecting the corresponding HNE-Michael adducts in the crude biological matrix by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Finally, we set-up and validated a sensitive, selective and specific LC-ESI-MS/MS method for the determination of HD and of the corresponding HNE-Michael adducts to monitor their profile in physiological (aging) and pathological conditions (diabetes, atherosclerosis) characterized by a carbonyl-mediated degenerative overload. The results obtained, beside to give a contribution to the understanding of the biochemical role of histidine-dipeptides, provide a strong rational to the design of novel derivatives, active as exogenous agents able to detoxify carbonyl compounds.     

Carnosine,homocarnosine and anserine in vertebrate retinas

The dipeptides carnosine, homocarnosine and anserine are differentially distributed among the retinas of several vertebrate species. Retinas of birds are rich in anserine while those of frogs have primarily carnosine. Several mammalian species contain only very low levels of homocarnosine. The biological function of these dipeptides is unknown but their presence and synthesis in retina may confound studies of uptake, metabolism and cellular localization of their component amino acids β-alanine, gamma-aminobutyric acid and histidine.     

Carnosine synthase deficiency is compatible with normal skeletal muscle and olfactory function but causes reduced olfactory sensitivity in aging mice

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.     

Carnosine and cancer: a perspective

The application of carnosine in medicine has been discussed since several years, but many claims of therapeutic effects have not been substantiated by rigorous experimental examination. In the present perspective, a possible use of carnosine as an anti-neoplastic therapeutic, especially for the treatment of malignant brain tumours such as glioblastoma is discussed. Possible mechanisms by which carnosine may perform its anti-tumourigenic effects are outlined and its expected bioavailability and possible negative and positive side effects are considered. Finally, alternative strategies are examined such as treatment with other dipeptides or β-alanine.