The identification of the N tau-methyl histidine-containing dipeptide, balenine, in muscle extracts from various mammals and the chicken

The occurrence of the dipeptide, balenine (beta-alanyl-N tau-methyl histidine), has been identified by comparative chromatography in extracts of muscles from 9 species of mammal, including man, and the chicken. The identity of the dipeptide from 6 mammalian species and chicken was confirmed by preparative isolation prior to determination of the amino acid composition and N-terminal residue. In all cases, the dipeptide contained equimolar amounts of beta-alanine and N tau-methyl histidine with beta-alanine as the N-terminus. The concentration of the dipeptide varied dramatically between species, being just detectable in the muscle of man and rat while present at concentrations of up to 14 mumol/g wet muscle in adult pigs. It is proposed that balenine, like carnosine and anserine, should be regarded as a normal constituent of muscle.     

Gender-related differences in carnosine,anserine and lysine content of murine skeletal muscle

Summary. The aminoacyl-imidazole dipeptides carnosine (-alanyl-L-histidine) and anserine (-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.     

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.     

Effects of carnosine and anserine on muscle and non-muscle phosphorylases

Rabbit muscle phosphorylases a and b are activated by carnosine, whereas potato and yeast phosphorylases are inhibited at the same concentration of dipeptide. Rabbit muscle phosphorylase a is activated by anserine whereas the b form enzyme and the potato and yeast enzymes are inhibited by the dipeptide. The dipeptides affect the Vmax values for the enzymes rather than the substrate Km values. Kinetic analysis suggested that, for rabbit muscle phosphorylase, both dipeptides compete for occupancy of the same binding site(s) on the enzyme.     

Carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans

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.     

The effect of reserpine, chlorpromazine and neumbutal on levels of dipeptides in rat brain and muscle

Rat brain levels of histidine, carnosine, and homocarnosine were determined after intraperitoneal injection of chlorpromazine (CPZ), sodium pentobarbital (PB), or reserpine (RSP). At the same time, rat muscle levels of histidine, carnosine, and anserine were determined. RSP, CPZ, and PB significantly lowered brain homocarnosine levels and RSP raised histidine levels. RSP, CPZ, and PB significantly lowered levels of muscle carnosine and anserine. PB and CPZ also lowered levels of muscle histidine.     

Histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles.

1. In rat skeletal muscles (longissimus dorsi and quadriceps femoris), carnosine and anserine levels decreased 35-50% during senescence, and were 35-45% lower in hypertensive rats compared to normotensive levels. 2. In rat left ventricular cardiac muscle, although no free carnosine and anserine were detected, the total level of histidine dipeptides declined 22% during senescence and in hypertensive animals decreased 35% compared to normotensive levels. 3. The significance of these changes in relation to the possible antioxidant roles of histidine dipeptides in muscle is discussed.     

Homocarnosinosis: Hypercarnosinuria

Abstract: Homocarnosine (γ-aminobutyrylhistidine) is a brain-specific dipeptide. Homocarnosinosis is a familial metabolic disorder in which spastic paraplegia, progressive mental deficiency, and retinal pigmentation coexist with increased CSF homocarnosine levels, i.e. approximately 20 times higher than the mean control level. In the present study, the urinary excretion of carnosine ( β -alanylhistidine) and anserine ( β -alanyl-1-methylhistidine) was determined in patients with homocarnosinosis and in their close relatives. Both the patients and their relatives were also loaded with chicken meat, which is rich in anserine and carnosine. The results were compared with those obtained in childhood hypercarnosinuria with serum carnosinase deficiency. Somewhat surprisingly, patients with homocarnosinosis were also shown to have hypercarnosinuria. Chicken meat loading in healthy individuals results in increased excretion of carnosine and anserine and, in addition, 1-methylhistidine in the urine. Homocarnosinosis patients and patients with serum carnosinase deficiency also showed an increased excretion of carnosine and anserine, but 1-methylhistidine was not detected in serum carnosinase deficiency, and it was present only in very small amounts in homocarnosinosis. This defect seems to be due to lack of the hydrolyzing enzyme activity. The biochemical and clinical similarities between adult homocarnosinosis and infantile serum carnosinase deficiency are intriguing. A complete insight into the relationship between them must await further investigation.     

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.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Changes in carnosine levels in muscles working in different regimens of stimulation

Carnosine content in muscles functioning under single or tetanic (both direct and indirect) contractions, in the period of active contractility (within the first 10 min of experiment) and in fatigued muscles was determined. In exercising muscle, carnosine content was shown to decrease. The loss of the dipeptide during active contractions was, on the average, 10.5%; that at fatigue--13.8%. At exercise (single contractions), the decrease of carnosine was higher than in the muscles functioning in a short tetanus regime. It was shown that the previously described phenomenon of fatigue elimination by carnosine addition to the Ringer solution washing the muscle is concomitant with the elevation (by 12%) of the intramuscular concentration of exogenous carnosine.     

Specificity and distribution of mammalian carnosinase.

Hog kidney carnosinase (EC 3.4.13.3) was found to have a narrow specificity; it hydrolyzed carnosine, anserine and glycyl-L-histidine, but did not split L-alanyl-L-histidine or homocarnosine. The isoelectric point of this enzyme was 5.8 and its molecular weight was about 84 000. Carnosinase was found to be widely distributed in various tissues of the rat. Uterus, kidney, liver and lung contained high levels of carnosinase, whereas moderate concentrations were found in spleen, heart and brain, with low levels in small intestine, skeletal muscle and stomach, and none in blood.     

Interorgan transport and catabolism of carnosine and anserine in rainbow trout.

1. After large amounts of carnosine or anserine were injected into rainbow trout white muscle, they were promptly washed out into blood and incorporated mainly into kidney. 2. These dipeptides were transported only a little to the other portions of white muscle but significantly to red muscle. 3. After anserine administration, pi-methyl-L-histidine, a constituent of anserine, increased largely in the kidney, followed by liver and muscles. 4. Histidine, a decomposed product of carnosine, increased in muscles after carnosine administration prior to the increase in kidney and liver.     

Vascular smooth muscle actions of carnosine as its zinc complex are mediated by histamine H(1) and H(2) receptors

The endogenous dipeptide carnosine (beta-alanyl-L-histidine), at 0.1-10 mM, can provoke sustained contractures n rabbit saphenous vein rings with greater efficacy than noradrenaline. The effects are specific; anserine and homocarnosine are ineffective, as are carnosine's constituent amino acids histidine and beta-alanine. Zinc ions enhance the maximum carnosine-induced tension (to 127 +/- 13% of control at 10 microM Zn(total)) and muscle sensitivity is potentiated (mean K(0.5) reduced from 1.23 mM to 17 microM carnosine with 15 microM Zn(total)). The dipeptide acts as a Zn-carnosine complex (Zn. Carn). The effects of carnosine at 1 microM-10 mM (total) in the presence of 1-100 microM Zn(2+) (total) can be described as a unique function of [Zn.Carn] with an apparent K(0.5) for the complex of [7.4)(10(-8)] M. Contractures are reduced at low [Ca(2+)], unaffected by adrenoceptor antagonists, but can be blocked by antagonists to several receptor types. The most specific effect is by mepyramine, the H(1) receptor antagonist. With Zn present, carnosine can inhibit the H(1)-specific binding of [(3)H]mepyramine to isolated Guinea pig cerebella membranes. This effect of carnosine can be described as a function of the concentration of Zn.Carn with an apparent IC(50) of 2.45 microM. Like histamine, carnosine evoked an H2-mediated (cimetidine-sensitive) relaxation in the presence of mepyramine, but was less potent (10.8 +/- 3.1% of initial tension remaining at 10 mM carnosine compared with 13.4 +/- 7.5% remaining at 0.1 mM histamine). Preliminary studies with a Zn-selective fluorescent probe indicate that functionally significant levels of Zn can be released from adventitial mast cells that could modulate actions of carnosine in the extravascular space as well as those of histamine itself. We conclude that carnosine can act at the smooth muscle H(1)-receptor to provoke vasoconstriction and that it also has the potential to act at H(1)-receptors in the central nervous system. Carnosine's mode of action is virtually unique: a vascular muscle receptor apparently transduces the action of a dipeptide in the form of a metal chelate. The functional relationship of carnosine with histamine and the possible physiological relevance of Zn ions for the activity of both agents have not previously been reported.     

Hydrolysis of carnosine and related compounds by mammalian carnosinases

Comparative study of hydrolysis of carnosine and a number of its natural derivatives by human serum and rat kidney carnosinase was carried out. The rate of carnosine hydrolysis was 3–4-fold higher then for anserine and ophidine. The rate of homocarnosine, N-acetylcarnosine and carcinine hydrolysis was negligible by either of the enzymes used. Our data show that methylation, decarboxylation or acetylation of carnosine increases resistance of the molecule toward enzymatic hydrolysis. Thus, metabolic modification of carnosine may increase its half-life in the tissues.     

Interactions among carnosine,anserine, ophidine and copper in biochemical adaptation

Recent findings indicate that carnosine, anserine and ophidine should chelate copper in the tissues where these dipeptides are present in high concentration. The observations that carnosine, anserine and ophidine are located in skeletal muscles exhibiting active oxidative metabolism and/or glycolysis and that their accumulation appears to occur ontogenetically at the same time as these tissues begin to function suggests that these dipeptides may be involved in the intracellular transport of copper for activation of cytochrome oxidase at the end of the electron transport chain and in the regulation of anaerobic glycolysis. This hypothesis provides explanations for the presence of ophidine in the skeletal muscle of whale, the presence of anserine in the flight muscles of birds, the regulatory mechanism that permits orderly replacement of the primary olfactory neuron within the nasal olfactory epithelium, and the high activity of carnosinase in the uterus.     

Carnosine Synthesis in Olfactory Tissue During Ontogeny: Effect of Exogenous β-Alanine

Carnosine has now been demonstrated by chemical analysis to be present in rat olfactory mucosa on day 16 of gestation. The tissue content of this dipeptide then increases progressively during fetal and postnatal life. Radioactive carnosine can be isolated from cultured embryonic rat olfactory mucosa incubated with [14C]beta-alanine as early as 13-14 days of gestation. The amount of incorporation also increases progressively with the initial age of the explant and with time in culture indicating in vitro maturation of the carnosine synthesis capability of olfactory tissue. To test whether the level of beta-alanine was limiting the synthesis of carnosine, we evaluated the effect of elevated beta-alanine levels on tissue carnosine content. Exogenous beta-alanine caused an increase in the tissue content of carnosine at several ages in vivo and in vitro. In adult animals this increase was observed in olfactory bulb, olfactory mucosa, and skeletal muscle. However, there was no associated alteration in carnosine synthetase activity. In addition, the different half-lives of carnosine in olfactory tissue and muscle seemed unaltered, arguing against any effect on degradative enzymes. Thus, tissue carnosine levels are regulated, at least in part, by substrate availability. The early appearance of carnosine synthetic capacity during prenatal development indicates that this enzyme activity should be a valuable aid in studying early events in olfactory neuron maturation.     

Imidazole dipeptides can quench toxic 4‐oxo‐2(E)‐nonenal: Molecular mechanism and mass spectrometric characterization of the reaction products

Imidazole dipeptides, such as carnosine (β‐alanyl‐l ‐histidine) and anserine (β‐alanyl‐N^π‐methyl‐l ‐histidine), are highly localized in excitable tissues, including skeletal muscle and nervous tissue, and play important roles such as scavenging reactive oxygen species and quenching reactive aldehydes. We have demonstrated several reactions between imidazole dipeptides (namely, carnosine, and anserine) and a lipid peroxide‐derived reactive aldehyde 4‐oxo‐2(E)‐nonenal. Seven carnosine adducts and two anserine adducts were characterized using liquid chromatography/electrospray ionization‐multiple‐stage mass spectrometry. Adduct formation occurred between imidazole dipeptides and 4‐oxo‐2(E)‐nonenal mainly through Michael addition, Schiff base formation, and/or Paal‐Knorr reaction. The reactions were much more complicated than the reaction with a similar lipid peroxide‐derived reactive aldehyde, 4‐hydroxy‐2(E)‐nonenal.     

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 compounds protect the double-chain DNA from oxidative damages

Carnosine and its derivatives in the concentrations corresponding to their level in excitable tissues have been shown to protect DNA from oxidative damages. Their efficiency (5 mM) was of the following order: ophidine > carnosine ≈ anserine > homocarnosine > N-acetylcarnosine. β-Alanine and gamma-aminobutyric acid (GABA) did not have any capability for protection. The revealed effect can be one of the causes of oxidative stability of the brain and muscle tissue in vertebrate animals.