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.     

Monoclonal Antibodies to Mammalian Carnosine Synthetase

A set of mouse monoclonal antibodies has been generated against rabbit muscle carnosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross-reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low-abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.     

beta-Alanine supplementation augments muscle carnosine content and attenuates fatigue during repeated isokinetic contraction bouts in trained sprinters.

Carnosine (beta-alanyl-l-histidine) is present in high concentrations in human skeletal muscle. The ingestion of beta-alanine, the rate-limiting precursor of carnosine, has been shown to elevate the muscle carnosine content. We aimed to investigate, using proton magnetic resonance spectroscopy (proton MRS), whether oral supplementation with beta-alanine during 4 wk would elevate the calf muscle carnosine content and affect exercise performance in 400-m sprint-trained competitive athletes. Fifteen male athletes participated in a placebo-controlled, double-blind study and were supplemented orally for 4 wk with either 4.8 g/day beta-alanine or placebo. Muscle carnosine concentration was quantified in soleus and gastrocnemius by proton MRS. Performance was evaluated by isokinetic testing during five bouts of 30 maximal voluntary knee extensions, by endurance during isometric contraction at 45% maximal voluntary contraction, and by the indoor 400-m running time. beta-Alanine supplementation significantly increased the carnosine content in both the soleus (+47%) and gastrocnemius (+37%). In placebo, carnosine remained stable in soleus, while a small and significant increase of +16% occurred in gastrocnemius. Dynamic knee extension torque during the fourth and fifth bout was significantly improved with beta-alanine but not with placebo. Isometric endurance and 400-m race time were not affected by treatment. In conclusion, 1) proton MRS can be used to noninvasively quantify human muscle carnosine content; 2) muscle carnosine is increased by oral beta-alanine supplementation in sprint-trained athletes; 3) carnosine loading slightly but significantly attenuated fatigue in repeated bouts of exhaustive dynamic contractions; and 4) the increase in muscle carnosine did not improve isometric endurance or 400-m race time.     

Biosynthesis, release, and uptake of carnosine in primary cultures

Biosynthesis, release, and uptake of carnosine (beta-alanyl-L-histidine) in highly enriched primary cell cultures of skeletal muscle and CNS tissue have been investigated. The synthesis is restricted to muscle cells, oligodendrocytes, and ensheathing cells of olfactory bulb and increases during differentiation of these cells. Astrocytes, in contrast, do not synthesize carnosine but are equipped with a dipeptide transporter by which carnosine is taken up very efficiently.     

Beta-alanine (Carnosyn?) supplementation in elderly subjects (60–80?years): effects on muscle carnosine content and physical capacity

The aim of this study was to investigate the effects of beta-alanine supplementation on exercise capacity and the muscle carnosine content in elderly subjects. Eighteen healthy elderly subjects (60-80 years, 10 female and 4 male) were randomly assigned to receive either beta-alanine (BA, n=12) or placebo (PL, n=6) for 12 weeks. The BA group received 3.2 g of beta-alanine per day (2×800 mg sustained-release Carnosyn? tablets, given 2 times per day). The PL group received 2× (2×800 mg) of a matched placebo. At baseline (PRE) and after 12 weeks (POST-12) of supplementation, assessments were made of the muscle carnosine content, anaerobic exercise capacity, muscle function, quality of life, physical activity and food intake. A significant increase in the muscle carnosine content of the gastrocnemius muscle was shown in the BA group (+85.4%) when compared with the PL group (+7.2%) (p=0.004; ES: 1.21). The time-to-exhaustion in the constant-load submaximal test (i.e., TLIM) was significantly improved (p=0.05; ES: 1.71) in the BA group (+36.5%) versus the PL group (+8.6%). Similarly, time-to-exhaustion in the incremental test was also significantly increased (p=0.04; ES 1.03) following beta-alanine supplementation (+12.2%) when compared with placebo (+0.1%). Significant positive correlations were also shown between the relative change in the muscle carnosine content and the relative change in the time-to-exhaustion in the TLIM test (r=0.62; p=0.01) and in the incremental test (r=0.48; p=0.02). In summary, the current data indicate for the first time, that beta-alanine supplementation is effective in increasing the muscle carnosine content in healthy elderly subjects, with subsequent improvement in their exercise capacity.     

Mise en Evidence de la Carcinine Synthétase, une Nouvelle Enzyme Catalysant le Métabolisme de l''Histamine dans le Systeme Nerveux Central de Carcinus maenas

Carcinine biosynthesis was induced in vitro from its two components, beta-alanine and histamine. The reaction was catalyzed by muscle, heart, and CNS extracts from Carcinus maenas. The specific activity of the enzyme, carcinine synthetase, was 15 times higher in CNS than in other organs. Only CNS extracts induced biosynthesis of carcinine from histidine, and only in the presence of pyridoxal-5'-phosphate. Hence the seat of carcinine biosynthesis seems to be the CNS. It is highly probable that in the CNS, histidine is transformed into histamine, which is then catabolized into carcinine. The latter would then be transported and accumulated in the cardiac tissue. Thus histamine--the metabolism of which takes place totally within the CNS--would be implicated as a participant in the neuronal activity of Carcinus maenas. Carcinine synthetase is a soluble enzyme that requires the presence of ATP, beta-alanine, and histamine. Mg2+ and dithiothreitol are also essential for activity. Optimum pH is approximately 7.6. Carcinine synthetase differs from carnosine synthetase and gamma-glutamylhistamine synthetase in that it does not catalyze synthesis of beta-alanylhistidine or gamma-glutamylhistamine.     

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.     

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.     

Existence of carcinine, a histamine-related compound, in mammalian tissues

Carcinine (beta-alanylhistamine) was synthesized in vitro from histamine and beta-alanine. It was detected quantitatively using an HPLC method previously described for the quantification of the related compounds histamine, histidine, carnosine and 3-methylhistamine. Carcinine was identified in several tissue of the rat, guinea pig, mouse and human, and was then shown to be metabolically related in vivo to histamine, histidine, carnosine and 3-methylhistamine through radioisotopic labeling. The results demonstrate that carcinine may be concurrently quantitated using the same HPLC method as that used to measure histamine, histidine, carnosine and 3-methylhistamine. These findings suggest a role for carcinine in the carnosine-histidine-histamine metabolic pathway and in the mammalian physiologic response to stress.     

Biosynthesis of carcinine (beta-alanyl-histamine) in vivo

Carcinine was biosynthesized by Carcinus maenas from [14C]beta-alanine, [14C] histidine and [14C] histamine. Since carnosine (beta-alanyl-histidine) could not be detected in crab tissues, biosynthesis of carcinine could only be effected by direct coupling of beta-alanine and histamine resulting from histidine decarboxylation. Biosynthesis of carcinine was weak when [14C]beta-alanine and [14C] histidine were used as precursors. On the contrary when [14C] histamine was used, synthesis was important. Thus carcinine appears to be a product of histamine catabolism. After injecting [14C] histamine, radioactive carcinine was concentrated mainly in the heart and nervous system; nonmetabolized [14C] histamine was recovered mainly in the latter. The nervous system might therefore be the seat of carcinine biosynthesis and thus the site of action of histamine.     

Carnosine and anserine in working muscles--study using proton NMR spectroscopy]

NMR spectroscopy was used to study carnosine and anserine metabolism in rat tissues under intensive muscle loading. Muscle loading was accompanied by the dipeptide (predominantly anserine) accumulation in muscle tissues. Preliminary per os administration of carnosine (250 mg/kg of body mass) did not increase the dipeptide content in muscle tissues but diminished the lactate content in rat muscles under intensive muscle loading.     

Vegetarianism,female gender and increasing age,but not <Emphasis Type="Italic">CNDP1</Emphasis> genotype,are associated with reduced muscle carnosine levels in humans

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects’ age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.     

Development of cholinergic neurons of the rat retina

Biochemical and electrophysiological features of cholinergic neurons from the developing rat retina were analyzed in an attempt to identify important periods in the maturation of these neurons. Acetylcholine synthesis was assayed in intact, isolated retinas; choline acetyltransferase activity was measured in homogenates. Spontaneous and evoked acetylcholine release and certain aspects of synapse formation were examined in a retina-muscle cell culture system. In this system, retinal cells from rats of different ages were dissociated with trypsin and added to previously cultured rat striated muscle cells which served as postsynaptic targets for cholinergic neurons. The results indicate that two developmental periods can be described. The first stage occurs during the final week of gestation well before morphological signs of synapses appear. During this early period, cholinergic neurons acquire the ability to synthesize and release acetylcholine and to form functional synapses. These developing neurons progressively lose their ability to form and to maintain synapses with an inappropriate target. A second stage of maturation begins at the end of the first postnatal week. In this phase, dramatic increases in acetylcholine synthesis, choline acetyltransferase activity, and high-affinity choline uptake are coupled temporally with the morphological differentiation of synapses.     

Axoplasmic transport of carnosine (β-alanyl-L-histidine) in the mouse olfactory pathway

Carnosine in the chemoreceptor neurons of the olfactory epithelium can be labeled in vivo by intranasal irrigation with either¹⁴C--alanine or¹⁴C-L-histidine. This newly synthesized carnosine (but not the precursor amino acids) is translocated to the olfactory bulb, where the olfactory chemoreceptor axons synapse with the dendrites of mitral cells and other second-order neurons. Labeled carnosine arrives in the bulb several hours after intranasal administration of precursor. Similar arrival time is seen for macromolecules after intranasal administration of [³H]L-fucose, [¹⁴C]L-proline, or [¹⁴C]L-histidine. Macromolecules labeled with [³H]uridine take much longer to reach the bulb. Carnosine is also labeled after [³H]uridine administration. No labeling of macromolecules is observed after administration of 1-[¹⁴C]--alanine. Oral administration of the same dose of [¹⁴C]--alanine gives almost no labeled carnosine in bulb or epithelium. This method has permitted us to estimate that the half-life of labeled carnosine in both the bulb and epithelium is about 20 h. This method provides a means of selectively prelabeling the olfactory chemoreceptor neurons in the olfactory epithelium and their synapses in the olfactory bulb prior to cellular and subcellular separation procedures, and may also enable us to monitor the influences of olfactory stimulation on synthesis and transport of carnosine.     

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.     

Enzymatic and immunological evidence for two forms of carnosinase in the mouse.

Carnosinase (aminoacyl-L-histidine hydrolase, EC 3.4.13.3) hydrolyzes the dipeptide carnosine (beta-alanyl-L-histidine), which is thought to play a role in cerebral and skeletal muscular function and has been implicated as a neuroaffector in the olfactory bulb. Carnosinase activity is present in many tissues of the mouse including heart, liver and lung, but it is most active in kidney, uterus and nasal olfactory mucosa. Kinetic measurements with 1H-NMR spectroscopy indicate that the enzyme is stereospecific and can hydrolyze L-but not D-carnosine. Anserine is a poorer substrate, while homocarnosine is essentially a non-substrate. However, these two dipeptides are effective inhibitors of the hydrolysis of L-carnosine. Carnosinase activity is unaffected when assayed in 2H2O at 99% isotopic purity. From considerations of the effect of Mn2+ on (1) substrate concentration velocity curves; (2) thermostability, and (3) inhibitor behavior, tissues with carnosinase can be divided into two groups. Kidney, uterus and olfactory mucosa represent one group, while central nervous system, muscle, spleen, etc. represent the second. The validity of this classification is confirmed by immunological evidence. Antiserum prepared against carnosinase purified from kidney cross-reacts with and inhibits the activity of olfactory mucosa, kidney and uterus but not that from central nervous system, heart or liver.     

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.     

Extrahepatic sites of metabolism of carbon tetrachloride in rats

Rats were injected i.v. and i.p. with [14C]carbon tetrachloride and the localization and binding of metabolites in the tissues were studied by autoradiography. Based on the autoradiographic findings, various tissues were tested for their capacity to form 14CO2 from [14C]carbon tetrachloride in vitro. Autoradiography in vitro was used to localize the sites of [14C]carbon tetrachloride metabolism under in vitro conditions. The results showed that several tissues accumulating metabolites in vivo had an ability to form 14CO2 in vitro, and accumulation of metabolites was observed also under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. The structures identified to have a marked carbon tetrachloride-metabolizing capacity were, besides the liver, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, the oesophageal mucosa, the mucosa of the larynx, the tongue and the cheeks, the lateral nasal gland and the kidney cortex. It is well established that the degradation of carbon tetrachloride involves the cytochrome P-450 system, and the metabolism of the substance in the mentioned tissues is probably correlated to high concentrations of cytochrome P-450. The nasal olfactory mucosa was found to be the tissue with the highest capacity to form 14CO2 from the [14C]carbon tetrachloride and microautoradiography indicated that in this tissue the cells of the subepithelial glands of the lamina propria mucosae are most actively engaged in the metabolism. It was also shown that cytochrome P-450 is present in the nasal olfactory mucosa.     

PURIFICATION AND CHARACTERIZATION OF CARNOSINE SYNTHETASE FROM MOUSE OLFACTORY BULBS

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 K_i values which were at least 40 times the K_m 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 K_i values for 1–methyl histidine than the K_m value for histidine. In contrast, carnosine-anserine synthetase from chicken pectoral muscle gave K_m 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.