Vitamin D3 and 1,25-dihydroxyvitamin D3 stimulate the skeletal muscle-calcium mobilization in rachitic chicks

The Ca content in skeletal muscle relative to vitamin D3 intake was studied in chicks. It was found that the Ca content in rachitic chick muscle was significantly higher than normal and it decreased with vitamin D3 treatment. In 4-week-old chicks fed a vitamin D-deficient diet, the Ca content in leg muscle reached 9.86 +/- 1.07 mg/100 g wet wt, although in chicks receiving vitamin D3 in doses of 100 and 500 IU/kg diet, it was 7.80 +/- 0.72 and 6.08 +/- 0.61 mg/100 g wet wt, respectively. A single i.m. dose of 0.50 micrograms of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or vitamin D3 caused a dramatic decrease in the muscle Ca content by 3 to 6 h after the injection. A simultaneous rise in the Ca level in blood serum was observed. However, at this time the Ca binding protein content in duodenal mucosa and the stimulation of Ca absorption were negligible. These findings allow the conclusion that the vitamin D deficiency in chicks leads to a surplus Ca accumulation in skeletal muscle. The administration of vitamin D3 or its metabolites causes rapid Ca release during the first 6 h. This may be the source of the Ca level increase in blood serum. In this respect 1,25(OH)2D3 was much more effective than vitamin D3.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Concentrations and antioxidative activity of anserine and carnosine in poultry meat extracts treated with demineralization and papain

Anserine and carnosine found in animal skeletal muscle are capable of inhibiting the catalysis of lipid oxidation by heme and non-heme iron. A demineralization technique and a proteolytic enzyme (papain) were used in this research in order to reduce the levels of proxidants while maintaining high levels of anserine and camosine in poultry (chicken, duck and turkey) meat extracts. Undemineralized poultry meat extracts contained larger amounts of anserine, camosine, heme and non-heme iron (p < 0.05) than did demineralized poultry meat extracts. Both undemineralized and demineralized breast meat extracts of chicken, duck and turkey contained higher concentrations of anserine and camosine, but lower amounts of heme and non-heme iron than did thigh meat extracts. In chicken, duck and turkey meat (breast and thigh) extracts (undemineralized and demineralized), the anserine concentrations were greater (p < 0.05) than the camosine concentrations. The hydrogen-donating ability of undemineralized and demineralized chicken breast meat extracts was not significantly different (p > 0.05): however, demineralized chicken breast meat extracts showed higher (p < 0.05) ferrous chelating ability than did undemineralized meat extracts. The concentrations of anserine, camosine, heme and non-heme iron in chicken breast meat extracts increased (p < 0.05) with the addition of papain (1%) to the meat mixture before extraction. Heme and non-heme iron in the chicken breast meat extracts increased as the reaction time for papain increased from 30 to 120 min, but the concentrations of anserine and camosine were not significantly affected by the longer reaction time for papain. The hydrogen-donating ability and ferrous chelating ability of demineralized chicken breast meat extracts were not significantly affected by papain. The ratios of carnosine/anserine were very specific in the chicken, duck and turkey meat extracts (breast and thigh); and the turkey meat extracts had lower (p < 0.05) camosine/anserine ratios than did the chicken and duck meat extracts. The camosine/anserine ratios of undemineralized and demineralized poultry meat extracts were not significantly different (p > 0.05). This suggests that the carnosine/anserine ratios of undemineralized chicken (0.62 - 0.80), duck (0.75 - 0.77) and turkey (0.15 - 0.16) meat extracts could be used to estimate the single meat species in uncooked or cooked meat products.     

Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks

1. The behaviour of total acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of chick embryos between 11th and 21st day of development and of 8 and 180-day-old chicks is described. 2. Total acid soluble carnitine fluctuates around the same levels in the brain, liver and muscle until 18th day of development, whereas it attains a peak on that day in the heart. At hatching compared to 18th day, it suddenly increases three times in the muscle, drops not significantly in the heart and brain, but sharply in the liver (-40%). However the levels are always higher than those of the grown chick in the brain but lower in the other tissues. 3. Free carnitine levels are almost constant in all tissues during the embryonic life; if compared to adult ones, they are very much lower in the liver, heart and muscle, but higher in the brain, even in 8 day-old chick. 4. Short chain esterified, carnitine reaches a maximum on 18th day of egg incubation in the liver, brain and heart; in the muscle it stays on constant levels until this day and then rapidly increases so that at hatching it doubles the values. 5. The short chain esterified to free carnitine percentage ratio peaks in all tissues on 18th day of development, attaining figures which are well above those determined in the grown chick.     

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.     

The absorption of orally supplied β-alanine and its effect on muscle carnosine synthesis in human vastus lateralis

Summary. β-Alanine in blood-plasma when administered as A) histidine dipeptides (equivalent to 40 mg · kg⁻¹ bwt of β-alanine) in chicken broth, or B) 10, C) 20 and D) 40 mg · kg⁻¹ bwt β-alanine (CarnoSyn™, NAI, USA), peaked at 428 ± SE 66, 47 ± 13, 374 ± 68 and 833 ± 43 μM. Concentrations regained baseline at 2 h. Carnosine was not detected in plasma with A) although traces of this and anserine were found in urine. Loss of β-alanine in urine with B) to D) was <5%. Plasma taurine was increased by β-alanine ingestion but this did not result in any increased loss via urine. Pharmacodynamics were further investigated with 3 × B) per day given for 15 d. Dietary supplementation with I) 3.2 and II) 6.4 g · d⁻¹ β-alanine (as multiple doses of 400 or 800 mg) or III) L-carnosine (isomolar to II) for 4 w resulted in significant increases in muscle carnosine estimated at 42.1, 64.2 and 65.8%.     

Effect of starvation and sea-water acclimation on the concentration of free l-histidine and related dipeptides in the muscle of eel,rainbow trout and Japanese dace

• 1.1. During the starvation of the eel (200 days), the rainbow trout (62 days) and the Japanese dace (75 days), white muscle free l-histidine decreased rapidly in every species, while carnosine and anserine levels in the eel and trout, respectively, exhibited relatively smaller percentage changes.
• 2.2. Accompanying sea-water acclimation, l-histidine in skeletal muscle of the ell and trout increased 2- and 5-fold, respectively, but in dace muscle no significant chenge occured. The concentration of carnosine in the eel and anserine in the trout remained almost at constant levels even in sea-water.

     

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.     

Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase.

1. The brain and ocular fluid of skipjack tuna (Katsuwonus pelamis) contained high levels of N-acetylhistidine deacetylase. 2. This enzyme had a molecular weight of about 120,000 and was activated by zinc or cobaltous ions. 3. Cod (Gadus callarias) brain, ocular fluid and muscle contained a similar metal-activated thiol hydrolase, the muscle enzyme being known as anserinase. 4. The purified enzymes hydrolyzed N-acetylhistidine, carnosine, homocarnosine, anserine and certain other dipeptides. 5. Their specificity resembled that of hog kidney homocarnosinase. 6. In both fish, brain and ocular fluid were rich sources of this hydrolase, whereas muscle contained only trace amounts.     

Effect of two β-alanine dosing protocols on muscle carnosine synthesis and washout

Carnosine (β-alanyl-L-histidine) is found in high concentrations in skeletal muscle and chronic β-alanine (BA) supplementation can increase carnosine content. This placebo-controlled, double-blind study compared two different 8-week BA dosing regimens on the time course of muscle carnosine loading and 8-week washout, leading to a BA dose-response study with serial muscle carnosine assessments throughout. Thirty-one young males were randomized into three BA dosing groups: (1) high-low: 3.2 g BA/day for 4 weeks, followed by 1.6 g BA/day for 4 weeks; (2) low-low: 1.6 g BA/day for 8 weeks; and (3) placebo. Muscle carnosine in tibialis-anterior (TA) and gastrocnemius (GA) muscles was measured by 1H-MRS at weeks 0, 2, 4, 8, 12 and 16. Flushing symptoms and blood clinical chemistry were trivial in all three groups and there were no muscle carnosine changes in the placebo group. During the first 4 weeks, the increase for high-low (TA 2.04 mmol/kgww, GA 1.75 mmol/kgww) was ~twofold greater than low-low (TA 1.12 mmol/kgww, GA 0.80 mmol/kgww). 1.6 g BA/day significantly increased muscle carnosine within 2 weeks and induced continual rises in already augmented muscle carnosine stores (week 4-8, high-low regime). The dose-response showed a carnosine increase of 2.01 mmol/kgww per 100 g of consumed BA, which was only dependent upon the total accumulated BA consumed (within a daily intake range of 1.6-3.2 g BA/day). Washout rates were gradual (0.18 mmol/kgww and 0.43 mmol/kgww/week; ~2%/week). In summary, the absolute increase in muscle carnosine is only dependent upon the total BA consumed and is not dependent upon baseline muscle carnosine, the muscle type, or the daily amount of supplemented BA.     

Metabolic Transformation of Neuropeptide Carnosine Modifies Its Biological Activity

1. The ability of carnosine and carnosine-related compounds (CRCs) to interact with several free oxygen radicals is analyzed.2. Carnosine, the CRCs (imidazole, histidine, anserine), and ergothioneine were found to be equally efficient in singlet oxygen quenching. During generation of hydroxyl radicals from hydrogen peroxide in the Fenton reaction, carnosine was found to be more effective than the CRCs tested.3. By measuring the chemiluminescence produced by carnosine and CRCs in rabbit leukocytes in the presence of luminol or lucigenin, we conclude that carnosine and other CRCs play a stimulating role in superoxide oxygen production while suppressing the myeloperoxidase system.4. ADP-induced aggregation of human platelets is slightly stimulated by carnosine but is inhibited by acetylanserine.5. The following rank order of efficiency of CRCs was demonstrated while measuring the oxidation of human serum lipoproteins: acetylcarnosine < acetylanserine < homocarnosine = ophidine < carnosine < anserine.6. The results obtained demonstrate that metabolic transformation of carnosine into CRCs in tissues may play an important role in regulating the native antioxidant status of the organism.