The effect of 4 weeks β-alanine supplementation and isokinetic training on carnosine concentrations in type I and II human skeletal muscle fibres

Seven male students were supplemented with β-alanine (β-ALG) for 4 weeks (6.4 g day⁻¹) and seven with a matching placebo (PLG). Subjects undertook 4 weeks of isokinetic training with the right leg (T) whilst the left leg was untrained (UT), serving as a control. Each training session consisted of 10 × 10 maximal 90° extension and flexion contractions at 180°/s using a Kin-Com isokinetic dynamometer, with 1 min rest between bouts. Muscle biopsies were taken from the vastus lateralis immediately before and at the end of the supplementation period. Following freeze drying muscle fibres were dissected and characterised by their MHC profile, as type I, IIa, IIx, or as hybrids of these. Carnosine was measured by HPLC. There was a significant increase in carnosine in both T and UT legs of the β-ALG (9.63 ± 3.92 mmol kg⁻¹ dry muscle and 6.55 ± 2.36 mmol kg⁻¹ dry muscle respectively). There was a significant increase in the carnosine content of all fibre phentotypes, with no significant difference between types. There were no significant differences in the changes in muscle or in fibres between the T and UT legs. In contrast there was no significant change in the carnosine content in either the T or UT legs with placebo. The results indicate that 4 weeks training has no effect on the muscle carnosine content. Whilst an increase was seen with β-alanine supplementation, this was not further influenced by training. These findings suggest that β-alanine availability is the main factor regulating muscle carnosine synthesis.     

Inhibition of 6-hydroxydopamine-induced endoplasmic reticulum stress by l-carnosine in SH-SY5Y cells

Conditions that cause endoplasmic reticulum malfunction (ER stress) play a key role in the development of various human diseases including neurodegenerative diseases. Carnosine is an endogenous peptide, present in excitable tissues such as brain and skeletal muscle. Although there are reports suggesting that carnosine has a biological role independent of its antioxidant activity, there have been no reports of the effects of carnosine on the ER stress response. We investigated the effects of carnosine on 6-hydroxydopamine (6-OHDA)-induced cell death and ER stress in SH-SY5Y cells. After assessing control cell viability in serum-free conditions for 24 h (100% viability), we found that 50 μM 6-OHDA reduced cell viability to 76.4% of control values, whereas addition of 10 mM carnosine significantly reduced cell death to 96.1% viability in a dose-dependent manner. Consistent with its cytoprotective action, carnosine markedly inhibited subsequent ER stress responses, including phosphorylation of eukaryotic initiation factor 2alpha (eIF2α) and c-jun, expression of glucose regulatory protein 78 and C/EBP homologous protein, and mRNA splicing of X-box protein 1. The measurement of reactive oxygen species (ROS) generation by 6-OHDA showed that addition of 10 mM carnosine slightly but obviously inhibits the 6-OHDA-induced ROS production. In conclusion, our results show that carnosine almost completely inhibits 6-OHDA-induced ER stress responses and cytotoxicity, and that slight antioxidant activity of carnosine against 6-OHDA is observed. Further in vivo studies are needed to investigate clinical uses for carnosine.     

Hand-head coordination changes from discrete to reciprocal hand movements for various difficulty settings

l-Carnosine (β-alanyl-l-histidine), a dipeptide of the amino acids β-alanine and histidine, is found in mammalian tissues including those in the central nervous system and in skeletal muscles. In the present study, we examined the effects of intraduodenal (ID) injection of l-carnosine on splenic sympathetic nerve activity (splenic-SNA) in urethane-anesthetized rats and found that ID injection of 3.3 mg/kg of body weight of l-carnosine significantly suppressed splenic-SNA. Since it has been suggested that splenic-SNA reduction increases natural killer (NK) activity of splenic cells, which in turn elevates tumor immunity, we then investigated the effect of l-carnosine on the proliferation of human colon cancer cells transplanted into athymic nude mice. The findings of this study revealed that 1 mg/mL of l-carnosine solution given as the only drinking water inhibited tumor proliferation. These results suggest that l-carnosine suppresses splenic-SNA and inhibits cancer cell proliferation, probably by elevating NK activity.     

Carnosine,taurine and enzyme activities of human skeletal muscle fibres from elderly subjects with osteoarthritis and young moderately active subjects

Ageing is associated with a reduction in muscle carnosine (β-alanyl-l-histidine), but there are no data on the changes specifically in type I and type II muscle fibres. Given the higher carnosine content of type II fibers, changes observed in whole muscle may be secondary to a shift in fibre composition. Carnosine, β-alanine, histidine, taurine, and citrate synthase (CS) and glycogen phosphorylase (Phos), were measured in pools of single muscle fibres from freeze-dried muscle biopsies of vastus lateralis of nine elderly sedentary subjects (65–80 years) with osteoarthritis of the knee and undergoing total knee replacement, and nine young moderately active healthy subjects (20–35 years). Fibres were characterised as type I or II by myosin ATPase activity. Carnosine was 53.2% lower in type II fibres of older subjects resulting in an estimated 7% (and most probably still higher) decline in intracellular physico-chemical buffering capacity. Younger subjects showed higher CS activities in type I and higher Phos activities in type II fibres. These differences were less apparent in elderly subjects. Possible causes for the change in the carnosine content are reduced physical activity, reduced meat intake, or the result of progressive denervation.     

Administration of carnosine in the treatment of acute spinal cord injury

l-Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and l-histidine. It acts as a free radical scavenger and possesses antioxidant properties. l-Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha in different experimental settings. In the present study, we investigated the efficacy of l and d-carnosine on the animal model of spinal cord injury (SCI). The spinal cord was exposed via a four-level T5–T8 laminectomy and SCI was produced by extradural compression of the spinal cord at level T6–T7 using an aneurysm clip with a closing force of 24 g. Treatment with d-carnosine (150 mg/kg administered i.p., 1 h and 6 h, after SCI), but not l-carnosine significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation, inducible NO synthase (iNOS) and Hsp70 expression, (d) proinflammatory cytokines, and (e) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Furthermore, d-carnosine (150 mg/kg administered i.p., 1 h and 6 h, after SCI) significantly ameliorated the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate the strong difference between l-carnosine and d-carnosine. The result strongly suggests that d-carnosine treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

高效毛细管电泳法测定肌肽含量方法的研究

目的建立肌肽原料药的含量测定和杂质检查方法,为其质量控制提供快速、有效的分析手段。方法色谱柱为河北永年光导纤维厂的未涂层石英毛细管（75 m I.D.×65 cm）,流动相：100 mmol.L-1 Na2B4O7-H3BO3缓冲溶液,重力进样,检测波长为200 nm。考察了不同流动相下药物的色谱峰,同时进行了专属性、线性关系、精密度等项的考察。结果肌肽在在40.0～100.0 g.mL-1范围内线性关系良好（r=0.9991）,RSD为1.2%,最低检测限为2 ng。相关杂质、中间体与肌肽分离良好。结论该法专属性强,结果准确,重现性好,可用作肌肽含量测定和相关物质控制的方法。     

Electrophysiological evidence of biphasic action of carnosine on long-term potentiation in urethane-anesthetized rats

Carnosine is a dipeptide synthesized by the carnosine synthetase from β-alanine and l-histidine. The well-known effects of carnosine may be related with mechanisms producing long-term potentiation which is one of the electrophysiological signs of memory. In the present study we aimed to investigate the effect of four different doses of carnosine on long-term potentiation in urethane-anesthetized rat. A bipolar stimulating electrode was placed in the medial perforant path and a double-barrel glass micropipette was placed in the dentate gyrus as the recording electrode. Artificial cerebrospinal fluid (in the control group) or carnosine (0.1, 1, 10, and 100 μg/μL) was infused into the dentate gyrus. Our results showed that the I/O curve of the excitatory postsynaptic potential slope or population spike amplitude was not significantly shifted by carnosine. We found that population spike amplitude increased to 244% and 287% at the dose of 100 μg/μL in the post-tetanic and induction phases, respectively, but decreased to 163% and 186% at the dose of 0.1 μg/μL and to 145% and 162% at the dose of 1 μg/μL when compared with 203% and 232% of the control values. However, there were no significant differences for the slope of excitatory postsynaptic potential. Carnosine had no effect on the EPSP slope or PS amplitude recorded from the dentate gyrus in response to test stimuli when high-frequency stimulation was not delivered. In the present study, we speculated that the effects of carnosine in lower or higher doses could be explained by its effect on different processes, such as soluble guanylyl cyclase inhibition or the conversion of carnosine into histamine.     

蛋白质氧化羰基化和肌肽的保护作用

目的 探讨肌肤的抗氧化、抗衰老作用的途径和机制。方法用H2O2诱导小鼠新鲜脑组织使其产生氧化碳基化蛋白，用DNPH法和定磷法检测碳基化蛋白的含量和氧化磷酸化水平。结果 在加入肌肤的保护组，碳基化蛋白含量低于损伤组，氧化磷酸化水平高于损伤组，各组差异明显。结论 肌肤对蛋白质氧化碳基化有明显的保护作用。     

On the enigma of carnosine’s anti-ageing actions

Carnosine (β-alanyl-l-histidine) has described as a forgotten and enigmatic dipeptide. Carnosine’s enigma is particularly exemplified by its apparent anti-ageing actions; it suppresses cultured human fibroblast senescence and delays ageing in senescence-accelerated mice and Drosophila, but the mechanisms reponsible remain uncertain. In addition to carnosine’s well-documented anti-oxidant, anti-glycating, aldehyde-scavenging and toxic metal-ion chelating properties, its ability to influence the metabolism of altered polypeptides, whose accumulation characterises the senescent phenotype, should also be considered. When added to cultured cells, carnosine was found in a recent study to suppress phosphorylation of the translational initiation factor eIF4E resulting in decreased translation frequency of certain mRNA species. Mutations in the gene coding for eIF4E in nematodes extend organism lifespan, hence carnosine’s anti-ageing effects may be a consequence of decreased error-protein synthesis which in turn lowers formation of protein carbonyls and increases protease availability for degradation of polypeptides altered postsynthetically. Other studies have revealed carnosine-induced upregulation of stress protein expression and nitric oxide synthesis, both of which may stimulate proteasomal elimination of altered proteins. Some anti-convulsants can enhance nematode longevity and suppress the effects of a protein repair defect in mice, and as carnosine exerts anti-convulsant effects in rodents, it is speculated that the dipeptide may participate in the repair of protein isoaspartyl groups. These new observations only add to the enigma of carnosine’s real in vivo functions. More experimentation is clearly required.     

Glycation, ageing and carnosine: are carnivorous diets beneficial?

Non-enzymic protein glycosylation (glycation) plays important roles in ageing and in diabetes and its secondary complications. Dietary constituents may play important roles in accelerating or suppressing glycation. It is suggested that carnivorous diets contain a potential anti-glycating agent, carnosine (beta-alanyl-histidine), whilst vegetarians may lack intake of the dipeptide. The possible beneficial effects of carnosine and related structures on protein carbonyl stress, AGE formation, secondary diabetic complications and age-related neuropathology are discussed.