Antioxidant activity

Transcription

1 WHAT DOES IT DO? Antioxidant activity The term antioxidant refers to the ability of numerous vitamins, minerals and other phytochemical products to protect against the harmful effects of highly reactive molecules known as free radicals. Free radicals react with numerous structures of the body and can damage them. Cell membranes and DNA, the main source of our genetic heritage, are particularly prone to oxidative damage. The reactions of free radicals and damage due to oxidation have also been related to numerous senile diseases, such as neurodegenerative disorders, heart disease, diabetes and cancer. The best known biological antioxidants that exert preventive action against the oxidation of lipids, proteins and other essential macromolecules feature only a few characteristics in their mechanisms of action and provide only one of the following types of protection: inhibition of the formation of free radicals; removal of oxidizing agents; reaction with reactive species, preventing their natural evolution; transformation of a ROS (reactive oxygen species) into an anti-ros (no longer dangerous); membrane stabilisation; indirect action against the removal of substances that can catalyse the damage caused by free radicals (e.g. metal ions); stabilisation of iron. Firstly, since its interaction with many types of free radicals, including oxygen, hydrogen peroxide, and peroxide and hydroxylic radicals, has been demonstrated, L-carnosine can be defined as a broad-spectrum antioxidant. Secondly, being an extremely water-soluble antioxidant, L-carnosine is capable of inhibiting cell membrane oxidation due to the action of iron, zinc, copper, hydrogen peroxide, the oxygen radical, and the free peroxide and hydroxylic radicals. From a chemical point of view, the L-carnosine molecule owes its antioxidant activity to the presence of the peptide bond that stabilizes the imidazole radical. The antioxidant effect of L- carnosine has been shown to be much greater than that of the single or combined activity of the amino acids that constitute it, which would indicate that the peptide bond between alanine and histidine is involved in a special way in the overall antioxidant activity of L-carnosine. It has also been shown that the metabolic transformation of carnosine into imidazole, histidine and anserine may play a major role in regulating the native antioxidant status of the human body. The gradual accumulation of microscopic damage caused by free radicals to cell membranes, DNA, tissue structure and the enzyme system leads to a progressive weakening of the body and consequently increased susceptibility to disease.

2 The protective action exerted by antioxidant molecules such as L-carnosine can therefore be particularly useful in all those physiological situations in which the body is subjected to high oxidative stress. In the case of athletes or professional sportsmen, the damage caused by oxidation can be particularly significant due to the increased production of free radicals which occurs during intense physical activity. Although in such circumstances the body increases the production of endogenous antioxidant enzymes (glutathione, anti-peroxidase, catalase, superoxide-dismutase), an external supply of antioxidants in the diet can prevent excessive oxidation in the muscles and other tissues. In theory, the absence of oxidative damage during physical activity can result in an increase in resilience and consequently enhance athletic prowess. Anti-glycosylation activity L-carnosine inhibits the formation of substances known as AGEs, advanced glycosylation end products. Non-enzymatic glycosylation (referred to as Maillard reaction in food chemistry) is the reaction of amino groups with aldehydes, sugars or keto groups, leading to the production of reactive chemical entities. All this creates cross links and the possible formation of advanced glycosylation end products. Although the process of in-vivo glycosylation is slow, it becomes considerably important during ageing and in the presence of pathological conditions characterised by high blood sugar levels (e.g. diabetes). In such cases, abnormalities may occur in various tissues, especially in connective tissue, as a result of collagen cross links. An analysis of the glycosylation of the main protein sites has shown that the epsilon amino groups of lysine are the primary target, particularly in the proximity of histidine residues. The prevalence of this sequence closely resembles that of carnosine. Carnosine can react in vitro with sugars, such as glucose, lactose and dihydroxy acetone (DHA), to produce brown solutions, typical of glycosylation, as originally described by Maillard. DHA is the most reactive of these sugars. Carnosine reacts with DHA faster than with lysine, which means that, with glycosylation, dipeptides can compete with other amino group sources. It has also been noted that minor structural changes in carnosine (e.g. the addition of a methylene group) can reduce its reactivity. L-carnosine is capable of strongly inhibiting DHA glycation of the dipeptide Ac-Lys-His-NH2. Since this latter sequence resembles the preferred glycation site in proteins, this would indicate that L-carnosine is capable of inhibiting cross-linking of bovine serum albumin resulting from DHA protein glycation. All the in-vitro results were observed at relatively high levels of carnosine ( mm), at the same level of concentration as the sugars (0.2-2 M) or protein amino groups present in the reaction mixtures. This seems to be consistent with the mechanism of action supposed for carnosine, whereby it acts as a competitive acceptor in the glycation reaction. The concentration of carnosine required to inhibit protein damage in vivo is therefore probably dependent on the levels of reactive sugars being generated. Some glycated amino acids, such as lysine and arginine, have been reported to be mutagenic in the Ames test. Other glycated amino acids, such as proline and cysteine, do not exhibit mutagenicity. The formation of AGE products is of particular significance in cases where the blood glucose levels are periodically elevated (e.g. in diabetes). AGE levels are known to be highly involved in the development of degenerative changes, including cataract formation and atherosclerosis. Obtaining reliable data in this regard requires continuous research and extended periods of treatment. Some studies compare the inhibitory action exerted by L-carnosine in relation to glycosylation and aminoguanidine, the only well-documented inhibitor.

3 L-carnosine appears, however, to intervene at a previous step in the glycation process compared to aminoguanidine, thus diverting this reaction earlier towards the formation of non-damaging and rapidly cleared products. Unlike amino guanidine, carnosine is a natural product with extremely low toxicity. Although L-carnosine has been shown to be highly effective, even following oral intake, new and more extensive tests on animals are required to demonstrate its effectiveness, especially in cases of diabetes-associated degenerative changes. Against aging and diseases typical of old age It is well known that spontaneous modification of aldose-induced proteins is one of the main causes of age-related degeneration of proteins and cross-links, and it plays an important role in some oldage diseases, such as: inflammation of the joints arteriosclerosis diabetes Alzheimer s disease By preventing the accumulation of forms of oxidized, altered or cross-linked proteins (evident molecular signals of ageing), carnosine may actually help delay the aging process and reduce DNA oxidation. Numerous interesting studies have recently confirmed the beneficial effects of L-carnosine on the growth, morphology and longevity of cultures of human fibroblasts. In these experiments, researchers determined how treatment with 50mM of carnosine can maintain a juvenile phenotype in this type of cell. The removal of L-carnosine from the culture broth caused the immediate resumption of the ageing process. The same results were obtained when using the isomer D- carnosine. A. Hipkiss and co-workers (Institute of Gerontology, London) have shown that the relationship between carnosine, toxic aldehydes, ageing and age-related diseases is an interesting subject of study, combined with the potential therapeutic action of L-carnosine and associated structures against diseases resulting from aldehyde-mediated macromolecular changes. Besides being an antioxidant and free-radical scavenger, L-carnosine also reacts with harmful aldehydes, acting as a protector of susceptible macromolecules. In-vitro experiments have shown that carnosine inhibits non-enzymatic glycosylation and protein cross-links induced by reactive aldehydes. This research, too, has shown carnosine s inhibition of the formation of advanced glycation end products (AGEs) involving proteins and induced by MDA (malondialdehyde, a product of lipid peroxidation) and the formation of DNA-protein cross-linking induced by acetaldehyde and formaldehyde. The administration of 20 mm of L-carnosine has protected the cultures of fibroblasts and human lymphocytes (CHO) and those of endothelial cells of rat brain from the toxic effects of formaldehyde, acetaldehyde and MDA in addition to AGEs formed within a mixture of lysine and deoxyribose. L-carnosine has also effectively protected the cultivation of endothelial cells of rats from the toxicity of amyloid peptide. For these reasons, L-carnosine (which is non-toxic) could be tested as an adjuvant in the treatment of diseases which involve harmful aldehydes, such as diabetes (and its secondary complications), inflammatory phenomena, alcoholic liver damage and probably Alzheimer s disease. Carnosine s potential anti-glycation property suggests that this dipeptide can be taken into

4 consideration in the treatment of diabetics, where glycation represents the first step towards severe secondary pathological effects. Homocarnosine displays a lower reactivity than carnosine and indicates that some minor structural changes affect the relative levels of glycation. This would allow the development of substances similar to carnosine that have different reaction rates at various stages of the glycation process and, together with carnosine, can be used as a tool for further investigations. In-vitro glycation of other molecules, such as tripeptides, has been reported, but their susceptibility to peptidase in vivo restricts their potential use. L-carnosine, being a dipeptide, is not easily attacked by non-specific peptidases. It has also been shown that carnosine protects proteins against methylglioxal-mediated modifications, i.e. an endogenous metabolite present in high concentrations in diabetics and involved in the formation of advanced glycation end products and diabetic complications. The contrasting action exerted by L-carnosine against atherosclerosis has been shown in some studies conducted on animals. It is also known that the antioxidant power associated with carnosine can help prevent lipid peroxidation, a phenomenon that plays an important role in the onset of atherosclerosis. Since L-carnosine is absorbed rapidly and reaches the plasma intact, its antioxidant activity may also inhibit oxidation of low density lipoprotein (LDL) and also contribute in this respect to the prevention of arteriosclerosis. The introduction of antioxidant molecules such as L-carnosine in the diet can therefore favourably influence the antioxidant ability of serum and protect against lipid peroxidation and all the problems associated with it. The anti-glycation and antioxidant properties of carnosine also contribute to its inhibiting action against Alzheimer s disease. In this regard, the protective mechanism of carnosine is exerted at a neuronal and epithelial cell level against the activity of B-amyloid peptide. This substance is involved in vascular dysfunction of the brain and is considered a primary neurotoxin in Alzheimer s disease. It inhibits the proliferation of endothelial cells and is directly toxic on the peripheral and cerebral vascular endothelium. In the brain, this can give rise to weakening of the blood-brain barrier. It has been suggested that a prolonged reduction of this type can worsen (or even generate) Alzheimer s disease via chronic disorders in the extracellular fluid homeostasis in the brain which is one of the main functions of the blood-brain barrier and via an increase in the presence of the amyloid precursor protein. Neuroprotective action The specific concentration of the endogenous peptide L-carnosine in the brain as well as muscle has had led to the formulation of various hypotheses as to its biological function in this field. It has been found that carnosine is often associated with neurons which, though totally dependent on the glucose, are more durable. This could be a consequence of the known anti-ageing and antiglycosylation action of carnosine. If carnosine underwent in-vivo glycosylation, the resulting product, due to its very non-mutagenicity, would not only inhibit the homeostatic functions that preserve protein integrity, but it would also lower the production of endogenous mutagens. Antioxidant action, which has been studied with in-vitro and in-vivo models, has been shown to protect against the damage caused to neurons by free radicals, especially the hydroxyl type. The mechanism involved is thought to include activation of Na,K-ATP-ase and reduce the activity of tyrosine-hydrolase (an enzyme normally activated by the presence of free radicals).

5 It should also be noted that L-carnosine is a water-soluble dipeptide, so it is able to capture the products of lipid peroxidation. In the process of cerebral ischemia, for instance, carnosine acts as a neuroprotector, helping improve blood supply to the brain, normalize the electroencephalogram, reduce the accumulation of lactate and protect against ROS (reactive oxygen species). L-carnosine can therefore be considered a specific regulator of the main metabolic pathways whereby neurons maintain homeostasis of the brain under favourable conditions. L-carnosine has an antioxidant that includes metal chelation. In a recent study, carnosine was proposed as a neuromodulator. Researchers used the correlation between the cell current and the voltage applied to determine the direct effects and neuromodulating action of carnosine on the neurons of rat olfactory bulb in primary cultures. Copper and zinc inhibited N-methylaspartate and the currents mediated by GABA receptors and synaptic transmission. Carnosine played a preventive role against the action of copper and reduced the effects of zinc. These results show that carnosine can indirectly influence neuronal excitability by modulating the effects of zinc and copper. L-carnosine, being able to neutralize the excess protons, can further defend nerve cells against the deleterious effects of, among other things, this type of environmental influence. Protection against alcohol-induced muscle disease Prolonged and chronic use of alcohol is associated with reduced protein synthesis in skeletal muscles. Type II skeletal muscle fibres (i.e. those with an anaerobic glycolytic metabolism) are reduced. The chronic form of alcohol-induced skeletal myopathy is characterized by selective atrophy of type II fibres and affects up to 2/3 of those who abuse the use of alcohol. The subspecies of type IIb fibres (which have few or no mitochondria) are particularly affected. The subspecies of Type IIa fibres, and especially slow-contraction type I fibres (aerobic-oxidative), are relatively more resistant. In myopathic alcoholics, L-carnosine present in the plasma and the muscle is reduced. Alcohol abusers also reveal an increase in lipid peroxidation indices. Furthermore, the indisputable presence of a high number of free radicals (i.e. unpaired electrons or oxygen-reactive species) plays an important role in the pathogenesis of alcohol-induced muscle disease. The mechanisms concerning the involvement of ROS (reactive oxygen species) damage in the aetiology of disorders due to the abuse of alcohol in non-muscle tissues have been studied frequently. ROS may also be involved in the pathogenesis of alcohol-induced myopathy and cardiomyopathy. Two interesting studies found that, in the presence of L-carnosine, the erythrocytes of alcoholics suffering withdrawal symptoms significantly increased their ability to resist acidic haemolysis. In these patients, carnosine produced beneficial effects on erythrocyte morbidity, normalizing cell morphology. In summary, L-carnosine has been reported to have a significant stabilizing and regenerative effect on the erythrocytes of alcohol abusers. For the skin Thanks to its natural antioxidant properties, L-carnosine can effectively counteract the body s natural ageing process. The skin, which is known to be one of the first organs in which the action of radical degeneration is visible, is positively influenced by the action of L-carnosine. It has also been shown that L-carnosine as port of the diet or applied topically can preserve the skin s immune defences in the presence of exposure to ultraviolet rays (UVB) or chemical agents such as urocanic acid.

6 The formation of crosslinks between adjacent collagen fibres is also known to be one of the processes that cause the characteristic appearance of wrinkles and hence loss of elasticity. This phenomenon occurs naturally with ageing and can be accelerated by exposure to sunlight. Given its antioxidant and anti-glycosylating properties, L-carnosine is likely to contribute positively in preventing the formation of cross-links in collagen and other skin proteins. Carnosine can also help in the healing of wounds, as shown by numerous studies in vivo and in vitro. Immunostimulant action The immunoregulatory effect attributed to carnosine still requires an in-depth analysis of its precise mechanism of action. According to research conducted so far, it appears to involve the activation of T and B cells and also stereo stimulation specific to peritoneal macrophages. A recent study has shown that anserine and carnosine can modulate the cell function of neutrophils and U937 cells, especially in respect of the respiratory act, the production of interleukin-1-beta and apoptosis. Both peptides, in fact, have led to increased respiratory capacity and the production of interleukin-1-beta of human neutrophils but not of U937 cells. The results obtained suggest that carnosine and anserine are able to modulate the immune response at least at the level of human neutrophils. Action on the muscles Thanks to its many properties, L-carnosine can be considered a new-generation ergogenic aid. Similar to what has been observed in brain tissue, the natural concentration of L-carnosine in muscles has numerous functional explanations, many of which are truly surprising and can with good reason guide the use of this substance in the field of sport. With regard to the muscles, the known properties of L-carnosine are beneficial at several levels. In particular, the intake of this molecule has the theoretical potential to: increase strength due to its ability to stimulate muscle contraction, by activating the enzymes responsible for producing these contractions (myofibrillar ATPase); increase the sensitivity of contractile proteins present in muscle tissue to Ca2+ ions; increase resistance to muscular effort, contrasting the effects of lactic acid (buffer action) and free radicals; protect the body from the damage caused by free radicals resulting from strenuous exercise (antioxidant); probably decrease injury recovery time. L-carnosine is an endogenous dipeptide that contributes to the skeletal muscle s antioxidant defence system. It can inhibit the oxidation of lipids in concentrations similar to those present in the skeletal muscle (5-25 mm). It has also been found that the antioxidant mechanism of carnosine is multifunctional in that it can chelate metals and eliminate free radicals. The primary role of carnosine at a muscle level is to eliminate the hydrogen ions produced during periods of rapid glycolysis, which normally lead to an accumulation of lactic acid which occurs mostly in conjunction with short, intense physical effort, such as rapid acceleration or exercise close to limit levels. In practice, L-carnosine acts as an intramuscular scavenging agent that delays the accumulation of lactic acid. It has been estimated that muscle dipeptides (mainly L-carnosine) determine 10-40% of the overall

7 buffering capacity of the muscle tissue. During intense exercise, L-carnosine can play a major role in preventing a reduction in ph caused by the accumulation of lactic acid and improving overall physical performance. Although this theory has not been backed up by sufficient clinical studies, research conducted on race horses has shown that concentrations of muscle L-carnosine are greater in muscles with a high percentage of fast-twitch glycolytic fibres and lower in muscles with a prevailing number of slowtwitch oxidative fibres. In addition to the potential effects on anaerobic metabolism (lactic acid), L- carnosine can increase oxidative (aerobic) metabolism by enhancing the mitochondria s efficiency producing cellular energy. The levels of muscle L-carnosine in athletes are known to be higher among those who practise sports with the highest anaerobic requirements (canoeists and sprinters). Athletes engaged in prolonged stress activities (marathon runners) have lower muscle concentrations of L-carnosine, though still higher than those found in untrained subjects. The intake of L-carnosine is associated with an increase in physical performance, especially with anaerobic activities. It is also an effective retardant of the sensation of muscle pain that accompanies intensive training, and it improves the speed of recovery and healing times. The muscle content of L-carnosine can be influenced by the amount introduced via the diet in terms of the dipeptide itself and the amino acids forming it (histidine in particular). The use of L-carnosine as a dietary supplement mediates the damage caused by free radicals and also helps eliminate lactic acid in the muscle tissue and blood and delay the onset of localized pain.