Carnosine and Related Peptides: Therapeutic Potential in Age-Related Disorders.

Imidazole dipeptides (ID), such as carnosine (ß-alanyl-L-histidine), are compounds widely distributed in excitable tissues of vertebrates. ID are also endowed of several biochemical properties in biological tissues, including antioxidant, bivalent metal ion chelating, proton buffering, and carbonyl scavenger activities. Furthermore, remarkable biological effects have been assigned to such compounds in age-related human disorders and in patients whose activity of serum carnosinase is deficient or undetectable. Nevertheless, the precise biological role of ID is still to be unraveled. In the present review we shall discuss some evidences from clinical and basic studies for the utilization of ID as a drug therapy for age-related human disorders.

Energy metabolism is compromised in skeletal muscle of rats chronically-treated with glutaric acid.

Glutaric acidemia type I (GA I) (GA I, McKusick 23167; OMIM # 231670) is an autosomal recessive metabolic disorder caused by glutaryl-CoA dehydrogenase deficiency (EC 1.3.99.7). Clinically, the disease is characterized by macrocephaly, hypotonia, dystonia and diskinesia. Since the pathophysiology of this disorder is not yet well established, in the present investigation we determined a number of energy metabolism parameters, namely (14)CO(2) production, the activities of the respiratory chain complexes I-IV and of creatine kinase, in tissues of rats chronically exposed to glutaric acid (GA). High tissue GA concentrations (0.6 mM in the brain, 4 mM in skeletal muscle and 6 mM in plasma) were induced by three daily subcutaneous injections of saline-buffered GA (5 micromol x g(-1) body weight) to Wistar rats from the 5th to the 21st day of life. The parameters were assessed 12 h after the last GA injection in cerebral cortex and middle brain, as well as in skeletal muscle homogenates of GA-treated rats. GA administration significantly inhibited the activities of the respiratory chain complexes I-III and II and induced a significant increase of complex IV activity in skeletal muscle of rats. Furthermore, creatine kinase activity was also inhibited by GA treatment in skeletal muscle. In contrast, these measurements were not altered by GA administration in the brain structures studied. Taken together, it was demonstrated that chronic GA administration induced an impairment of energy metabolism in rat skeletal muscle probably due to a higher tissue concentration of this organic acid that may be possibly associated to the muscle weakness occurring in glutaric acidemic patients.

Inhibition of the electron transport chain and creatine kinase activity by ethylmalonic acid in human skeletal muscle.

Ethylmalonic aciduria is a common finding in patients affected by short-chain acyl-CoA dehydrogenase (SCAD) deficiency and other diseases characterized by encephalopathy, muscular symptomatology, and lactic acidemia. Considering that the pathophysiological mechanisms of these disorders are practically unknown and that lactic acidosis suggest an impairment of energy production, the objective of the present work was to investigate the in vitro effect of ethylmalonic acid (EMA), at concentrations varying from 0.25 to 5.0 mM, on important parameters of energy metabolism in human skeletal muscle, such as the activities of the respiratory chain complexes and of creatine kinase, which are responsible for most of the ATP produced and transferred inside the cell. We verified that EMA significantly inhibited the activity of complex I-III at concentrations as low as 0.25 mM, complex II-III at 1 mM and higher concentrations, and complex II at the concentration of 5 mM. In contrast, complex IV was not inhibited by the acid. Finally, we observed that the activity of creatine kinase was significantly inhibited by EMA at the concentrations of 1 and 5 mM. These results suggest that EMA compromises energy metabolism in human skeletal muscle. In case the in vitro effects detected in the present study also occur in vivo, it is tempting to speculate that they may contribute, at least in part, to explain the hypotonia/myopathy, as well as the increased concentrations of lactic acid present in the patients affected by illnesses in which EMA accumulates.

Effect of in vivo administration of ethylmalonic acid on energy metabolism in rat tissues.

High concentrations of ethylmalonic acid (EMA) occur in tissues and biological fluids of patients affected by deficiency of short-chain acyl-CoA dehydrogenase activity, as well as in other illnesses characterized by neurological and muscular symptoms. Considering that the pathophysiological mechanisms responsible for the clinical manifestations of these diseases are virtually unknown, in the present work we developed a chemical in vivo model of ethylmalonic acidemia in young Wistar rats for neurochemical and behavioral studies through subcutaneous administration of EMA to young rats. The doses of EMA administered subcutaneously varied according to the age of the animals, being injected 3, 4, and 6 micromol g(-1) of body weight in rats of 7, 14, and 21 days, respectively. The concentrations of the acid were measured in blood and brain at regular intervals after a single injection (30-120 min) and reached the highest concentrations (3.0 mM and 0.5 micromol g(-1), approximately 0.5 mM), respectively, after 30 and 60 min of EMA injection. Next, we investigated the effects of acute EMA administration on the activities of complexes I-III, II, II-III, and IV of the respiratory chain in cerebral cortex and skeletal muscle, as well as on the activity of creatine kinase in cerebral cortex, striatum, skeletal muscle, and cardiac muscle of rats of 14 days of life. Control rats were treated with saline in the same volumes. We verified EMA administration did not change these enzymatic activities in all tissues studied. Although transient high concentrations of EMA did not alter important parameters of energy metabolism, it cannot be ruled out that chronic administration of this organic acid would disrupt energy metabolism.

Differential inhibitory effects of methylmalonic acid on respiratory chain complex activities in rat tissues.

Methylmalonic acidemia is an inherited metabolic disorder biochemically characterized by tissue accumulation of methylmalonic acid (MMA) and clinically by progressive neurological deterioration and kidney failure, whose pathophysiology is so far poorly established. Previous studies have shown that MMA inhibits complex II of the respiratory chain in rat cerebral cortex, although no inhibition of complexes I-V was found in bovine heart. Therefore, in the present study we investigated the in vitro effect of 2.5mM MMA on the activity of complexes I-III, II, II-III and IV in striatum, hippocampus, heart, liver and kidney homogenates from young rats. We observed that MMA caused a significant inhibition of complex II activity in striatum and hippocampus (15-20%) at low concentrations of succinate in the medium, but not in the peripheral tissues. We also verified that the inhibitory property of MMA only occurred after exposing brain homogenates for at least 10 min with the acid, suggesting that this inhibition was mediated by indirect mechanisms. Simultaneous preincubation with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) and catalase (CAT) plus superoxide dismutase (SOD) did not prevent MMA-induced inhibition of complex II, suggesting that common reactive oxygen (superoxide, hydrogen peroxide and hydroxyl radical) and nitric (nitric oxide) species were not involved in this effect. In addition, complex II-III (20-35%) was also inhibited by MMA in all tissues tested, and complex I-III only in the kidney (53%) and liver (38%). In contrast, complex IV activity was not changed by MMA in all tissues studied. These results indicate that MMA differentially affects the activity of the respiratory chain pending on the tissues studied, being striatum and hippocampus more vulnerable to its effect. In case our in vitro data are confirmed in vivo in tissues from methylmalonic acidemic patients, it is feasible that that the present findings may be related to the pathophysiology of the tissue damage characteristic of these patients.

Glutaric acid administration impairs energy metabolism in midbrain and skeletal muscle of young rats.

A genetic mice model of glutaric acidemia type I (GAI) has recently been developed, however affected animals do not develop the striatal damage characteristic of patients with this disorder. Therefore, the initial aim of the present work was to induce high glutaric acid (GA) concentrations in rat brain similar to those found in GAI patients through subcutaneous injection of GA. High brain GA concentrations (up to 0.60 micromol/g congruent with 0.60mM) were achieved by a single subcutaneous injection of saline-buffered GA (5 micromol/g body weight) to Wistar rats of 7-22 days of life. GA brain levels were about 10-fold lower than in plasma and 5-fold lower than in skeletal and cardiac muscles, indicating that the permeability of the blood brain barrier to GA is low. We also aimed to use this model to investigate neurochemical parameters in the animals. Thus, we evaluated the effect of this model on energy metabolism parameters in midbrain, in which the striatum is localized, as well as in peripheral tissues (skeletal and cardiac muscles) of 22-day-old rats. Control rats were treated with saline in the same volumes. We verified that CO2 production from glucose was not altered in midbrain of rats treated with GA, indicating a normal functioning of the tricarboxylic acid cycle. Creatine kinase activity was also not changed in midbrain, skeletal and cardiac muscles. In contrast, complex I-III activity of the respiratory chain was inhibited in midbrain (25%), while complexes I-III (25%) and II-III (15%) activities were reduced in skeletal muscle, with no alterations found in cardiac muscle. These data indicate that GA administration moderately impairs cellular energy metabolism in midbrain and skeletal muscle of young rats.