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Carnitine, mitochondrial function and therapy

Carnitine is important for cell function and survival primarily because of its involvement in the multiple equilibria between acylcarnitine and acyl-CoA esters established through the enzymatic activities of the family of carnitine acyltransferases. These have different acyl chain-length specificities and intracellular compartment distributions, and act in synchrony to regulate multiple aspects of metabolism, ranging from fuel-selection and -sensing, to the modulation of the signal transduction mechanisms involved in many homeostatic systems. This review aims to rationalise the extensive range of experimental and clinical data that have been obtained through the pharmacological use of L-carnitine and its short-chain acylesters, over the past two decades, in terms of the basic biochemical mechanisms involved in the effects of carnitine on the various cellular acyl-CoA pools in health and disease.

Glutathione peroxidase, glutathione reductase, glutathione S-transferase, and γ-glutamyltranspeptidase activities in the human early pregnancy placenta

GSH peroxidase, GSSG reductase, GSH S-transferase, and gamma-glutamyltranspeptidase activities were measured in the supernatant of 13 human early pregnancy placenta homogenates. From measurements of GSH peroxidase activity with both H2O2 and cumene hydroperoxide as second substrate it was deduced that immature placenta contains only the Se-dependent form. All the specimens investigated exhibited GSSG reductase and gamma-glutamyltranspeptidase activities. GSH S-transferase activity was noted only using 1-chloro-2,4-dinitrobenzene as electrophilic substrate, while no detectable activity was found with 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(p-nitrophenoxy) propane, and p-nitrobenzylchloride. It is concluded that human placenta is equipped, from early pregnancy, with the enzymatic systems which are involved in GSH-mediated cellular detoxication and in preserving the integrity of the sulfhydryl status of the cells.

Carnitine in metabolic disease : Potential for pharmacological intervention

L-carnitine (LC) deficiency is commonly observed in chronic hemodialysis (HD) patients. As a result of this and other causes of secondary LC deficiencies, LC has been described as a conditionally essential nutrient or conditional vitamin. Although a large number of clinical trials regarding the beneficial effects of LC administration in HD patients have been published, some controversy about its use in this indication persists. In this article, we will review the use of LC in dialysis patients, by focussing mainly on those experimental and clinical data supporting the notion that supra-physiological concentrations of LC in plasma and target organs may exert beneficial effects on several metabolic parameters that have derangements of a common origin (e.g. insulin resistance, type 2 diabetes, dyslipidemia) and which are frequently present in end-stage renal disease (ESRD) patients undergoing dialysis.

Superoxide dismutase, reduced glutathione and TBA-reactive products in erythrocytes of patients with multiple sclerosis.

Superoxide dismutase, reduced glutathione and lipid peroxides levels were determined in the erythrocytes of multiple sclerosis patients. Superoxide dismutase activity and the malonyldialdehyde production rate were found to be significantly enhanced. The isoelectric focusing pattern of superoxide dismutase from multiple sclerosis and normal subjects erythrocytes was substantially overlapping. Our results indicate the occurrence of a higher susceptibility of multiple sclerosis erythrocytes to lipid peroxidation.

Involvement of phosphatidylserine exposure in the recognition and phagocytosis of uremic erythrocytes

Cell surface-exposed phosphatidylserine (PS) represents a signal for macrophage recognition and cell phagocytosis. This study examines PS exposure and susceptibility to erythrocyte phagocytosis in patients with chronic uremia in an attempt to assess the possible pathogenic mechanism behind cell removal in a condition associated with shortened erythrocyte life. Both PS-expressing erythrocytes and erythrophagocytosis (human monocyte-derived macrophages ingesting one or more erythrocytes) were significantly increased in uremic patients compared with healthy controls. Phagocytosed uremic erythrocytes appeared intact, suggesting they were identified before lysis through some surface change recognized by the macrophages. The degree of phagocytosis was markedly greater for PS-positive than PS-negative fluorescence-activated cell sorter (FACS)-sorted uremic erythrocytes. A significant correlation (r = 0.655) was found between the percentage of PS-expressing red blood cells (RBCs) and the percentage of phagocytosing macrophages in uremic patients. Reconstitution experiments showed the ability of uremic plasma to promote both PS exposure and erythrophagocytosis, the latter without direct interaction with the macrophage population. Phagocytosis of uremic erythrocytes was strongly inhibited when the macrophages were preincubated with glycerophosphorylserine (GPS), a structural derivative of PS, but this was not the case with the equivalent derivative of phosphatidylethanolamine, glycerophosphorylethanolamine. This inhibition appeared to be specific because GPS failed to inhibit the phagocytosis of opsonized uremic erythrocytes that occurs through an Fc receptor-mediated pathway. These findings suggest that a PS-recognition mechanism may promote the susceptibility of uremic RBCs to phagocytosis and thus be involved in the shortened erythrocyte life span of uremia.

Carnitine palmitoyltransferase (CPT) modulators: a medicinal chemistry perspective on 35 years of research.

The metabolism of fatty acids, including their absorption, storage, mobilization, synthesis, and catabolism, has been the origin and target of innumerable drugs and pharmacological tools and the focus of countless research programs. The oxidation of fatty acids (FAO) is one of the most important cellular energy sources, and a pharmacological control on this process could have interest for a variety of therapeutic applications. In diabetes, the reduced sensitivity to insulin causes excessive release of fatty acids from the adipose tissue and increases their oxidation rate. This compounds the defects in tissue glucose uptake by promoting underutilization of glucose in the cells and overproduction of glucose by the liver, particularly due to excessive gluconeogenesis. In the failing, overloaded heart, generation of energy by FAO poses heavy demands on the diminishing oxygen supply and promotes accumulation of potential toxic metabolites. FAO inhibition in cancer cells that are heavily dependent on lipids as energy source has the potential to shut down their growth. In dislipidemic and/or obese patients, on the other hand, increasing the FAO rate could curb the levels of circulating lipids and reduce tissue lipid storage, impacting favorably on body weight and the development of insulin resistance. Finally, elements of the FAO machinery in the brain contribute to central control of energy homeostasis and feeding behavior. Most of the oxidation of long chain fatty acids (LCFAs) to acetyl-CoA occurs in the mitochondrial matrix. LCFAs are first converted to their CoA esters by the ATP dependent acyl-CoA synthases in the outer mitochondrial membrane, the cytosol, and the endoplasmic reticulum; however, themitochondrialmembrane is not permeable to long chain acyl-CoA (over ∼C12). The mechanism by which LCFA access the mitochondrial matrix was elucidated by the pioneering studies of Fritz andYue andMcGarry and Foster and is illustrated in Figure 1. Acyl-CoAs are converted to acylcarnitine derivatives by the enzyme carnitine palmitoyltransferase 1 (CPT1) on the cytosolic face of the external mitochondrial membrane. Acylcarnitines are substrates for the shuttle-transporter carnitine acylcarnitine translocase (CACT), which mediates the transit of acylcarnitines from the cytosol to the matrix and the transport of free carnitine in the opposite direction. Once inside the mitochondrial membranes, acylcarnitines are reconverted to acyl-CoA by the enzyme carnitine palmitoyltransferase 2 (CPT2) and can thus enter the FAO cycle. McGarry and Foster established in 1980, on the basis of careful experimental data, that CPT1 is the controlling element of FAO rate and ketogenesis. The term “rate-controlling” is more appropriate than “rate-limiting”, which has been used by some authors. Indeed, CPT1 activity is regulated by a considerable number of signals, which deliver feedback on the energy requirements of the organism and can vary over an extremely wide range. The variable basal activity of CPT1 and the difficulties in obtaining the isolated enzyme in active form have made it difficult to dissect the contribution of the various elements of the CPT system to FA transport and oxidation rate. FAO can also occur in microsomes and peroxisomes, which process LCFAdown to theC8 length, after which they can enter the mitochondria via passive transport. Microsomes and peroxisomes possess their own CPTs, enzymes that are not well characterized but possibly are very similar if not identical to the mitochondrial CPT enzymes. Peroxisomal oxidation can be induced in situations where themitochondrial oxidation does not have sufficient capacity (for example, by high fat diet), and it has been demonstrated that the peroxisomal CPT is subject to similar controlling elements as the mitochondrial CPT system. The contribution of peroxisomal CPT activity to observed FAO rates in cellular systems upon CPT inhibition is an incognita. The lack of molecular modulators with well-defined activity and selectivity has been an added hurdle. Since the late 1970s, a few small molecules affecting the CPT enzymes, particularly inhibitors, have been identified. The oxirane carboxylic acids described by Tutwiler, Wolf, Sherratt, and Eistatter (BGLCF GmbH and McNeil) and the aminocarnitine derivatives described by Giannessi (Sigma Tau), Gandour, Anderson, and Griffith have been the most prominent and well characterized examples. These compounds have been used in an impressive number of in vitro and in vivo assays (including human clinical studies), assessing in particular potential effects in diabetes and cardiac failure. Nevertheless, or perhaps for this reason, a quantitative, consistent picture of relative and absolute activity, in vivo potency, selectivity, safety, and therapeutic potential of the various molecular entities is difficult to draft. In view of the complexity of the system, its sensitivity to a great number of factors, and the number of different setups that have been used to investigate its behavior, this is perhaps not surprising. It is important to stress at this stage that all compounds for which data are reported in the literature are nonselective inhibitors and affect more than one isoform of CPT (besides known or unknown off-target effects). Interest in this target, as documented by scientific literature, has been slowly but steadily growing in the past decades (Figure 2). A few reviews have been compiled that cover CPT inhibitors. This review will attempt to appraise the wide and tangled field of CPT modulators from the perspective of medicinal chemistry, using the chemotypes of CPT-interacting agents that have been described in the literature as a guiding beacon. Given the difficulty in obtaining quantitative comparable

Molecular and catalytic properties of purified glutathione S-transferase from human placenta

Abstract Glutathione S-transferase from human placenta has been purified with a simple and rapid method. The protein has an acidic isoelectric point (pI 4.65) and a molecular weight of 45,000, and is composed of two subunits. Evidence for the existence of two active forms, interconvertible by treatment with disulfide reducing agents, has been obtained on disc gel electrophoresis. Its amino acid composition is quite similar to that of erythrocyte glutathione S-transferase. The steady-state kinetics follow Michaelis-Menten kinetics and the conjugation reaction with 1-chloro-2,4-dinitrobenzene displays a random sequential mechanism. The purified enzyme is not able to catalyze the reduction of organic hydroperoxides. Bilirubin and sulfobromophthalein competitively inhibit transferase activity. The effect of sulfhydryl reagent was also studied.

Reversible Carnitine Palmitoyltransferase Inhibitors with Broad Chemical Diversity as Potential Antidiabetic Agents

A series of carnitine related compounds of general formula XCH(2)CHZRCH(2)Y were evaluated as CPT I inhibitors in intact rat liver (L-CPT I) and heart mitochondria (M-CPT I). Derivative 27 (ZR = -HNSO(2)R, R = C(12), X = trimethylammonium, Y = carboxylate, (R) form) showed the highest activity (IC(50) = 0.7 microM) along with a good selectivity (M-CPT I/L-CPTI IC(50) ratio = 4.86). Diabetic db/db mice treated orally with 27 showed a significant reduction of serum glucose levels.

Cell age-related monovalent cations content and density changes in stored human erythrocytes

Conversion of erythrocyte membrane protein 4.1b to 4.1a occurs through a non-enzymatic deamidation reaction in most mammalian erythrocytes, with an in vivo half-life of approximately 41 days, making the 4.1a/4.1b ratio a useful index of red cell age [Inaba and Maede, Biochim. Biophys. Acta 944 (1988) 256-264]. Normal human erythrocytes distribute into subpopulations of increasing cell density and cell age when centrifuged in polyarabinogalactan density gradients. We have observed that, when erythrocytes were stored at 4 degrees C under standard blood bank conditions, the deamidation was virtually undetectable, as cells maintained the 4.1a/4.1b ratio they displayed at the onset of storage. By measuring the 4.1a/4.1b values in subpopulations of cells of different density at various time points during storage, a modification of the normal cell age/cell density relationship was observed, as erythrocytes were affected by changes in cell volume in an age-dependent manner. This may stem from a different impact of storage on the imbalance of monovalent cations, Na(+) and K(+), in young and old erythrocytes, related to their different complement of cation transporters.

Toward personalized hemodialysis by low molecular weight amino-containing compounds: future perspective of patient metabolic fingerprint

BACKGROUNDnL-carnitine deficiency is commonly observed in chronic hemodialysis patients, and this depletion may cause clinical symptoms like muscle weakness, anaemia, and hypotension.nnnMATERIALS AND METHODSnWe pursued a targeted metabonomics investigation in 28 hemodialysis patients (13 non diabetics and 15 diabetics) and in 10 age-matched healthy controls, on plasma levels of all carnitine esters and of several amino acids. Samples were taken before and after the first hemodialysis treatment of the week. Multiplexed data were collected in LCMRM (Multiple Reaction Monitoring) and analysed by unsupervised multivariate analysis.nnnRESULTSnIn diabetic uremic patients, we observed lower values of propionylcarnitine than in other groups, while acylcarnitine concentration was higher in uremics compared to controls. The hemodialysis session induced a decline in free, short-chain, medium-chain and dicarboxylic acylcarnitines, whereas the long chain acylcarnitines remained unaffected. Plasma levels of amino acid proline, ornithine, citrulline and serine were significantly elevated in uremic patients before dialysis compared to controls. For most tested plasma amino acids, a significant reduction after hemodialysis session was found.nnnDISCUSSIONnOur study is the first that investigated on possible modifications of the system of carnitine in diabetic patients in hemodialysis not only in relation to the condition of deficiency but also compared to lipid and glucose homeostasis alteration typical of diabetics. We proposed the application of targeted metabolic fingerprint in the management of the hemodialysis patients.