Centella asiatica and lipoic acid, or a combination thereof, inhibit monocyte adhesion to endothelial cells from umbilical cords of gestational diabetic women.

BACKGROUND AND AIMS: Diabetes mellitus is associated with inflammatory endothelial activation and increased vascular leukocyte adhesion molecule expression, both playing a prominent role in the development of vascular complications. Centella asiatica (CA) and Lipoic Acid (LA) have shown anti-inflammatory and anti-oxidant properties in a variety of experimental models; however, their action on human umbilical vein endothelial cells (HUVECs), chronically exposed to hyperglycemia and pro-inflammatory environment during pregnancy, is still unknown. METHODS AND RESULTS: In HUVECs from umbilical cords of gestational diabetic (GD) or healthy (C) women, both CA and LA affected tumor necrosis factor-α (TNF-α)-induced inflammation, being associated with a significant decrease in vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression (western blot) and exposure (flow cytometry), as well as monocyte-HUVECs interaction (adhesion assay). Notably, this was associated with a significant reduction of an index of nitro-oxidative stress, such as the intracellular peroxynitrite levels (fluorescence detection by cytometric analysis), Mitogen-Activated Protein kinase (p44/42 MAPK) expression/phosphorylation levels and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) cytoplasm-nucleus translocation (flow cytometry). Overall our results indicate that both CA and LA used separately, and even better when combined, are effective to reduce the inflammatory response in TNF-α-treated HUVECs. Notably, this was more significant in GD than in C-HUVECs and also evident at baseline. CONCLUSION: In conclusion, our in vitro study demonstrates that both CA and LA, or a combination thereof, are able to mitigate the potentially dangerous effects on the endothelium of chronic exposure to hyperglycemia in vivo.

Prevention of peritoneal sclerosis: a new proposal to substitute glucose with carnitine dialysis solution (biocompatibility testing in vitro and in rabbits).

AIM: Commercial glucose peritoneal dialysis solutions expose the peritoneum to hyperosmolar glucose containing variable amounts of non-enzymic breakdown products of glucose. These solutions are toxic for the peritoneum. The aim of the present study is to compare in vitro and in vivo characteristics of a new dialysis solution containing carnitine, a naturally occurring compound, as substitute of glucose. MATERIAL AND METHODS: We compared in vitro and in the rabbit a new peritoneal dialysis solution containing carnitine, with two standard bicarbonate glucose peritoneal dialysis solutions and a solution containing icodextrin. RESULTS: In vitro and in vivo the solution containing carnitine seems to be more biocompatible than standard glucose solutions and those containing icodextrin. CONCLUSIONS: In our study the peritoneal dialysis solution containing carnitine seems to prevent the mesothelial changes observed with solutions containing glucose. Since carnitine has been extensively studied and seems to be well tolerated by hemodialysis patients, even at high doses for long periods, clinical trials in humans may be planned in the near future.

Increased platelet phosphatidylserine exposure and caspase activation in chronic uremia.

Platelet activation is associated with exposure of the aminophospholipid phosphatidylserine (PS) to the outer hemi-leaflet of the plasma membrane bilayer, which seems to be involved in the coagulation process. Because platelet activation may occur in patients suffering from chronic uremia, which is frequently associated with a thrombophilic tendency, we studied whether uremic platelets show an increased propensity to expose PS on the outer membrane leaflet and whether this process is linked with important functional and molecular changes. Flow cytometric percentage of annexin V-positive platelets, a measure of PS externalization, was significantly elevated (P < 0.001) in uremic patients when compared to normal controls under both unstimulated and agonist-stimulated conditions. Uremic platelet procoagulant activity, as measured by thrombin generation, was more than twice as high (4.13 +/- 0.3 micro mL(-1)) as that found in normal controls (1.86 +/- 0.2 micro mL(-1)). Two independent assays showed that the enzymatic activity of caspase-3, a protease involved in the loss of membrane PS asymmetry, was significantly greater in the platelets of uremic subjects than in those of healthy controls. PS exposure in agonist-stimulated platelets was markedly reduced by inhibition of caspase-3 activity but was not affected by inhibition of calpain activity. These results support the view that the thrombophilic susceptibility of uremic patients may be partly ascribed to increased PS exposure to the outer membrane leaflet of platelets. This process seems to be causally linked to an increase in caspase-3 activity, particularly during platelet activation.

Anion allosteric effect in the recognition of tetramethylammonium salts by calix[4]arene cone conformers.

Rigid calix[4]arene cone conformers, which are efficient receptors for quaternary ammonium salts, are usually obtained through the functionalization of their lower rim with suitable groups. Using flexible cone conformer of calix[4]arene, bearing four 4-hydroxybenzyl groups as cooperative and rigidifying structural elements at the upper rim of the calix, which act as anion binding groups, a new heteroditopic cavitand, 7, was synthesized. Whereas the tetramethoxy derivative 8 does not show any complexing ability, its tetrahydroxy analogue 7 recognizes tetramethylammonium salts with high efficiency. The binding abilities of this new receptor toward a series of tetramethylammonium salts (tosylate, chloride, acetate, trifluoroacetate, and picrate) have been investigated in CDCl(3) solution and compared to the monotopic and rigidified, through the lower rim, cone biscrown-3-calix[4]arene 9. The results obtained confirmed that in CDCl(3) ion pairing strongly affects binding. In particular, the rigid monotopic receptor 9 experiences good efficiency toward tetramethylammonium salts having anions with low ion-pairing ability such as trifluoroacetate or picrate. On the contrary, for the new heteroditopic cavitand 7, a reverse order of efficiency was found. In the latter case a different complexation mode was hypothesized in which the tetramethylammonium cation is deeply entrapped into the host cavity and its counteranion participates to the recognition process by coordination via hydrogen bonding by the four OH groups. To further support the role of the anion in the recognition process, a "dual host" approach, employing 7 or 9 in the presence of a specific receptor for chloride anion (10), was utilized. Molecular modeling studies confirmed that in the complexes formed by 7 and TMA salts the counteranion is involved in hydrogen bonding with the host OH groups and that the guests are bound as ligand-separated ion pairs.

The effect of pivalate treatment of pregnant rats on body mass and insulin levels in the adult offspring.

Pivalic acid is used as a prodrug to increase gut absorption of a variety of different antibiotics. Pivalic acid is also known to induce a number of metabolic aberrations which may be in part explained by concurrent mild carnitine depletion. Rat pups (5 days old) born to mothers treated throughout their pregnancy and lactation period with sodium pivalate, showed an increase in liver and muscle triglycerides and elevated plasma ketone bodies, compared to controls. A reduction of free carnitine content in liver, muscle and plasma was also observed in the pivalate treated group. In a second study, pups were treated with either pivalate for 24 days (females), or pivalate for 120 days (males). Both groups were fed standard diets. In both groups (male and female), the pivalate treatment showed a statistically significant hyperinsulinaemia and an increase of body mass compared with that of age- and sex-matched control groups. In addition, after a glucose loading, significantly higher levels of insulin in the pivalate-treated group (male) with respect to controls were observed. In conclusion, our data suggest that maternal pivalate treatment may predispose adult offspring to developing insulin-resistance and obesity.

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.

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.

Participation of carnitine palmitoyltransferase in the synthesis of dipalmitoylphosphatidylcholine in rat alveolar type II cells.

We have investigated the role of carnitine palmitoyltransferase (EC 2.3.1.21) in pulmonar type II pneumocyte, a lung cell responsible for the synthesis of surface active lipids. Adult type II pneumocytes were isolated from rat lung and purified by differential adherence. When these lung cells were incubated with radioactive palmitate, the percentage of radioactivity recovered into dipalmitoylphosphatidylcholine (DPPC), a major surface active lipid, was almost 60% with respect to total phosphatidylcholine (PC) molecular species. Cellular lysates from type II pneumocytes contained detectable amount of carnitine palmitoyltransferase (CPT) activity (1 nmol/min/mg). Most of the CPT activity found in these cells could be inhibited by incubating them for 60 min with 5 microM tetradecylglycidic acid (TDGA), a specific and irreversible CPT inhibitor of the malonyl-CoA sensitive CPT isoform (CPT I). TDGA treatment of adult type II pneumocytes caused a significant reduction in the incorporation of radioactive palmitate into PC, though this effect did not seem to be specific for DPPC. TDGA affected the incorporation of radioactive palmitate at the sn2 rather than the sn1 position of the glycerol backbone of PC. The incorporation of radioactive palmitate into DPPC was also observed when these lung cells were incubated with palmitate-labeled palmitoyl-L-carnitine. Our data suggest that type II pneumocyte CPT may play an important role in remodelling PC fatty acid composition and hence DPPC synthesis.

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.

L-carnitine decreases glycolysis in liquid-stored platelets.

BACKGROUND: The platelet storage lesion is characterized metabolically by a pH decrease associated with lactic acid generation; a change in platelet morphology from discoid to spherical; a diminished response to in vitro challenge tests, such as the hypotonic shock response (HSR) and extent of shape change (ESC); increased surface P-selectin expression; and decreased in vivo recovery and survival. Altering storage conditions to improve these measures could allow for extension of the duration of in vitro storage. STUDY DESIGN AND METHODS: ABO-identical paired platelet concentrates were pooled and then equally divided into two plastic bags. Either L-carnitine (LC) or an equal volume of saline (control) was added to one container of each pair. Platelets were stored at 20 to 24 degrees C for 5 to 10 days or at 1 to 6 degrees C for 5 days at various concentrations of LC between 0.1 and 15 mM: At the end of storage, pH, glucose consumption, lactate generation, HSR, ESC, and surface P-selectin expression were measured. In different experiments, paired platelet concentrates were spiked with a Staphylococcus epidermidis suspension in the presence and absence of L-carnitine at a concentration of 5 mM: RESULTS: At 20 to 24 degrees C and concentrations of LC between 0.1 and 5 mM:, there was evidence of better pH preservation, less glucose consumption, and less lactate generation. Only with storage beyond 5 days was a difference present in either surface P-selectin expression or HSR. An L-carnitine concentration of 5 mM: appeared optimal. L-carnitine did not enhance the growth of bacteria after 7 to 8 days of storage. CONCLUSION: LC at 5 mM: may improve the quality of platelet concentrates that are stored beyond 5 days. There was no indication that LC at this concentration would promote bacterial growth. It may be a useful additive to platelet preservation.

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions.

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.

Rigidified calixarenes bearing four carbamoylmethylphosphineoxide or carbamoylmethylphosphoryl functions at the wide rim

Conformationally rigidified tetraCMPO derivatives have been prepared from calix[4]arene bis(crown ether) 4a in which adjacent oxygens are bridged at the narrow rim by two diethylene glycol links. Acylation of the tetraamine 4c with the CMPO-active ester 5b gave the tetraphosphine oxide 6a, while the tetraphosphinate 6b and the tetraphosphonate 6c were obtained by Arbuzov reaction of tetrabromoacetamido derivative 7 with PhP(OEt)2 or P(OEt)3. The extraction ability of these CMPO derivatives was checked for selected lanthanides and actinides and compared with the analogous compounds 1b, 10b and 10d derived from calix[4]arene tetrapentyl ether. All rigidified bis(crown ether) ligands are more effective extractants than their pentyl ether counterparts and require only 1/10 of the concentration (cL= 10 4M) to obtain the same distribution coefficients, while with CMPO itself a 2,000-fold concentration is necessary. This could be a consequence of a better preorganisation of the ligating functions owing to the rigidity which on the other hand did not change the observed selectivity for americium (DAm/DEu=9-19) and for light lanthanides over heavy ones. NMR relaxivity titration curves show that the complex of Gd3+ with ligand 6a is highly oligomerised in anhydrous acetonitrile over a large range of ligand:metal concentration ratios. Nuclear magnetic relaxation dispersion (NMRD) profiles also showed that large oligomers were formed, and their mean tumbling times were deduced from the Solomon-Bloembergen-Morgan equations. The NMR spectra of dia- and paramagnetic lanthanide complexes with 6a agreed with the presence of two conformers with an elongated calix[4]arene skeleton in which the distances between opposite methylene groups are different. Contrary to what was observed with ligand 2a, the addition of nitrate ions does not labilize the metal complexes, presumably because of the rigidification effect of the ether bridges. Single-crystal X-ray structures were obtained for the active ester 5b and for diphenylphosphorylacetic acid 5a.

Dimeric capsules by the self-assembly of triureidocalix

A number of calix[6]arenes bearing ureas at the upper rim positions of alternate rings 1, 3 and 5 were prepared and studied in detail by NMR spectroscopy and gel permeation chromatography. N-Unsubstituted ureas were shown to dimerize through a cyclic array of hydrogen bonds to give cylindrical cavities capable of encapsulating small molecules such as dichloromethane, benzene and fluorobenzene. Slow equilibria between dimer and monomer were observed in [D6]DMSO-CDCl3 mixtures. By contrast, N-substituted ureas are monomeric. All urea monomers with bulky O-substituents display a solvent-dependent, slow equilibrium between C3v and Cs cone conformations.

Recognition of amides by new rigid Calix

The synthesis of new hosts specifically designed for the recognition of amides, characterized by two binding regions: a rigid calix[4]arene cavity and a sidearm, inserted at its rim, able to form strong hydrogen bonds, is described. The binding abilities of the new receptors toward amides of general structure R(1)CONR(2)R(3) have been investigated in CDCl(3) solution by (1)H NMR spectroscopy. When the additional binding site is the N-phenylureido group spaced by a methylene unit from the apolar cavity, binding constants up to 756 M(-)(1) were measured. Neither the two separate potential binding sites, nor the model host, where the calix[4]arene skeleton is flexible show detectable binding ability toward the series of guests examined. The rigidity of the calix[4]arene apolar cavity is the key control element in determining the efficiency of these molecular recognition processes. The presence of NH groups in the guest controls the efficiency and selectivity of binding.

Increased erythrocyte phosphatidylserine exposure in chronic renal failure.

The appearance of phosphatidylserine, an aminophospholipid normally confined to the inner monolayer, at the outer leaflet of red cell membrane may have several pathophysiologic implications. This study examines erythrocyte phosphatidylserine exposure in chronic renal failure (CRF) patients on conservative treatment or on dialysis, to assess possible alterations to phospholipid asymmetry in a condition associated with a state of deranged red cell function. A significant increase in phosphatidylserine-expressing erythrocytes was found in undialyzed patients with CRF (2.32%) and patients on hemodialysis (3.06%) and on peritoneal dialysis (2.14%) compared with control subjects (0.68%). In undialyzed CRF patients, a strong correlation (r = 0.903) was found between the percentage of phosphatidylserine-expressing red cells and the serum creatinine concentration. The increased exposure of phosphatidylserine in uremic erythrocytes may be due to inhibition of phosphatidylserine transport from the outer to the inner leaflet of plasma membrane and may promote an increased erythrophagocytosis. In reconstitution experiments, normal erythrocytes showed an increase in phosphatidylserine-expressing cells when incubated in uremic plasma (3.2% after 2 h versus 1.1% at beginning of incubation), whereas phosphatidylserine-positive uremic erythrocytes decreased when resuspended in normal plasma (2.03% after 2 h and 1.65% after 8 h versus 2.9% at beginning of incubation). Preliminary characterization of the putative uremic compound(s) indicates a molecular weight between 10,000 and 20,000, as well as heat instability. These findings show an impairment of erythrocyte membrane phospholipid asymmetry in CRF patients, regardless of the dialysis treatment. Such abnormality seems related to the uremic state and could contribute to the red cell pathology present in CRF.

Entry of [(1,2-13C2)acetyl]-L-carnitine in liver tricarboxylic acid cycle and lipogenesis: a study by 13C NMR spectroscopy in conscious, freely moving rats.

The biochemical pathways involved in acetyl-L-carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4-h [(1,2-13C2)acetyl]-L-carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl-L-carnitine from tissues was observed. [(1,2-13C2)Acetyl]-L-carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3-hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2-13C2)acetyl]-L-carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C-labelling pattern in 3-hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl-L-carnitine-derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra- and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA-activated mitochondria-derived ketone bodies into 3-hydroxy-3-methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.

Role of acetyl-L-carnitine in rat brain lipogenesis: implications for polyunsaturated fatty acid biosynthesis.

This study was undertaken to explore the metabolic fate of acetyl-L-carnitine in rat brain. To measure the flux of carbon atoms into anabolic processes occurring at regional levels, we have injected [1-(14)C]acetyl-L-carnitine into the lateral brain ventricle of conscious rats. After injection of [1-(14)C]acetyl-L-carnitine, the majority of radioactivity was recovered as 14CO2 expired (60% of that injected). The percentage of radioactivity recovered in brain was 1.95, 1.60, 1.30, and 0.93% at 1, 3, 6, and 22 h, respectively. Radioactivity distribution in various lipid components indicated that the fatty acid moiety of phospholipid contained the majority of radioactivity. The radioactive profile of these fatty acids showed that the acetyl moiety of acetyl-L-carnitine was incorporated into saturated (60%), monounsaturated (15%), and polyunsaturated (25%) fatty acids [mainly present in 20:4 (5.2%) and 22:6 (7.8%)]. Injection in the brain ventricle of radioactive glucose, the major source of acetyl-CoA in the CNS, revealed that glucose was a precursor of saturated (85%) and monounsaturated (15%) but not of polyunsaturated fatty acids. Thus, this study demonstrated distinct fates of glucose and acetyl-L-carnitine following intracerebroventricular injection. In summary, these data implicate acetyl-L-carnitine as an important member of a complex acetate trafficking system in brain lipid metabolism.