Effects of long-chain acyl carnitine on membrane fluidity of human erythrocytes

Amphiphilic compounds such as long-chain acyl carnitines accumulate in ischemic myocardium and potentially contribute to the myocardial damage. To characterize alterations in membrane molecular dynamics produced by palmitoylcarnitine, human erythrocytes were spin-labeled with 5-doxylstearic acid, and membrane fluidity was quantified by measuring the changes in the order parameter derived from ESR spectra. Palmitoylcarnitine induced triphasic alterations in membrane fluidity of human erythrocytes. The membrane fluidity increased for 5 min, then decreased in a concentration-dependent manner. At higher concentrations (100 and 150 microM) of palmitoylcarnitine, membrane fluidity increased again after 30 and 20 min of the incubation, respectively. Addition of 2.8 mM CaCl2 resulted in a significant decrease in membrane fluidity and enhanced the alterations in membrane fluidity caused by palmitoylcarnitine. The results suggest that alterations in molecular dynamics are one mechanism through which long-chain acyl carnitine could play an important role in ischemic injury.     

Effects of long-chain acyl carnitine on membrane fluidity of human erythrocytes

Amphiphilic compounds such as long-chain acyl carnitines accumulate in ischemic myocardium and potentially contribute to the myocardial damage. To characterize alterations in membrane molecular dynamics produced by palmitoylcarnitine, human erythrocytes were spin-labeled with 5-doxylstearic acid, and membrane fluidity was quantified by measuring the changes in the order parameter derived from ESR spectra. Palmitoylcarnitine induced triphasic alterations in membrane fluidity of human erythrocytes. The membrane fluidity increased for 5 min, then decreased in a concentration-dependent manner. At higher concentrations (100 and 150 μM) of palmitoylcarnitine, membrane fluidity increased again after 30 and 20 min of the incubation, respectively. Addition of 2.8 mM CaCl₂ resulted in a significant decrease in membrane fluidity and enhanced the alterations in membrane fluidity caused by palmitoylcarnitine. The results suggest that alterations in molecular dynamics are one mechanism through which long-chain acyl carnitine could play an important role in ischemic injury.     

A fixation procedure suitable for autoradiography of endogenous long-chain acyl carnitine

A tissue processing procedure was evaluated for fixation of endogenous long-chain acyl carnitine (LCA) to facilitate autoradiographic subcellular localization of this amphiphile. Suspensions of neonatal rat myocytes labeled with exogenous 14C-palmitoyl carnitine retained 85.2% of the radiolabel after tissue processing. Autoradiography demonstrated no significant translocation of radiolabeled LCA from myocytes to unlabeled sheep erythrocytes mixed in equal proportions and processed together. To evaluate endogenous LCA fixation, cultured myocytes were incubated for 3 days with 3H-carnitine. Radioactivity was distributed in LCA, short-chain acyl carnitine, and free carnitine pools in proportion to the physiological concentrations of the metabolites traced. Before tissue processing, LCA contained 4.5% of total radioactivity. After tissue processing, labeled water-soluble components were lost and 88% of the retained radioactivity was in the LCA pool. The enrichment of endogenous LCA radioactivity was attributable to the selective extraction of endogenous short-chain and free carnitine. Nearly 75% of endogenous LCA was preserved. In contrast, 99.5% of both endogenous short-chain and free carnitine were extracted. Thus, endogenous LCA can be selectively preserved, permitting quantitative subcellular localization of this amphiphile with ultrastructural autoradiography.     

Preparation of long-chain acyl thioesters - thio wax esters - by the use of lipases

Long-chain acyl thioesters have been prepared by lipase-catalyzed thioesterification of lauric, myristic, palmitic and stearic acids with decanethiol, dodecanethiol, tetradecanethiol, hexadecanethiol and octadecanethiol. Lipase from Candida antarctica was more effective than that from Rhizomucor miehei. The extent of thioesterification increased with increasing chain length of the fatty acids and decreasing chain length of the alkanethiols. Lipase-catalyzed transthioesterification of fatty acid methyl esters with alkanethiols was less effective than thioesterification for the preparation of acyl thioesters. © Rapid Science Ltd. 1998     

Effects of long-chain fatty-acyl esters of coenzyme A and carnitine on cell-free rat heart preparations

The purpose of this study was to investigate the effects of fatty acyl CoA and carnitine esters on the glycolytic system of the rat heart. Using a respiring incubation mixture containing a whole-heart homogenate it was observed that oleoyl-CoA slowed down the glucose disappearance whereas lactate accumulation did not change. Experiments were also performed by means of an incubation mixture prepared with a soluble heart extract, considered to contain all glycolytic enzymes present in heart fibres. Palmitoyl-CoA or oleoyl-CoA as well as palmitoyl carnitine, added separately or together, were unable to alter the glucose disappearance and lactate accumulation in this mixture. These data suggest that long chain acyl-esters have not direct inhibitory actions on the heart glycolytic activity. However, CoA esters seem to exert indirect inhibitory effects which may be relevant to the myocardium under oxygen restriction situations.     

Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters

A procedure for the quantitative determination of the acyl chain composition of cellular long-chain acyl-CoA esters in subnanomole amounts is described. The abundant cellular lipids of samples are removed by extraction with organic solvents, and the proteins are precipitated from the aqueous phase by the addition of acetonitrile. The CoA thiolesters are adsorbed on neutral aluminum oxide and reduced with sodium borohydride to the corresponding alcohols that are then converted to t-butyldimethylsilyl ethers and analyzed quantitatively by gas chromatography. Saturated and unsaturated acyl chains behaved similarly throughout the procedure, and the common lipid esters do not interfere with the analysis of the CoA esters in the final assay procedure described. This simple and relatively rapid method is suitable for analyzing a large number of samples at a time.     

Investigation of the effect of theophylline administration on total,free, short-chain acyl and long-chain acyl carnitine distributions in rat renal tissues

This study is conducted to investigate the effect of oral theophylline administration on total (TC), free (FC), short- (SC), long-chain acyl (LC), acyl (AC) carnitine distributions as well as the ratio of acyl to free carnitine (AC/FC) in rat renal tissues. Theophylline was administrated at 100 mg kg⁻¹ body weight day⁻¹, and effects were monitored after a treatment period that lasted between 1 week and 5 weeks. The results indicated that theophylline administration leads to significantly higher concentrations of TC, FC, SC, L and AC in renal tissues as compared to those of control and placebo groups (P<0·001). Moreover, the ratio of AC/FC was significantly increased (P<0·001) as compared to either control or placebo groups. These changes may result from theophylline-enhanced mobilization of lipids from adipose tissues, which consequently stimulates an increased carnitine transport into the renal tissues to form acylcarnitines for subsequent β-oxidation inside the renal mitochondria. © 1998 John Wiley & Sons, Ltd.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

The interaction of ferrocytochrome c with long-chain fatty acids and their CoA and carnitine esters.

Non-covalent modification of cytochrome c may have implications for electron transport and energy metabolism. We examined the interaction of various fatty acids (FAs), their coenzyme A and carnitine esters, and fatty alcohols with horse heart ferrocytochrome c. A comparison of FAs indicated a minimum chain length of 14 carbons was required for significant effect on the ferroheme chromophore and major changes in electronic spectra. Coenzyme A and carnitine esters interacted less strongly than FAs whereas long-chain alcohols did not interact with the protein. We found a single, saturable FA binding site with Kd (oleate) of 23.1 microM (by stopped-flow kinetics), 30 microM (by radiochemical binding assay), and 29 microM (by spectrophotometric assay). The binding stoichiometry was 1:1. We present evidence from electronic spectra, and proton NMR (nuclear magnetic resonance) that the S-Fe coordination (methionine 80) was disrupted by ligand binding. From molecular modeling we identify a putative binding channel flanked by lysines 72 and 73.     

Lysis of erythrocytes by sepharose-staphylococcal delta toxin complex.

Purified delta toxin covalently attached to large polymers of sepharose retained 12.5% to 25% of its hemolytic activity against human erythrocytes. The lysis of human erythrocytes by such a complex indicates that the interaction of delta toxin with superficial membrane structures alone may suffice to initiate the lysis of erythrocytes.     

Control of cardiac sodium pump by long-chain acyl coenzymes A

Since we had shown recently that fatty acyl-CoA derivatives stimulate (Na+ + K+)-ATPase activity at suboptimal ATP concentrations, we used sealed vesicles of beef heart sarcolemma to examine the effects of these compounds on the transport function of the enzyme. The sodium pump was detected in inside-out vesicles as a component of Na+ uptake that was dependent on intravesicular (extracellular) K+ and extravesicular (intracellular) ATP and was sensitive to vanadate and digitoxigenin. The pump flux was stimulated without a lag by palmitoyl-CoA (K0.5 = 3 microM) when ATP concentration was 50 microM, but not when it was 2 mM. Saturating palmitoyl-CoA reduced the K0.5 of ATP for the pump by a factor of 3-6. Raising the intracellular K+ concentration increased the K0.5 of ATP, and this effect of K+ was antagonized by palmitoyl-CoA. At concentrations up to 0.5 mM, palmitoyl-CoA had no effect on ATP-independent (passive) Na+ uptake. All tested long-chain acyl-CoA derivatives had effects similar to that of palmitoyl-CoA; but CoA, acetyl-CoA, and palmitic acid were ineffective. Palmitoyl carnitine and docosahexanoic acid, amphiphilic compounds with inhibitory and biphasic effects on the hydrolytic activity of purified (Na+ + K+)-ATPase, had purely inhibitory effects on the pump at high concentrations that also affected the passive fluxes. The data support the proposition that fatty acyl-CoA derivatives mimic the effect of ATP at a regulatory site and suggest that these intracellular liponucleotides may be involved in the control of the pump.     

Uptake of long-chain fatty acid methyl esters by mammalian cells

Albumin-bound long-chain fatty acid methyl esters (ME) were taken up and utilized by Ehrlich ascites tumor cells and slices of rat heart, liver, and kidney. Much more ME than albumin was taken up by the tumor cells, indicating that ME dissociated from the carrier protein during their uptake. 70-80% of the radioactivity associated with the cells after 1 min of incubation at 37 degrees C remained as ME. The results of studies with metabolic inhibitors and glucose suggest that uptake of ME is an energy-independent process. Changes in incubation medium pH between 7.8 and 6.5 did not markedly alter uptake of ME. Cells incubated with FFA and methanol did not synthesize ME. These findings indicate that ME are taken up intact, and they suggest that the presence of an anionic carboxyl group is not essential for the binding of a long-chain aliphatic hydrocarbon to a mammalian cell. When incubation with labeled ME was continued for 1 hr, increasing amounts of radioactivity were recovered in FFA, phospholipids, neutral lipid esters, and CO(2). ME radioactivity associated with the cells after a brief initial incubation was released in the form of ME and FFA when the cells were incubated subsequently in a medium containing albumin. If the second incubation medium contained no albumin, most of the ME radioactivity initially associated with the cells was incorporated into phospholipids, neutral lipid esters, and CO(2). These results suggest that much of the ME which is taken up, is hydrolyzed to FFA, and that the fatty acids derived from ME are available for further metabolism.     

Lysis of C1Q-coated chicken erythrocytes by human lymphoblastoid cell lines

Human lymphoblastoid cells lysed chicken erythrocytes (E) that carried cell surface bound human C1q. Antibody to E(A) was not required for the C1q-dependent reaction. The effect of C1q was inhibited by Fab'2 anti-C1q and by the serum C1q inhibitor. The action of the lymphoblastoid cells was inhibited by anti-metabolites and by pretreatment of the cells with trypsin which is known to destroy their C1q receptor. Lymphoblastoid cell lysate was inactive. The time course of the C1q-dependent lysis was comparable to that of the antibody-dependent cellular cytotoxic reaction of human K-cells. Lysis of EA by human peripheral lymphocytes was enhanced up to 50% by human C1q.     

Role of carnitine esters in brain neuropathology

L-Carnitine (L-C) is a naturally occurring quaternary ammonium compound endogenous in all mammalian species and is a vital cofactor for the mitochondrial oxidation of fatty acids. Fatty acids are utilized as an energy substrate in all tissues, and although glucose is the main energetic substrate in adult brain, fatty acids have also been shown to be utilized by brain as an energy substrate. L-C also participates in the control of the mitochondrial acyl-CoA/CoA ratio, peroxisomal oxidation of fatty acids, and the production of ketone bodies. Due to their intrinsic interaction with the bioenergetic processes, they play an important role in diseases associated with metabolic compromise, especially mitochondrial-related disorders. A deficiency of carnitine is known to have major deleterious effects on the CNS. Several syndromes of secondary carnitine deficiency have been described that may result from defects in intermediary metabolism and alterations principally involving mitochondrial oxidative pathways. Mitochondrial superoxide formation resulting from disturbed electron transfer within the respiratory chain may affect the activities of respiratory chain complexes I, II, III, IV, and V and underlie some CNS pathologies. This mitochondrial dysfunction may be ameliorated by L-C and its esters. In addition to its metabolic role, L-C and its esters such as acetyl-L-carnitine (ALC) poses unique neuroprotective, neuromodulatory, and neurotrophic properties which may play an important role in counteracting various disease processes. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach, which may limit further developmental damage, cognitive loss, and improve long-term therapeutic outcomes. The neurophysiological and neuroprotective actions of L-C and ALC on cellular processes in the central and peripheral nervous system show such effects. Indeed, many studies have shown improvement in processes, such as memory and learning, and are discussed in this review.     

Lysis of erythrocytes by the secreted cysteine proteinase of Porphyromonas gingivalis W83

The cysteine proteinase produced in the culture supernatant of Porphyromonas gingivalis was extensively purified. Haemagglutination type assays in which the enzyme was titrated against a fixed concentration of erythrocytes, showed that low levels of enzyme directly caused lysis of the red blood cells. However, using the same assay, the presence of stoichiometric amounts of the thiol blocking agent, 2,2'-dipyridyl disulphide (2-PDS) specifically inhibited the action of the enzyme or its haemagglutination with W83 cells or vesicles. In all cases, electron micrographs revealed that in the presence of 2-PDS the erythrocytes remained intact. Thiol activator free enzyme or aerated, inactivated enzyme had no effect on the red blood cells. These results show conclusively that the secreted cysteine proteinase of P. gingivalis causes lysis of erythrocytes and must now be regarded as a potent virulence determinant of P. gingivalis.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Separation of long-chain fatty acid esters of retinol by high-performance liquid chromatography

Individual long-chain fatty acid esters of retinol can be resolved by high-performance liquid chromatography using an octyl- or phenyl-substituted reverse-phase column and mixtures of acetonitrile with water as mobile phase. This simple procedure provides good resolution of biologically important retinyl esters including retinyl palmitate and retinyl oleate. Using an isocratic elution system, it is shown that nine synthetic esters of retinol, ranging in fatty acyl chain length from 12 to 20 carbons, each elute with a unique elution volume and produce an absorbance signal at 340 nm proportional to molar concentration. The method is suitable for analysis of various esters of retinol in biological samples including lymph chylomicrons and blood plasma. The octyl-substituted reverse-phase column can also be used to separate more polar neutral retinoids including retinol and retinaldehyde.     

Mass spectrometric analysis of long-chain esters of diols

Homologous series of synthetic long-chain monoesters and diesters of 1,2-ethanediol were analyzed by mass spectrometry, and the patterns of fragmentation were studied. Under electron impact saturated ethanediol monoesters yielded prominent ions characteristic of the short-chain diol, such as the rearranged ion formed by 2,3-cleavage (m/e 104) and the ion caused by 3,4-cleavage (m/e 117). Fragments characteristic of the constituent long-chain moieties were the acylium ions [RCO](+), [RCO - 1](+), and the ions [RC(OH)(2)](+). The mass spectra of ethanediol diesters exhibited very intense peaks due to the ions formed by loss of the acyloxy group, [M - RCOO](+), or one carboxylic acid, [M - RCOOH](+). These ions are distinctive for diol diesters. Precise mass measurements by high resolution mass spectrometry verified the composition of the ion fragments. Spectral studies of some monoesters and diesters of 1,3-propanediol, 1,4-butanediol, 2,3-butanediol, and also of some monounsaturated homologues, demonstrated that mass spectrometry is very suitable for the identification, distinction, and analysis of diol lipids.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.