Fatty Acid Side Chain Research Articles

A Differential Scanning Calorimetry Study of the Interaction of the Antimalarial Agent Halofanthrine with Dipalmitoyl Phosphatidyl Choline Bilayers.

The influence of the antimalarial agent halofanthrine on the phase transition temperature (Tm) of dipalmitoyl phosphatidyl choline (DPPC) liposomes was investigated by differential scanning calorimetry (DSC). The effects of increasing drug content and varying pH conditions (to give predominantly charged and uncharged states of the drug) on the interaction were considered and compared with those caused by structurally related antimalarials, mefloquine and quinine, under similar conditions. Increasing concentrations of halofanthrine resulted in the gradual disappearance of the original transition endotherm and the appearance of a new drug-induced endotherm at a lower temperature and with a decreased enthalpy of transition. Qualitatively similar observations were made at different pH conditions, suggesting little difference in the mode of interaction of charged and uncharged halofanthrine molecules with DPPC. Increasing concentrations of mefloquine and quinine also caused a decline in Tm but neither drug-induced endotherms was associated with a decreased enthalpy of transition. The results indicate that halofanthrine, unlike mefloquine and quinine, intercalates in the hydrophobic interior of the phospholipid. Its localization among the fatty acid side chains of the phospholipid is possibly related to its greater hydrophobic character compared to mefloquine and quinine. The disruption of phospholipid packing and the consequential effects on membrane permeability and integrity may be relevant to the greater in vitro antimalarial activity of halofanthrine and should be considered in any delibration of its pharmacodynamic action.

Caloric availability of SALATRIM in rats and humans

SALATRIM is a reduced-calorie fat substitute composed of structured triacylglycerols. These structured triacylglycerolB are composed of long-chain fatty acids (predominantly stearic) and short-chain aliphatic acids (acetic, propionic, and/or butyric). It has been demonstrated in rat studies and in a clinical study that SALATRIM with various combinations of these aliphatic acid and fatty acid side chains delivers fewer calories per gram than conventional triacylglycerols such as corn oil. The reduced calories are accounted for by the lower caloric value of the short-chain aliphatic acids and limited absorption of the stearic acid which is freed by enzymatic hydrolyse in the gastrointestinal tract. In caloric availability studies with rats SALATRIM was found to deliver between 4.5 and 6.0 kcal/g

Multicentric Evaluation of a New PT Reagent Based on Recombinant Human Tissue Factor and Synthetic Phospholipids

A new PT reagent based on recombinant human tissue factor and synthetic phospholipids (phosphatidyl choline and phosphatidyl serine) with defined fatty acid side chains was calibrated against BCT/253 and CRM 149R. A small but consistent bias in the International Sensitivity Index (ISI) value was obtained using either the human or rabbit brain reference material. ISI values were around 1.0 or slightly lower depending on the respective instrument. Mixing studies with factor deficient plasmas showed a high factor sensitivity especially for factor VII as compared to commercial rabbit brain or human placenta thromboplastin. In an international field trial the reagent was tested using fully or semi automated Electra coagulometers in 4 different laboratories. Results with normal samples were in excellent agreement among the different laboratories. Mean values were 10.9, 10.9, 11.0, 11.2 s with a range of 9.5 to 12.5 s. Results of males and females were not different. In patients with liver disease very similar PT activities were found as compared to sensitive rabbit brain or human placental thromboplastins. In normals and patients with oral anticoagulation INR values correlated very well against BCT (r = 0.98, regression line y = -0.07 + 0.9 x). The distribution of samples was linear over the whole range. In the comparison against sensitive rabbit brain thromboplastin or human placental thromboplastin similar correlations were found. In a few cases higher INR values were observed for the recombinant reagent especially in patients with intensive treatment. Factor assays in those patients showed at least the strong reduction of one vitamin K-dependent coagulation factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrodioxyl (Perhydroxyl), Peroxyl, and Hydroxyl Radical-Initiated Lipid Peroxidation of Large Unilamellar Vesicles (Liposomes): Comparative and Mechanistic Studies

The mechanisms and relative efficiencies of lipid peroxidation initiation by biological O 2-derived oxidants were studied using large unilammellar vesicle (LUV) liposomes, structural models for biological membranes, as targets for oxidation. LUVs, when prepared from dilinoleoylphosphatidylcholine (DLPC) containing either 0 or 5 mol% hydroperoxide (LOOH, added either as a linoleic acid or DLPC hydroperoxide), maintained structural integrity, which enabled evaluation of the relative ability of oxidants to initiate lipid peroxidation when generated outside of the bilayer. LUVs were more oxidizable than multilamellar vesicles or lipids dispersed in solution, supporting their appropriateness as biological membrane models. In parallel to previous results using lipid dispersions (J. Aikens and T. A. Dix, 1991, J. Biol. Chem. 266, 15091-15098), both perhydroxyl (HOO . ) and peroxyl (ROO . ) radicals initiated lipid peroxidation in LUVs. Oxidants that did not initiate included H 2O 2, organic hydroperoxides, and, most notably, superoxide (O − 2). HOO . and ROO . initiated by different mechanisms: HOO . required the presence of the preexisting LOOHs for efficient initiation, indicating the direct reaction of HOO . with LOOH, whereas ROO . initiated by hydrogen atom abstraction at the bisallylic site of unsaturation on the fatty acid side chain of the PCs. Hydroxyl radicals (HO . s) were poor initiators in comparison to ROO . s (and, indirectly, HOO . s), which might be considered surprising as the latter species are chemically weaker oxidants. The decreased activity of HO . was not due to decreased access to the LUVs; rather, this oxidant appears to react to generate less viable lipid peroxidation propagating species. It was also demonstrated that the fluidity of the LUV membrane had little effect on the relative initiating activity of each oxidant. It is argued that HO . may initiate lipid peroxidation only indirectly in vivo (through the generation of carbon-based peroxyl radicals, ROO . s) and that greater effort should be made to understand the roles of HOO . and ROO . at lipid peroxidation initiation.

Differential Solubilization of Membrane Lipids by Detergents: Coenrichment of the Sheep Brain Serotonin 5-HT 1A Receptor with Phospholipids Containing Predominantly Saturated Fatty Acids

Most membrane receptors lose binding activity during purification and we studied the correlation between this event and differential solubilization of membrane lipids by detergents. Both 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate [Chaps; high critical micellar concentration (cmc) ∼0.5%] and n-dodecyl β-D-maltoside (DDM; low cmc ∼0.009%) solubilized the 8-[ 3H]hydroxy-2-(di- n-propylamino)tetralin ([ 3H]8-OH-DPAT)-binding serotonin 5-HT 1A receptor (5-HT 1A-R) optimally at 2% (w/v) detergent concentration despite the widely differing cmc of the two detergents. In contrast, n-octyl-β-D-glucopyranoside (octyl glucoside; high cmc ∼0.7%), Thesit (low cmc ∼0.005%), and Triton X-100 (low cmc ∼0.013%) solubilized virtually no [ 3H]8-OH-DPAT-binding activity. The total mass of solubilized lipids was always low at 0.5% detergent concentration and attained a plateau between 1 and 2.5% for all detergents except octyl glucoside. The mass of octyl glucoside-solubilized lipids showed an increasing trend even at 3.0% detergent concentration. The total amount of solubilized lipid is unrelated to the amount of 5-HT 1A-R solubilized but the species of lipid is important. Thus Chaps and DDM, with diverse structures and cmc, both preferentially solubilized phospholipids enriched in saturated fatty acids (67 and 72%, respectively). In contrast, octyl glucoside, Triton X-100, and Thesit showed no preference in solubilizing phospholipids. Octyl glucoside, which solubilized significantly higher proportions of saturated fatty-acid-containing phosphatidylethanolamine (slightly higher saturated fatty acids in total phospholipids), also produced more (twofold higher) solubilized 5-HT 1A sites than Triton X-100 and Thesit. This suggests an optimum involvement of saturated fatty acid side chains in forming tightly packed vesicles which stabilize the 5-HT 1A-R more than the vesicles of higher fluidity formed by phospholipids containing higher proportions of cis-double-bonded unsaturated fatty acids. Indeed, delipidation of the 1.5% Chaps-solubilized receptor preparations by Sephacryl S-200 column chromatography eliminated essentially all [ 3H]8-OH-DPAT-binding activity. Therefore, for efficient recovery during solubilization and reconstitution of a prototypic heptahelical receptor (5-HT 1A), it is essential to stabilize the receptor protein through association with saturated phospholipids.

Lipopolysaccharide epitope specificity and binding cross reactivity of the human IgM anti-lipid a monoclonal antibody SdJ5-1.17.15

The binding properties and specificity of the SdJ5-1.17.15 (SdJ5) human IgM monoclonal antibody (mAb), prepared against S. minnesota R595 heat killed whole organisms, were assessed by dot blot assay in vitro. In that assay, lipopolysaccharide (LPS) related antigens, immobilized on nitrocellulose membrane, were reacted with the test and control human IgM antibodies. Results indicated the SdJ5 mAb reacted specifically with lipid A, a component of LPS, from a variety of bacterial species, but not with the whole LPS molecule. The inability of the mAb to react with whole LPS in dot blot assay was attributed to the possible effect of the solid phase on epitope exposure or structure. This hypothesis was tested by inhibition studies which indicated that liquid phase adsorption with LPS abolished or greatly reduced the specific recognition of solid phase lipid A by the mAb. These results indicated that LPS-associated antigens were recognized by the SdJ5 anti-lipid A mAb in liquid, but not solid, phase. In an attempt to identify the SdJ5-specific epitope on lipid A, the pattern of reactivities of different lipid A analogues with the mAb were examined by dot blot assay. Results indicated that a combination of the fatty acid side chains and the phosphate groups of lipid A (both groups being implicated as important for sepsis mediation) were either directly involved in the epitope structure or affected the exposure of epitope in solid phase. Because SdJ5 recognizes an LPS epitope on lipid A closely associated with endotoxin activity, this mAb could potentially be a useful therapeutic agent against septic shock.

High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions.

The structure of E. coli-derived rat intestinal fatty acid-binding protein has recently been refined to 1.2 A without bound fatty acid and to 2.0 A and 1.75 A with bound hexadecanoate (palmitate) and 9Z-octadecenoate (oleate), respectively. The structure of E. coli-derived human muscle fatty acid-binding protein has also been solved to 2.1 A with a C16 bacterial fatty acid. Both proteins contain 10 anti-parallel beta-strands in a +1, +1, +1... motif. The strands are arranged in two beta-pleated sheets that are orthogonally oriented. In each case, the fatty acid is enclosed by the beta-sheets and is bound to the proteins by feeble forces. These feeble forces consist of (i) a hydrogen bonding network between the fatty acid's carboxylate group, ordered solvent, and side chains of polar/ionizable amino acid residues; (ii) van der Waals contacts between the methylene chain of the fatty acid and the side chain atoms of hydrophobic and aromatic residues; (iii) van der Waals interactions between the omega-terminal methyl and the component methenyls of the phenyl side chain of a Phe which serves as an adjustable terminal sensor situated over a surface opening or portal connecting interior and exterior solvent; and (iv) van der Waals contacts between methylenes of the alkyl chain and oxygens of ordered waters that have been located inside the binding cavity. These waters are positioned over one face of the ligand and are held in place by hydrogen bonding with one another and with the side chains of protein's polar and ionizable residues. Binding of the fatty acid ligand is associated with minimal adjustments of the positions of main chain or side chain atoms. However, acquisition of ligand is associated with removal of ordered interior solvent suggesting that the free energy of dehydration of the binding site may be as important for the energy of the binding reaction as the free energy of stabilization of the fatty acid: protein complex.

Diacylglycerol is an effector of the clonal expansion of cells containing activated Ha-ras genes.

Diacylglycerols (DAG) are lipid second messengers which are generated during phospholipase-catalyzed hydrolysis of phospholipids. The model DAG, sn-1,2-didecanoylglycerol (DIC10), is an effective topical tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. We now report that 11/12 of DMBA-initiated/DIC10-promoted papillomas examined contain an A-->T mutation in the 61st codon of the Ha-ras gene, suggesting that DAGs affect the clonal expansion of activated Ha-ras-containing cells. To explore further the DIC10-induced clonal expansion of activated Ha-ras-containing cells, we have examined the tumor-promoting effect of DIC10 in the skin of transgenic TG.AC mice, which harbor a v-Ha-ras transgene. By 9 weeks of promotion, 100% of the TG.AC mice developed squamous papillomas and by 15 weeks these mice developed > 20 papillomas/mouse. Because fatty acids are known to participate in signal transduction pathways, and since cellular lipases could cleave the fatty acid side chains present in DIC10, we have examined the tumor promoting activity of n-decanoic acid to verify the specificity of promotional activity of DIC10. n-Decanoic acid did not function as a tumor promoter. These data implicate DAG as an effector of the clonal expansion of mutated Ha-ras-containing cells, and support a mechanism whereby an increase in endogenous DAG could contribute to the clonal expansion of cells containing a Ha-ras oncogene.

Electrospray mass spectrometry for characterization of lipid a fromEnterobacter agglomerans

Negative ion and positive ion electrospray mass spectrometry have been employed to characterize the lipid A mixture produced by hydrolysis of lipopolysaccharides from Enterobacter agglomerans, a Gram-negative bacterium commonly found in field cotton. Neutral monophosphoryl lipid A molecules form stable anions via deprotonation, but adduct formation via cation attachment occurs in low yield. Dephosphorylated lipid A molecules, on the other hand, readily form adducts with Na+, whereas deprotonation occurs in low yield. The mass spectra of lipid A produced by E. agglomerans reveal the presence of lipid A ions which differ in the nature of attached fatty acid side chains. At least two heptaacyl forms of lipid A are present, one of which has a structure which appears to be the same as the structure of heptaacyl lipid A produced by Salmonella minnesota. The second structure differs only by the nature of the side chain at position 3' of the disaccharide backbone where a hydroxymyristoyloxymyristoyl group replaces the myristoyloxymyristoyl substituent. Collisionally activated dissociations prior to mass analysis enable the identification of fragment ions which can be distinguished from at least eight intact deprotonated molecules present in crude lipid A.

Preparation and structure-activity relationships of simplified analogues of the antifungal agent cilofungin: a total synthesis approach.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.

Stereochemical requirements for the modulation by retinoic acid of thyroid hormone activation of Ca(2+)-ATPase and binding at the human erythrocyte membrane.

Physiological concentrations of retinoic acid can block the activation of human erythrocyte Ca(2+)-ATPase in vitro by thyroid hormone [Smith, Davis & Davis (1989) J. Biol. Chem. 264, 687-689]. The present studies were undertaken to ascertain the nature of this blockade. Two binding sites for L-thyroxine (T4) were demonstrated on washed erythrocyte membranes; the high-affinity site had a Kd value of 2.7 x 10(-10)M and a Bmax. of 76 fmol/mg of protein. The lower-affinity site possessed a Kd of 1 x 10(-8) M. Retinoic acid was as potent a displacer of radiolabelled T4 as was the unlabelled hormone. Certain retinoic acid analogues with either ring or fatty acid side chain modifications retained some ability to displace [125I]T4 binding and to block iodothyronine activation of Ca(2+)-ATPase. The side chain terminal carboxyl group was essential for full activity of the retinoic acid molecule. Its absence or replacement with an ethylsulphone group rendered the molecule considerably less active in the ATPase model. Retinol, 13-cis-retinoic acid, benzene-substituted all-trans-retinoic acid and polyprenoic acid all failed to influence iodothyronine binding or to block activation of Ca(2+)-ATPase by T4. There was good agreement between the ability of an analogue to displace [125I]iodothyronine binding and its ability to inhibit the T4-dependent activation of the Ca(2+)-ATPase. It would appear from these observations that retinoic acid can modulate the activation of erythrocyte membrane Ca(2+)-ATPase by thyroid hormone through a mechanism which involves displacement of iodothyronine from binding sites. These activities apparently derive from both the ring structure and the fatty acid side chain of the retinoic acid molecule.

Interactions of saturated diacylglycerols with phosphatidylcholine bilayers: a deuterium NMR study

The interactions of a series of saturated diacylglycerols (DAGs) with fatty acid side chain lengths of 6-14 carbons with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts were studied by 2H NMR spectrometry. We found that the perturbation induced by the DAGs into the bilayer structure strongly depends on the length of the DAG fatty acid side chain. Shorter chain 1,2-sn-dihexanoylglycerol and, to a larger degree, 1,2-sn-dioctanoylglycerol (diC8) induce transverse perturbation of the bilayer structure: the order parameters of the phospholipid side chains are increased by the intercalating DAG molecules in the region adjacent to the phospholipid headgroups and decreased toward the terminal methyls, corresponding to the bilayer interior. The longer chain DAGs (C greater than or equal to 12) studied in this and previous [De Boeck & Zidovetzki (1989) Biochemistry 28, 7439] work induce lateral phase separation of the lipids into DAG-enriched gellike domains and relatively DAG-free regions in the liquid-crystalline phase. Each of the DAGs studied induces a decrease in the area per phospholipid molecule, and a corresponding increase in the lateral surface pressure of the bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeability Changes by Peroxidation of Unsaturated Liposomes with Ascorbic Acid/Fe2+

AbstractLiposomes composed of phosphatidylcholine having a polyunsaturated fatty acid side chain were peroxidized in ascorbic acid/Fe2+ solution. Lipid peroxidation and the change in membrane permeability were monitored by the formation of thiobarbituric acid reactive substance (TBARS) and the release of entrapped fluorescein isothiocyanate-labeled superoxide dismutase (FITC-SOD), respectively. Peroxidation of liposomes composed of dipalmitoylphosphatidylcholine and 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) having 4 double bonds on one fatty acid side chain showed high TBARS value and caused the release of FITC-SOD. This release started when TBARS reached a definite value. But liposomes composed of phosphatidylcholine having 1 or 2 double bond(s) on one fatty acid side chain caused little increase in lipid peroxidation and FITC-SOD release. During the peroxidation of PAPC-liposomes, the breakdown of PAPC and formation of lysophosphatidylcholine (or like substance) were detected by HPLC analysis...

Hydrolysis of glycerides by organic-solvent-soluble lipase

To investigate the structural properties of the active site in the lipase molecule, several glycerides have been hydrolysed by organic-solvent-soluble lipase in a homogeneous solvent system consisting of a buffer and tetrahydrofuran (1:4, v/v). 2-Monoacyl glyceride was not digested by this enzyme. For other glycerides, varying the acylated position in glycerol affects the Michaelis constant for interaction with the organic-solvent-soluble lipase, although the maximum velocity value between each glyceride and the organic-solvent-soluble lipase is similar. The values of Km for 1-monopalmitim, 1,2-dipalmitin, 1,3-dipalmitin and tripalmitin are 19.35, 11.69, 5.85 and 5.34 mmol dm–3, respectively. The effect of both the length and number of unsaturated bonds in the fatty acid chain in the glyceride has been investigated in detail. The results suggest that the three-dimensional structure of the fatty acid side chains of the glycerides does not affect the hydrolysis reaction and that the affinity of glycerides towards the enzyme depends on the conformation of the glycerol moeity.

The modulation of ferryl myoglobin formation and its oxidative effects on low density lipoproteins by nitric oxide

This study has investigated the interactions between nitric oxide and haem protein radicals. The results demonstrate that nitric oxide interacts with activated ferrylmyoglobin species with reduction to metmyoglobin, but the extent and duration of the reduction depends on the relative concentrations of nitric oxide and hydrogen peroxide. Ferryl myoglobin has a much greater relative potential for oxidising polyunsaturated fatty acid side chains in low density lipoproteins than in cell membranes. The peroxidative response can be modulated by nitric oxide: ferryl myoglobin-mediated peroxidation of LDL may be enhanced or suppressed by nitric oxide depending on the relative concentrations of NO and hydrogen peroxide.

Structure-activity relationships of retinoids as inhibitors of calmodulin-dependent human erythrocyte Ca2+-ATPase activity and calmodulin binding to membranes

All-trans retinoic acid displaces the binding of radiolabelled calmodulin to human erythrocyte membranes, and inhibits the activity of plasma membrane Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase; EC 3.6.1.3). This enzyme is dependent upon the action of calmodulin. In this study we explored the structural attributes of the retinoids which confer this ability to inhibit enzyme activity and calmodulin binding. With respect to the fatty acid side-chain, a clear requirement for inhibition is a trans-configuration of the polar end-group. The importance of the ring structure is indicated by the ineffectiveness of polyprenoic acid and a benzene ring retinoid analogue as inhibitors of enzyme activity and calmodulin binding. There was good correlation between the relative potencies of the analogues as enzyme inhibitors and as inhibitors of calmodulin binding. The ability of selected retinoid analogues, at physiological concentrations with respect to all-trans retinoic acid, to inhibit erythrocyte Ca(2+)-ATPase activity and membrane binding of calmodulin underscores the structurally specific effects of these compounds on the interaction of calmodulin with the membrane-bound enzyme.

Mass spectrometry studies of a novel digoxin-like substance (DLIS-2) isolated from human plasma ultrafiltrate

Two unique low molecular weight (531) compounds with both digoxin-like immunoreactivity and Na, K-ATPase inhibitory properties have been isolated from human plasma. One of these, digoxin-like substance 2, (DLIS-2), was studied by fast atom bombardment mass spectrometry and collisionally activated dissociation mass spectrometry/mass spectrometry. The fragment patterns were interpreted as being derived from a lysophosphatidyl serine containing a novel 19:4 fatty acid side chain. The molecular formula C25H42O9NP is consistent with these observations.

The formation of daptomycin by supplying decanoic acid to Streptomyces roseosporus cultures producing the antibiotic complex A21978C

Antibiotic substance A21978C is a complex of compounds having a common cyclic polypeptide nucleus and different fatty acid sidechains. Daptomycin is a semi-synthetic antimicrobial substance derived from the A21978C complex by a very elaborate chemical process. To obtain the daptomycin, the A21978C complex was first isolated from culture filtrates of Streptomyces roseosporus by several resin procedures. The resulting material was then ‘blocked’ and added to an Actinoplanes culture for deacylation. The protected A21978C nucleus was subsequently isolated by the same procedure as the parent complex and reacylated with the desired fatty acid (decanoic acid). The acylated compound was deblocked to yield daptomycin. This report describes the experimentation undertaken to establish a strategy to supply a very toxic precursor to cultures of S. roseosporus and, thereby, produce daptomycin biosynthetically.

Gamma-Aminobutyric acid esters. 3. Synthesis, brain uptake, and pharmacological properties of C-18 glyceryl lipid esters of GABA with varying degree of unsaturation.

A series of 14C-labeled and unlabeled di-gamma-aminobutyric acid esters of glyceryl lipids having zero to three double bonds (stearoyl, oleoyl, linoleoyl, and linolenoyl) were synthesized. Measurements of the octanol/water partition coefficients of the compounds showed an increase with decreasing number of double bonds (i.e., from linolenoyl to stearoyl). The brain-uptake index went up from 31.5 (linolenoyl) to 45.1 (stearoyl) and similarly the brain-penetration index went up from 15 (linolenoyl) to 28 (stearoyl). Intraperitoneal injections of these di-GABA lipid esters produced a substantial inhibition of the general motor activity in mice at a dose of 30 mg/kg; the most active molecules were those containing two and three double bonds, i.e., the linolenoyl and linolenoyl derivatives. This is in reverse order to that predicted by brain-uptake and lipid-solubility properties, suggesting that the structure of the fatty acid side chain may be an additional factor in influencing biological activity.

Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase.

The importance of the glutathione (GSH) redox cycle and of catalase as intracellular antioxidant defense systems in cultured endothelial cells against an extracellular flux of H2O2, a critical mediator of polymorphonuclear leukocyte-induced oxidant injury of endothelial cells, was examined. The activities of different parts of the GSH redox cycle were impaired by 1,3-bis(2-chloroethyl)-1-nitrosourea, buthionine sulfoximine, diethyl maleate and 2-cyclohexene-1-one. Catalase activity was inhibited by 3-amino-1,2,4-triazole. After an impairment of the GSH redox cycle, but not of catalase, the susceptibility of pulmonary artery endothelial cells to an attack by H2O2 was dramatically increased independent of the source of extracellularly generated hydrogen peroxide (i.e., glucose oxidase or stimulated polymorphonuclear leukocytes). Exogenous catalase, d-alpha-tocopherol, and particularly Trolox, the chroman compound of tocopherol, but not phytol, the fatty acid side chain of tocopherol, provided almost complete protection of the endothelial cells against a H2O2-mediated attack. Additional fluorometric studies suggested that H2O2 is scavenged by the antioxidants before it hits the target cells.