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Activation of myocardial cAMP-dependent protein kinase by lysoplasmenylcholine.

Plasmalogen-specific, calcium-independent phospholipase A2 (iPLA2) is activated during myocardial ischemia. Accordingly, we have assessed the activation of myocardial protein kinases by the iPLA2 product, lysoplasmenylcholine. Lysoplasmenylcholine-activated protein kinase activity from heart cytosol fractionated on a DE-52 column was identified as cAMP-dependent protein kinase (PKA) based on the following: (1) protein kinase activity stimulated by cAMP and lysoplasmenylcholine co-eluted on sequential chromatographic steps; (2) lysoplasmenylcholine-activated protein kinase activity was inhibited by the PKA inhibitor, PKI; and (3) the unprimed PKA form generated from the primed form of PKA was activated by cAMP and lysoplasmenylcholine. These results demonstrate a novel mechanism for PKA activation by lysoplasmenylcholine.

Animals↗

Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma.

Aerobic incubation of human spermatozoa in the presence of catalytic amounts of ascorbate and ferrous ion results in rapid peroxidative breakdown of sperm phospholipids and fatty acids; most strongly affected are phosphatidyl ethanolamine, ethanolamine plasmalogen, and docosahexanoic acid. Both peroxidation of the endogenous sperm phospholipid and the concurrent loss of motility can be fully prevented, but not reversed, by an "antiperoxidant" factor present in human seminal plasma. Exogenously applied lipid peroxides are powerfully spermicidal. Washed human spermatozoa, at a concentration normally present in semen, treated with as little as 30 nmoles of lipid peroxide/ml become irreversibly immotile within a few minutes. The antiperoxidant factor present in human seminal plasma effectively counteracts the toxic effect of exogenous peroxidized fatty acids upon human spermatozoa, but is unable to restore motility lost by lipid peroxide action.

Chemical Phenomena↗

Arachidonic acid of platelet phospholipids is decreased after extracorporeal removal of plasma low density lipoproteins in patients with familial hypercholesterolemia.

Platelet phospholipid composition was analyzed before and after extracorporeal removal of low density lipoproteins (LDL) by LDL apheresis in six patients with familial hypercholesterolemia. Elevated levels of total plasma cholesterol and the portion of plasma cholesterol carried by LDL were reduced by 56 and 66% after LDL apheresis. Platelet cholesterol contents remained unaffected. While the phosphatidylcholine (PC):sphingomyelin (SM) ratio in plasma lipoproteins was increased by 22% following apheresis, the same parameter was lowered by 14% in platelets. LDL apheresis induced decreases in the percentages of distinct molecular species containing arachidonic acid in platelet diacyl subgroups of PC, phosphatidylinositol (PI) and phosphatidylserine (PS) as well as in alkenylacyl (plasmalogen) phosphatidylethanolamine (PE). Directly after apheresis, the percentages of molecular species with arachidonic acid of diacyl PC, diacyl PI and alkenylacyl PE were reduced by 20, 23 and 8%, respectively. Two days after the procedure, total arachidonic acid of diacyl PC, diacyl PS and alkenylacyl PE was lowered by 11, 20 and 8%. Overall, the amount of phospholipid bound arachidonic acid was reduced by 16% after apheresis (from 79.1 to 66.4 nmol/10(8) platelets). The results are thus in agreement with previous data indicating decreased phospholipid bound arachidonic acid in red blood cells after apheresis (Engelman B. Bräutigam C, Kulschar R et al. Biochim Biophys Acta 1994:1196:154). Urinary 2,3-dinor thromboxane B2, an estimate of platelet thromboxane A2 (TXA2) production, tended to be decreased following the procedure. The percentage change in the TXA2 metabolite was positively related to the magnitude of change induced by apheresis in phospholipid bound arachidonic acid. In summary, the results suggest that in patients with hypercholesterolemia, the level of plasma LDL is an important determinant of the arachidonic acid content of several platelet phospholipids.

Adult↗

Measurement of ethanolamine- and serine-containing phospholipids by high-performance liquid chromatography with fluorescence detection of their Dns derivatives.

We describe a liquid chromatographic procedure for the analysis of amino group-containing phosphoglycerides in tissue. The total lipid extract is derivatized with Dns-chloride at 50 degrees C for 3 h. Dns derivatives of phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine and lysophosphatidylserine are separated by a silica gel column with gradient elution. The eluate is monitored by fluorescence detection at 342 nm (excitation) and 500 nm (emission). Ethanolamine and serine plasmalogens can be determined indirectly by converting their derivatives into Dns-lysophosphatidylethanolamine and Dns-lysophosphatidylserine with exposure to HCl fumes. The optimal sample size for derivative formation and analysis by this method is between 1 and 10 nmol of phospholipids (30 and 300 ng of lipid phosphorus), although the lower limit of detection is about 20 pmol. By analyzing the total lipid extract of rat brain we showed that the method was applicable to the quantitative analysis of amino group-containing phosphoglycerides in tissue samples.

Animals↗

Quantitative analysis of fatty acid methyl esters and dimethyl acetals on a polar (free fatty acid phase) capillary column.

Separation of fatty acid methyl esters and dimethyl acetals from complex biological samples has been achieved by gas-liquid chromatography on a capillary column coated with free fatty acid phase. Response-correcting factors were determined, showing rather large variations with fatty acid length. Polyunsaturated fatty acid methyl esters were shown to have lower responses than saturated species, whereas dimethyl acetals and equivalent methyl esters were found to give similar responses. Total fatty acid and aldehyde compositions of human and simian erythrocytes were determined and compared, showing a somewhat higher level of linoleate and arachidonate, and a lower level of plasmalogens in simian erythrocytes.

Acetals↗

Novel quantitative method for determination of molecular species of phospholipids and diglycerides.

A novel method is described for the quantitative analysis of subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) and molecular species within each subclass of glycerophosphatides. Diradylglycerols from phospholipase C hydrolysis of the phospholipids are converted to benzoate derivatives, the benzoates are separated into their respective subclasses by thin-layer chromatography, and quantitated by measuring absorbance at 230 nm. Molecular species within individual subclasses are separated using a combination of argentation thin-layer chromatography and reversed-phase high-performance liquid chromatography with direct, on-line quantitation at 230 nm. We applied the method to the analysis of ethanolamine phosphatides from beef brain and were able to quantitate the three diradylglycerol subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) as well as ca. 29 molecular species within each of these subclasses. This new quantitative approach for the analysis of specific molecular species of glycerolipids should be applicable to studies involving a variety of biologically important lipids, such as phosphatidylcholine, phosphatidylinositol, platelet activating factor, plasmalogens, and neutral type glycerolipids including diacylglycerols.

Animals↗

Pseudo-Zellweger syndrome: deficiencies in several peroxisomal oxidative activities.

We describe an infant girl with a clinical, chemical, and pathologic syndrome remarkably similar to Zellweger cerebrohepatorenal syndrome but whose liver parenchymal cells contained abundant peroxisomes. Peroxisomal L-alpha hydroxy acid oxidase, catalase, and the plasmalogen synthesizing enzyme dihydroxy acetone phosphate-acyl transferase activities were normal; other peroxisomal enzymatic activities, including fatty acyl-CoA oxidase and D-amino acid oxidase, were reduced by 80% to 85%. Oxidation of bile acids and pipecolic acid was also deficient. Autopsy revealed the presence of neuronal heterotopia, renal cortical cysts, adrenal atrophy, and accumulation of very long chain fatty acids. The clinical and pathologic features of this case of "pseudo-Zellweger syndrome" reflect a deficiency in multiple peroxisomal activities rather than a defect in peroxisomal biogenesis. The deficient enzymatic activities require flavin adenine dinucleotide, and the underlying defect may be in the utilization of this cofactor.

Acyl-CoA Oxidase↗

Rhizomelic chondrodysplasia punctata: clinical, pathologic, and biochemical findings in two patients.

The clinical, pathologic, and biochemical features of rhizomelic chondrodysplasia punctata are described in two patients. Although both patients had clinical and radiologic similarities, one patient survived for only 13 days and the other is still alive at 8 years. The most prominent pathologic feature was the marked degenerative change in the chondrocytes from resting cartilage. Fibroblast alkyldihydroxyacetone phosphate synthase activity was markedly reduced in both patients (approximately 10% of control mean); in contrast, dihydroxyacetone phosphate acyltransferase activity was only moderately reduced (50% of control mean). Alkyl and alk-l-enyl ether (plasmalogens) levels were very low in brain and liver. The accumulation of phytanic acid observed in plasma or liver was paralleled by a reduced ability of the patients' fibroblasts to oxidize phytanic acid. Our data indicate that the genetic defect in rhizomelic chondrodysplasia punctata results in abnormalities in two apparently unrelated pathways (i.e., phytanic acid oxidation and ether lipid biosynthesis.

Brain↗

Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups.

OBJECTIVE: To use the technique of complementation analysis to help define genotype and classify patients with clinical manifestations consistent with those of the disorders of peroxisome assembly, namely the Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). STUDY DESIGN: Clinical findings, peroxisomal function, and complementation groups were examined in 173 patients with the clinical manifestations of these disorders. RESULTS: In 37 patients (21%), peroxisome assembly was intact and isolated deficiencies of one of five peroxisomal enzymes involved in the beta-oxidation of fatty acids or plasmalogen biosynthesis were demonstrated. Ten complementation groups were identified among 93 patients (54%) with impaired peroxisome assembly and one of three phenotypes (ZS, NALD, or IRD) without correlation between complementation group and phenotype. Forty-three patients (25%) had impaired peroxisome assembly associated with the RCDP phenotype and belonged to a single complementation group. Of the 173 patients, 10 had unusually mild clinical manifestations, including survival to the fifth decade or deficits limited to congenital cataracts. CONCLUSIONS: At least 16 complementation groups, and hence genotypes, are associated with clinical manifestations of disorders of peroxisome assembly. The range of phenotype is wide, and some patients have mild involvement.

Acyltransferases↗

Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation.

Bovine seminal vesicles secrete a family of similar proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called bovine seminal plasma (BSP) proteins). The biochemical properties of these proteins are well documented and considerable progress has been made concerning their biological role. At ejaculation these BSP proteins bind to the sperm surface. The binding sites on the sperm surface have been identified as choline phospholipids (specifically phosphatidylcholine (PC), phophatidylcholine plasmalogen (PC plasm) and sphingomyelin (SPM)) composed of sperm plasma membrane. Our previous studies have shown that the BSP proteins interact specifically with heparin and high-density lipoproteins (HDL), the capacitation factors in bovine. In addition, we have shown that the BSP proteins potentiate epididymal sperm capacitation induced by heparin and HDL. Recently, we showed that the BSP proteins stimulated cholesterol and phospholipid efflux from the sperm membrane. Furthermore, the lipid efflux from sperm is dependent on BSP protein concentration and duration of incubation. The loss of membrane cholesterol is an important step in the capacitation process. These results together indicate that BSP proteins play an important role in sperm membrane lipid modification events that occur during sperm capacitation.

Amino Acid Sequence↗

Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase.

Alkyl dihydroxyacetone-phosphate synthase is the second enzyme of the ether-lipid biosynthetic pathway which is responsible for the introduction of the ether linkage between a fatty alcohol and a glycerol present in a subclass of phospholipids, the plasmalogens and possibly in glycolipid membrane anchors. In this study the gene coding for alkyl dihydroxyacetone-phosphate synthase was isolated from Trypanosoma brucei. Southern blot analysis of total genomic DNA suggested the presence of a single copy gene. The analysis, together with sequencing of different cDNA clones showed that the two alleles of the gene differ in only one nucleotide. The gene encodes a protein of 612 amino acids with a calculated molecular mass of 68,891, not counting the initiator methionine. It carries a type-1 peroxisomal targeting signal (a C-terminal tripeptide--AHL) and a calculated overall positive charge of +10. The gene was expressed in a bacterial system and the corresponding protein carrying a His-tag was purified. The recombinant alkyl dihydroxyacetone-phosphate synthase and the enzyme isolated directly from the glycosomes of bloodstream-form trypanosomes have comparable kinetics. The Km for hexadecanol was 42 microM, while approximately 100 microM of palmitoyl dihydroxyacetone phosphate (DHAP) was necessary for optimal activity. Sodium chloride inhibited both the His-tagged protein and the enzyme isolated from the glycosomes of bloodstream-form and insect stage T. brucei.

Alkyl and Aryl Transferases↗

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica.

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane--isopropanol--water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.

Animals↗

Thin-layer chromatographic analysis of myelin lipids, their differential O-deacylation by primary alkylamines and their selective staining by thionine. A limited phylogenetic study.

A silica gel thin-layer chromatographic procedure is described for the study of the myelin lipid patterns in a small phylogenetic series of nerve tissue specimens. It involves the selective staining by the thiazine dye thionine and the interpretations were facilitated by a preceding primary alkylamine O-deacylation step. Glycolipids, including sulfatides, and ethanolamine plasmalogens were the principal characterizing lipids.

Acylation↗

High-performance liquid chromatography of methylated phospholipids.

A rapid high-performance liquid chromatographic method for the separation of methylated phospholipids is described. The separation is accomplished on an amine column using acetonitrile--methanol--water as the eluting solvent and UV detection at 203 nm. The choice between gradient and isocratic elution for the separation depends upon the condition of column. The method is suitable for the isolation of phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and lysophosphatidylethanolamine from tissues. It is applicable to the study of reaction products in phosphatide methyltransferase assay mixtures. Choline and ethanolamine plasmalogens can be determined indirectly by converting them into lysophosphatidylcholine and lysophosphatidylethanolamine with exposure to hydrochloric acid fumes.

Animals↗

Quantitative analysis of aminophospholipids by high-performance liquid chromatography using succinimidyl 2-naphthoxyacetate as a fluorescent label.

A simple and rapid high-performance liquid chromatographic procedure for the quantitative analysis of ethanolamine- and serine-containing phospholipids in tissue is described. The technique involves reaction of lipid extracts with succinimidyl 2-naphthoxyacetate to give fluorescent derivatives of aminophospholipids. Reaction products are separated by a silica gel column with gradient elution. The eluate is monitored by fluorescence detection at 228 nm (excitation) and 342 nm (emission). Ethanolamine and serine plasmalogens can be measured indirectly by converting their derivatives into lysophosphatidylethanolamine and lysophosphatidylserine derivatives with exposure to hydrogen chloride fumes. The method is highly sensitive and selective.

Animals↗

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry.

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS2 or MS3, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1-5 ng of injected substance.

Chromatography, High Pressure Liquid↗

Gradient elution reversed-phase chromatographic isolation of individual glycerophospholipid molecular species.

We describe a gradient elution reversed-phase high-performance liquid chromatographic approach for isolation of individual glycerophospholipid molecular species which greatly improves resolution and reduces run time compared to isocratic techniques. Separations were optimized and elution order and retention time data established by synthesizing 37 different homogeneous phospholipids comprising the major alkylacyl, diacyl and plasmalogen molecular species in samples derived from mammalian sources. Empirical equations which predict the elution order of individual species were derived. The method was validated with the use of complex mixtures of choline and ethanolamine glycerophospholipid species from isolated rabbit cardiomyocytes and porcine endothelial cells.

Animals↗

Delayed myelination in a rhizomelic chondrodysplasia punctata case: MR spectroscopy findings.

Rhizomelic chondrodysplasia punctata is a member of genetic peroxisomal disorders. Delayed myelination, which is probably related to the inadequacy of plasmalogens biosynthesis, is an important feature of this disorder. Direct assessment of neuropathologic aspects of RCDP syndrome such as neuronal degeneration and delayed myelination is possible with MR spectroscopy. In this report, MR spectroscopy findings (decreased Cho/Cr and increased Ins-Gly/Cr ratios and increased levels of mobile lipids) of a rhizomelic chondrodysplasia punctata case supporting delayed myelination are presented. This is the second report of MR spectroscopy examination of the specific brain metabolic changes associated with rhizomelic chondrodysplasia punctata.

Aspartic Acid↗