Molecular species of phospholipid in rat hepatomas and in fetal, regenerating, and adult rat livers.

The molecular species of phospholipid in rat hepatomas were found to be different from those in normal adult liver. Phosphatidylcholine in Yoshida hepatoma contained phosphatidylcholine with 34:1 as the sums of the chain length and of the unsaturation of fatty acids esterified at C-1 and C-2 of glycerol, as an example (PC34:1), PC36:2, PC36:1, PC34:2, and PC36:3, and its phosphatidylethanolamine contained PE36:2, PE36:1, PE38:4, PE36:3, and PE34:1 as major species, whereas phosphatidylcholine in normal adult liver contained PC38:4, PC36:2, PC34:2, PC36:4, and PC34:1, and its phosphatidylethanolamine (PE) contained PE38:4, PE38:6, PE40:6, PE36:4, and PE36:2, with their level decreasing in that order. While Morris hepatoma also had significantly lower amounts of species containing polyunsaturated fatty acids, there were higher levels of those phosphoglycerides containing monoenoic and dienoic fatty acids. Regenerating liver showed similar patterns of molecular species of both phospholipids to those of normal liver. In contrast, fetal liver had similar molecular species of phosphatidycholine to those of hepatomas, but had similar species of phosphatidylethanolamine to those of normal and regenerating livers.

Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle.

Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.

Catalytic properties of rabbit kidney fatty acid omega-hydroxylase cytochrome P-450ka2 (CYP4A7).

We have examined in detail the substrate specificity of a rabbit kidney fatty acid omega-hydroxylase, designated cytochrome P-450ka2 (CYP4A7). The hydroxylation products were identified as omega- and (omega - 1)-hydroxy fatty acids mainly using gas chromatography-electron impact mass spectrometry. [1] Straight-chain saturated fatty acids ranging from 10 to 19 carbons were effectively hydroxylated at the omega- and (omega - 1)-position. The ratios of omega- to (omega - 1)-hydroxylation activity decreased with increasing the carbon chain length of fatty acids. [2] Both isomyristate and anteisomyristate, and isopalmitate were hydroxylated several fold more rapidly than myristate and palmitate, respectively, with iso-branched chain fatty acids being hydroxylated at the omega-position solely. [3] Both palmitoleate and palmitoelaidate, and both oleate and elaidate were hydroxylated much more rapidly than palmitate and stearate, respectively. [4] Linoleate, gamma-linolenate, and arachidonate were also excellent substrates for this enzyme. [5] Prostaglandin (PG) A1 and PGA2 were efficiently hydroxylated at the omega-position solely, with PGE1 and PGE2 being much less active. [6] Arachidonic acid not only showed a Km value significantly lower than those for lauric acid, gamma-linolenic acid and PGA1, but also it is a potent competitor for lauric acid and PGA1, showing a very high affinity for the enzyme. It is possible that arachidonic acid is the physiological substrate for kidney P-450ka2.

Changes in molecular species of rat liver choline glycerophospholipids with development.

Molecular species of rat liver choline glycerophospholipids were investigated at various stages of development by a gas chromatograph-mass spectrometer system. They were composed mainly by 1,2-diacyglycerophosphocholine and changed with development particularly during the perinatal period.. In the prenatal period, the major species were '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl species), '34 : 0' (mainly 16 : 0/18 : 0, palmitoystearoyl speciesY, '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl species) and '34 : 2' (mainly 16 : 0/18 : 2, palmitoyllinoleoyl speciesY and '32 : 0' decreased rapidly and '34 : 1' increased as the stage proceeded. After birth, however, polyunsaturated species such as '34 : 4' and '38 : 4-6' increased rapidly in contrast to the decrease of '32 : 1, 34 : 1, 36 : 1, and 34 : 0'. Moderate changes were observed in these species during subsequent development.

Studies on molecular species of choline glycerophospholipids of developing rat brain.

The chronological changes in molecular species of choline glycerophospholipids were studied for cerebra of 17-, 19- and 21-day-old rat fetuses, and 3-, 6-, 12-, 24- and 90-day-old rats. The molecular species found by gas chromatography-mass spectrometry and selected ion retrieval technique were phosphatidylcholines of '30 : 0, 32 : 0, 32 : 1, 34 : 0, 34 : 1, 34 : 2, 36 : 0, 36 : 1, 36 : 2, 36 : 3, and 36 : 4' where the larger number indicates the sum of chain lengths on positions C-1 and C-2; the smaller number is the total number of double bonds. Of these molecular species, '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl glycerophosphorylcholine), '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl glycerophosphorylcholine), '34 : 0' (16 : 0/18 : 0, palmitoylstearoyl glycerophosphorylcholine), '32 : 1' (mainly 16 : 0/16 : 1, palmitoylpalmitoleoyl glycerophosphorylcholine and '30 : 0' (14 : 0/16 : 0, myristoylpalmitoyl glycerophosphorylcholine) were main species. The '32 : 0' species increased to about 44% at around the 10th day and thereafter remained nearly constant. '34 : 1' and '34 : 0' decreased to about 17 and 6% at that time and then increased to about 30 and 14%, respectively. '30 : 0' increased from last stage of gestation to the 6th day and then decreased. '32 : 1' was about 16% for 17-day-old fetus and decreased grandually. '36 : 1' (18 : 0/18 : 1, stearoyloleoyl glycerophosphorylcholine) increased at the latter part of development.

Lysophosphatidylcholine from white muscle of bonito Euthynnus pelamis (Linnaeus): involvement of phospholipase A1 activity for its production.

A fairly large amount of lysophosphatidylcholine (LPC) was detected in the fresh muscle of bonito Euthynnus pelamis (Linnaeus). The major fatty acid esterified in LPC were highly unsaturated fatty acids, such as docosahexaenoic and eicosapentaenoic acids, and the form was mainly composed of 1-lyso-2-acyl-sn-glycero-3-phosphocholine (1-LPC). The content of this species continued to increase during 4 months of frozen storage and then decreased. Phospholipase A1 activity detected in the bonito muscle was supposed to be responsible for the accumulation of LPC.

Methylene-interrupted double bond in polyunsaturated fatty acid is an essential structure for metabolism by the fatty acid chain elongation system of rat liver.

Some plant oils contain non-methylene-interrupted polyunsaturated fatty acids (NMIFAs). Pinolenic acid (all cis delta-5,9,12/18:3) and columbinic acid (trans,cis,cis delta-5,9,12/18:3) are NMIFAs that exist in pine seed oil and columbine seed oil, respectively. We investigated the double bond position of fatty acid recognized by the fatty acid chain elongation system (FACES) of rat liver using NMIFAs as experimental tools. In the total elongation assay, amounts of C2 unit chain-elongated metabolites of pinolenic acid and columbinic acid were 32% and 11%, respectively, compared to that of gamma-linolenic (all cis delta-6,9,12/18:3) as the substrate. In the condensation reaction assay, the rate limiting step of FACES, the conversion rates of pinolenic acid and columbinic acid to the corresponding C20 beta-keto fatty acids were 19% and 9% of that of gamma-linolenic acid, respectively. The formation of elongated metabolite of podocarpic acid (all cis delta-5,11,14/20:3) was only 7% of that of arachidonic acid (all cis delta-5,8,11,14/20:4). From these results it was concluded that the condensing enzyme of FACES could recognize the methylene-interrupted cis double bond structure vicinal to the carboxyl group in the fatty acid molecule.

Cytosolic lysophosphatidylcholine/transacylase in the production of dipolyunsaturated phosphatidylcholine in bonito muscle.

Phosphatidylcholine with docosahexaenoic acid at both sn-1 and sn-2 positions occurs in relatively high abundance in bonito muscle. To explore a possible route for the dipolyunsaturated molecular species, phosphatidylcholine formation from 2-[1-14C]linoleoyl lysophosphatidylcholine was examined using a cytosolic fraction from bonito muscle. The formation of radiolabeled phosphatidylcholine was greatest at 15 degrees C and did not require the presence of cofactors such as CoA and calcium. By DEAE-cellulofine column chromatography, the activity to form phosphatidylcholine was separated from that of phospholipase A1, and the specific activity increased by about 100-fold. The possible involvement of cytosolic lysophosphatidylcholine/transacylase in synthesis of dipolyunsaturated phosphatidylcholine is discussed.

Application of selected ion monitoring to determination of platelet-activating factor.

The selected ion monitoring (SIM) technique was applied to determination of platelet-activating factor (PAF) or acetyl glyceryl ether phosphorylcholine (AGEPC). Two types of PAF, 1-hexadecyl- and 1-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (C16 = 0 AGEPC and C18 = 0 AGEPC), were found in human neutrophils on the challenge with ionophore A23187. The contents of C16 = 0 AGEPC in 1 X 10(7) neutrophil cells of four volunteers, respectively, were 47, 18, 59, and 73 ng and those of C18 = 0 AGEPC were 22, 4, 19, and 31 ng.

Polymorphonuclear leukocyte-platelet interactions: acetylglyceryl ether phosphocholine-induced platelet activation under stimulation with chemotactic peptide.

A mixture of rabbit polymorphonuclear leukocytes (PMNs) and platelets at concentrations of 5 X 10(6) PMN and 3.5 X 10(8) platelets/ml Tyrode's solution was stimulated with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). A micromolar concentration of FMLP elicited an immediate weak aggregation, followed by a strong aggregation with a time lag of about 1 min. Microscopic examination showed that the immediate aggregation was due to PMNs and the delayed one was more complex and involved platelets. The delayed aggregation was dependent upon the concentrations of both the PMNs and FMLP. The delayed aggregation was completely blocked by pretreatment of the PMN-platelet mixture with 8 microM CV-3988, a specific receptor antagonist of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), or by the application of platelets desensitized to AGEPC. The time course of AGEPC production by PMNs was well matched to that of the biphasic aggregation response. Furthermore, nordihydroguaiaretic acid inhibited both the AGEPC production by PMNs and the delayed aggregation in a similar dose-dependent manner. These result demonstrate that AGEPC, newly-generated by PMNs under FMLP-stimulation, is of primary importance in platelet aggregation in a PMN-platelet mixed system.

Properties of phospholipase A1/transacylase in the white muscle of bonito Euthynnus pelamis (Linnaeus).

The properties of phospholipase A1 (PLA1) obtained from the white muscle of bonito, Euthynnus pelamis (Linnaeus), were examined. The PLA1 activity had a pH optimum from 6.5 to 7.0 for phosphatidylcholine (PC), and calcium ion was not required. The optimum temperature was from 20 to 30 degrees C. When a fatty alcohol was used as an acceptor, a wax ester was produced by transferring a fatty acid at the sn-1 position of the donor's PC. The maximum production of lysophosphatidylcholine was shifted by 0.5 pH units to the acidic side and the pH optimum of wax ester synthesis was from 6.0 to 6.5. The synthesis was independent of calcium ion and Coenzyme A. The transacylation was also observed when 1-lyso-2-acyl-sn-glycero-3-phosphocholine was used as an acceptor. Fatty acid at the sn-1 position of the donor PC was transferred to the unoccupied hydroxy group of the acceptor at the sn-1 position. When 2,3-dipalmitoyl-sn-glycero-1-phosphocholine was used as the acyl donor, a similar amount of palmitic acid was transferred as in the case of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. However, 1-acyl-2-lyso-sn -glycero-3-phosphocholine, a positional isomer, was a poor acceptor. These results indicate that the transacylation by the PLA1 from bonito muscle is not stereospecific, but is position-specific both for the acyl donor and acceptor.

Different mechanisms of regioselection of fatty acid hydroxylation by laurate (omega-1)-hydroxylating P450s, P450 2C2 and P450 2E1.

P450 2C2 as well as P450 2E1 [Fukuda, T. et al. (1993) J. Biochem. 113, 7-12] catalyzed the hydroxylation of medium chain fatty acids, although the regioselectivity of substrates of the former contrasted with that of the latter. Whereas P450 2E1 hydroxylated C9-C18 fatty acids at the omega-1 position and to a much lesser extent at the omega and omega-2 positions, P450 2C2 hydroxylated C9-C13 fatty acids at different positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the omega-1 position almost exclusively by P450 2C2. The proportion of omega-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation positions were enlarged to the omega-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in different manners. The mutation of P450 2C2 did not change the hydroxylation positions of C9-C12 fatty acids, but caused a significant decrease in the proportion of the omega-1 hydroxy analog in the total products. In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. Thus, the products of palmitate hydroxylation were identified as omega-1, omega-2, omega-3, omega-4, omega-5, omega-6, and omega-7 monohydroxy isomers using gas chromatography-electron impact mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of Thr-303 of P450 2E1 with serine modifies the regioselectivity of its fatty acid hydroxylase activity.

Threonine-303 of rabbit P450 2E1, which is putatively located at the distal heme surface, was replaced by serine and valine via site-directed mutagenesis. In the oxidized state, the Ser-mutated P450 exhibited a low- and high-spin mixed-type (low > high) absorption spectrum, whereas the Val-mutated P450, like the wild-type P450, exhibited a nearly high-spin type spectrum. The reduced CO complexes of the Ser- and Val-mutated P450s, as well as that of the wild-type P450, showed a Soret absorption maximum at 452 nm. Both mutated P450s were active in the hydroxylation of C10 to C18 fatty acids at somewhat lower rates than the wild-type P450. The Val-mutated P450 gave the same two products (the major one is probably the omega-1 hydroxy analog) as the wild-type P450, while additional products were formed on incubation with C11 to C17 fatty acids as substrates of the Ser-mutated P450; a total of four products was detected for each of the C12 to C15 fatty acids, and three for each of the C11, C16, and C17 homologues. The metabolites of laurate were determined by GC-MS analysis to be the omega-1, omega-2, omega-3, and omega-4 hydroxy counterparts. The Ser-mutated P450 hydroxylated drug substrates at almost the same rates as the wild-type P450, while the mutation to valine significantly lowered the drug hydroxylase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in phospholipid composition of the mitochondrial membrane after orthotopic liver transplantation.

Changes in phospholipids and their fatty acid composition in liver mitochondria obtained from allogenic rats with orthotopic liver transplants were measured with and without immunosuppressive treatment. In untreated allogenic rats, mitochondrial phosphorylation activity was severely deteriorated at 8 days after transplantation. A significant change was also found in the amount of cardiolipin compared with other classes of phospholipids. Namely, cardiolipin decreased, and lysodiphosphatidylglycerol and phosphatidylglycerol increased concomitantly. Furthermore, the percentage of linoleic acid in cardiolipin decreased dramatically. Decrease in cardiolipin and changes in its fatty acid composition may be attributed to the deterioration of mitochondrial function upon acute rejection.

Endogenous platelet-activating factor and anti-platelet-activating factor in patients with renovascular hypertension.

Renovascular hypertension is relieved by percutaneous transluminal renal angioplasty. In four patients with renovascular hypertension, platelet-activating factor (PAF) was found to be released into the ipsilateral renal venous blood after percutaneous transluminal renal angioplasty, but was not found in the contralateral renal venous blood following this procedure. Anti-platelet-activating factor with a lipid-like property was also found, and its polarity was slightly lower than that of PAF judging by its behavior on thin layer chromatography. Anti-platelet-activating factor completely blocked the aggregation of rabbit platelets induced by PAF, ADP or arachidonic acid. These results indicate that PAF and anti-platelet-activating factor are released into renal venous blood following percutaneous transluminal renal angioplasty in patients with renovascular hypertension.

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine from stimulated human polymorphonuclear leukocytes.

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl GPC) was found in the fraction of platelet-activating factor obtained from stimulated human polymorphonuclear leukocytes (PMN). The amount of 1-acyl-2-acetyl GPC obtained from 1 X 10(7) PMN stimulated with ionophore A23187 at 37 C for 15 min ranged from 8 to 56 pmol (32 +/- 10 pmol, mean +/- standard error; n = 4). The main species was 16:0 palmitoyl (17 +/- 5 pmol), followed by 18:0 stearoyl (8 +/- 3 pmol) and 18:1 oleoyl (7 +/- 3 pmol). Although the physiological significance is unknown, 1-acyl-2-acetyl GPC was always detected when 1-alkyl-2-acetyl GPC was detected.

Phospholipids from the free-living nematode Caenorhabditis elegans.

The phospholipid and the fatty chain compositions of diacyl, alkylacyl and alkenylacyl glycerophospholipids of the free-living nematode, Caenorhabditis elegans, were investigated. The phospholipids were comprised of 54.5% ethanolamine glycerophospholipid (EGP), 32.3% choline glycerophospholipid (CGP), 8.1% sphingomyelin and 5.1% others. The most abundant fatty acid in CGP was eicosapentaenoic acid (20:5n-3). The fatty acids in CGP were more unsaturated than those in EGP. Alkenylacyl and alkylacyl subclasses accounted for 1.0 and 2.6%, respectively, of CGP and 14.0 and 19.6%, respectively, of EGP. At least 80% of the alkenyl and alkyl groups were 18:0 chains and the remaining were odd numbered chains. The potential presence of platelet-activating factor (PAF) was examined by bioassay, but PAF-like activity was not detected in the extracts of this nematode.

Molecular heterogeneity of platelet-activating factor (PAF) in rat glandular stomach determined by gas chromatography/mass spectrometry. PAF molecular species changes upon water-immersion stress.

The molecular heterogeneity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylacetyl-GPC) in normal rat glandular stomach was studied by gas chromatography/mass spectrometry (GC/MS) and tandem mass spectrometry. The percentage compositions of the molecular species of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the antrum were, respectively, 1-alkyl [16:0 (34%) and 18:0 (66%)]-2-acetyl-GPC and 1-acyl [16:0 (60%), 18:0 (14%) and 18:1 (26%)]-2-acetyl-GPC. The alkyl chain composition of 1-alkyl-2-acetyl-GPC was quite different from that of 1-alkyl-2-acyl-GPC in both the antrum and corpus, demonstrating a high degree of selectivity of alkyl chain utilization in PAF biosynthesis. The amount of 1-acyl-2-acetyl-GPC was much greater than that of 1-alkyl-2-acetyl-GPC. The molecular heterogeneity of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the corpus was similar to that in the antrum. Water-immersion stress affected not only the amount of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC, but also their molecular heterogeneity in the antrum and corpus. Whereas the amounts of 1-hexadecyl-2-acetyl-GPC and 1-acyl [16:0, 18:0 and 18:1]-2-acetyl-GPC decreased markedly (to less than one-fifth) in the antrum after such stress for 1 hr, the amount of 1-octadecyl-2-acetyl-GPC increased markedly (up to 4-fold) in the corpus and severe lesions were observed after stress for 7 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of growth temperature on the fatty acid composition of the free-living nematode Caenorhabditis elegans.

The effects of growth temperature on the fatty acid compositions of the phosphatidylcholine (PC), phosphatidylethanolamine (PE), and total lipid (TL) fractions of the free-living nematode Caenorhabditis elegans were investigated. A reduction in growth temperature from 25 to 15 degrees C caused the proportions of eicosapentaenoic acid (20:5n-3) to increase from 23.6 to 32.5% in the PC, from 7.4 to 10.8% in the PE, and from 12.9 to 19.9% in the TL fractions. Conversely, the levels of dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) in these phospholipid fractions and the TL fraction both decreased with decreasing growth temperature. Analysis of the positional distribution of fatty acids in the PC fraction revealed that the change in the composition of C20 polyunsaturated fatty acid was obvious in position sn-2. Lowering the growth temperature induced an increase in the level of the diacyl subclass of PE from 58% at 25 degrees C to 71% at 15 degrees C, with a concomitant decrease in the levels of the alkylacyl and alkenylacyl subclass of PE of C. elegans. These changes observed in the phospholipids of C. elegans might be one mechanism for adaptation to low temperature.

Platelet-activating factor detected in bronchoalveolar lavage fluids from an asthmatic patient.

It recently has been recognized that platelet-activating factor (PAF) may be a mediator of asthma exacerbation. We had the opportunity to analyze bronchoalveolar lavage fluids from an asthmatic infant, which were characterized by neutrophil infiltration. The patient's lungs were washed on three occasions with saline during asthmatic attacks. PAF was found in each case on the basis of its ability to cause the immediate aggregation of washed rabbit platelets. The PAF detected was equivalent to 1-1.4 pmol of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, three quarters of which were recovered in cell-associated form. By contrast, we did not detect PAF in bronchoalveolar exudates from patients with laryngeal stenosis or with respiratory distress syndrome. LysoPAF, the direct precursor as well as initial metabolite of PAF, was also analyzed after being converted to PAF by acetylation. There was a wide variation in the amount of lysoPAF present in individual patients, suggesting that lysoPAF levels cannot be taken as an indicator for the presence of PAF.