Separation of alkylacyl and diacyl glycerophospholipids and their molecular species as naphthylurethanes by high-performance liquid chromatography.
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Biomedical subjects
Publications and source records attributed to G Reichmann.
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Rat liver mitochondria were incubated with [3H]glycerol 3-phosphate, ATP, CTP and coenzyme A allowing acylatin of glycerophosphate with endogenous fatty acids and the further conversion of labelled phosphatidic acid (PA) to diacylglycerol (DG), CDP-diacylglycerol (CDP-DG) and phosphatidylglycerol (PG). In these glycerolipids, the distribution of label among the individual molecular species was found to be similar, with 16:0-18:1, 16:0-18:2 and 18:0-18:2/16:0-16:0 being the main species. It was concluded that mitochondrial enzymes involved in the de novo synthesis of these glycerolipids exhibited no acyl selectivity for their substrates. The pattern of molecular species of mitochondrial PA, DG and CDP-DG closely approached that of the same glycerolipids synthesized de novo in isolated rat liver microsomes.
The species pattern of phosphatidic acid was compared with that of CDP-diacylglycerol and diacylglycerol synthesized de novo by glycerol 3-phosphate acylation in a CoA ester-generating system in liver microsomes. The similarity of the species patterns of phosphatidic acid and CDP-diacylglycerol indicated that the CTP-phosphatidyl cytidylyltransferase showed no selectivity for individual species of its phosphatidic acid substrate. Since the species pattern of diacylglycerol deviated from that of phosphatidic acid, a slight acyl selectivity of the phosphatidic acid phosphohydrolase or a slight inhomogeneity of its substrate pool might be assumed. For the determination of the molecular species of CDP-diacylglycerol, a new method was developed. By incubation of CDP-diacylglycerol with oligonucleate 5'-nucleotidohydrolase (phosphodiesterase), phosphatidic acid was produced. The CDP-diacylglycerol-derived phosphatidic acid was methylated with diazomethane and then separated by reverse-phase HPLC in 15 molecular species.
Mammary-derived growth inhibitor (MDGI), a polypeptide growth inhibitor isolated from lactating bovine mammary tissue, previously shown to have extensive sequence homology with fatty acid-binding proteins, was demonstrated to meet the criteria of a fatty acid-binding protein. The protein was found to bind [3H]palmitic acid in a saturable manner and to be complexed with endogeneous free fatty acids. [3H]palmitic acid, when bound to the protein, was more rapidly taken up by the target cells (human mammary carcinoma cells [MaTu]) than was free [3H]palmitic acid, suggesting a lipid carrier function for the inhibitor. It is suggested that the fatty acid-binding properties of MDGI may relate to its ability to inhibit cell growth in vitro and to regulate other cellular functions.
The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant.
The species pattern of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from [14C]glycerol 3-phosphate was measured using a newly developed HPLC technique yielding 13 molecular species. A direct comparison of these species patterns presupposes determination of the lipolytic activity of lung microsomes. The lipolytic activity was quantitatively determined by measuring the changes of the endogenous concentration of diacylglycerol, triacylglycerol and free fatty acids. The species pattern of endogenous diacylglycerol measured in the time-course of lipolysis did not show any changes up to an incubation period of 20 min, suggesting that the lipolytic activity showed only a very low selectivity for individual substrate species. Diisopropylfluorophosphate (5 mumol/mg microsomal protein) strongly decreased the lipolytic activities as well as the microsomal phosphatidate phosphohydrolase activity, as measured by means of exogenous phosphatidic acid, and also the generation of phosphatidic acid from [14C]glycerol 3-phosphate. In lung microsomes, labeled phosphatidic acid and diacylglycerols were synthesized from the endogenous free fatty acids and sn-[14C]glycerol 3-phosphate, which had previously been added. By addition of CDPcholine to the prelabeled microsomes the synthesis of phosphatidylcholine was measured. After hydrolysis of phosphatidic acid and phosphatidylcholine with cytoplasmatic phosphatidate phosphohydrolase or phospholipase C, respectively, the de novo synthesized species patterns of these two lipids and of the diacylglycerol were determined. Comparison of the species pattern of de novo synthesized phosphatidic acid with that of diacylglycerol largely showed the same distribution of radioactivity among the individual species, except that the relative proportion of label was higher in the 16:0/16:0 and 16:0/18:0 species of phosphatidic acid and lower in the 16:0/20:4 and 18:0/20:4 species than in the corresponding species of diacylglycerol. The species pattern of de novo-synthesized diacylglycerol showed no differences from that of the phosphatidylcholine synthesized from it. From this result we concluded that the cholinephosphotransferase of lung microsomes is nonselective for individual species of the diacylglycerol substrate. The 16:0/18:1 and 16:0/18:2 species of phosphatidic acid, diacylglycerol and phosphatidylcholine showed a higher synthesis rate than their 18:0 counterparts, whereas the 16:0 or 18:0 analogues of species containing 20:4 and 22:6 fatty acids showed nearly the same synthesis rates.(ABSTRACT TRUNCATED AT 400 WORDS)
The lipid content, phospholipid composition and fatty acid composition of the predominant phospholipids were determined in cortical mitochondria and microsomes of rat kidney. Microsomes have a higher lipid content than mitochondria due to their higher content of phospholipid and cholesterol. Both subcellular fractions differ in their phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine form together about 80% and 60% of total phospholipid in mitochondria and microsomes, respectively. The fatty acid patterns of the major phospholipids in mitochondria and microsomes are very similar. Our detailed data will be a well founded basis for studying the influence of pathological events on the lipid composition of kidney.
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Comparisons of erythrocyte and plasma phospholipids made among adults, newborns, and a female patient with Diamond-Blackfan anaemia (DBA) revealed some indications for the continual existence of a neonatal phospholipid distribution in DBA. The relative percentage of phospholipids in erythrocytes and plasma were similar in newborns and in the female patient. The other peculiarities characteristic of newborns, such as deviations in the absolute phospholipid content, typical fatty acid patterns of phospholipids, could not be identified in DBA.
The concentrations of the free fatty acids, the triglycerides, the glycerol, and the phospholipids were measured in the perfusate of 10 dog kidneys after hypothermic pulsatile preservation of 48 hours duration. The concentration in free fatty acids decreased in which the fatty acid pattern is unchanged essentially. The free glycerol and the total phospholipids increased. These characteristics of the perfusate refer to an increased lipolysis of neutral fats and a changed membrane composition of the kidney during the preservation.
A prospective double-blind clinical trial was carried out to determine whether ambroxol (bromhexine metabolite VIII) treatment (1000 mg/day for a period of 5 days) reduces the risk of hyaline membrane disease (HMD) in potentially premature infants. Amniocentesis was performed before the first and 24 h after the last application of ambroxol or placebo to assess the development of the total phospholipid phosphorus content, the L/S ratio, the P/S ratio, and the properties of the surface tension of the amniotic fluid after ambroxol or placebo. There were 246 infants born to 224 mothers. Of the 116 infants with less than or equal to 36 completed weeks' gestation, 56 were in the ambroxol and 60 in the placebo group. No differences between groups occurred in risk factors for HMD (diabetes, asphyxia, male sex, cesarean section). Statistically significant differences in favor of the infants in the ambroxol group were found in the HMD incidence: 23.2% in the ambroxol group compared with 41.7% in the placebo group (p less than 0.05). There was no reduction of the HMD incidence in the less than or equal to 32-week gestational age category in the ambroxol group compared with the placebo group inspite of the fact that all the examined parameters for determining lung maturity reflected a stimulatory effect of ambroxol compared with the results of the placebo group, particularly before the 33rd week of gestation. Prolonged rupture of the membranes played no protective role against HMD.
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The content and distribution of phospholipids (PL) in red blood cells (RBC) of PK and of G6PD deficiency were found to differ in both disorders. RBC immaturity could not be excluded as reason for PL alterations in PK deficiency. The relative diminution of phosphatidylethanolamine in G6PD deficiency may be connected with the enzyme depletion in this disorder and its influence on the PL of RBC membranes. Moist or all fatty acid deviations seem to be conditioned by RBC immaturity. Nearly all PL fatty acid aberrations in G6PD deficiency RBC were observed also in PK deficiency RBC. Additional deviations of PL fatty acids in PK deficiency RBC may be caused by the stronger immaturity of the cells in this disorder.
Lipids in erythrocytes and plasma of children and adults with terminal renal insufficiency were determined and compared with those of normal controls. The erythrocyte phospholipids in uremic patients were altered in phosphatidylethanolamine (absolutely and relatively elevated) and in lecithin (relatively diminished). Sphingomyelin, phosphatidylserine, total erythrocyte phospholipid content and cholesterol were within the normal range. The detailed analysis of the distribution of plasma phospholipids showed an increase in all phospholipids except phosphatidylserine. The unesterified plasma cholesterol was also found to be elevated. The fatty acid distribution in the individual erythrocyte phospholipids showed the following variations in comparison with controls: one main fatty acid was significantly decreased in sphingomyelin and in lecithin and slightly decreased in phosphatidylethanolamine. It could be shown that the lipid alterations in uremic erythrocytes were quite different from those in plasma. There were not typical signs for an increased lipid peroxidation in the erythrocytes of patients with renal insufficiency.
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Monethanolamine, a frequent impurity of diethanolamine, inhibits the activity of the isoenzymes of alkaline phosphatase to various extents. Isoenzymes from liver and bone, in particular, are strongly inhibited. Inhibition is stronger at lower (25 degrees C) than at higher temperatures (37 degrees C).
The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.