Phosphatidyldimethylethanolamine Research Articles

Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties

In the yeast Saccharomyces cerevisiae, two membrane-associated enzymes catalyze the three-step methylation of Phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Phosphatidylethanolamine methyltransferase (PEMT) catalyzes the first methylation reactions (PE → phosphatidylmonomethylethanolamine (PMME)) and phospholipid methyltransferase (PLMT) catalyzes the second two methylation reactions (PMME → phosphatidyldimethylethanolamine (PDME) → PC). Using gene disruption mutants of the S. cerevisiae OPI3 and CHO2 genes, we independently studied the enzymological properties of microsome-associated PEMT and PLMT, respectively. The enzymological properties of the enzymes differed with respect to their pH optima, cofactor requirements and thermal lability. For the PEMT reactions, the apparent K m values for PE and S-Adenosylmethionine (AdoMet) were 57 μM and 110 μM, respectively. For the PLMT reactions, the apparent K m values for PMME and PDME were 380 μM and 180 μM, respectively. The apparent K m values for AdoMet were 54 μM and 59 μM with PMME and PDME as substrates, respectively. S-Adenosylhomocysteine ( AdoHcy) was a competitive inhibitor of PEMT ( K i = 12 μM) and PLMT ( K) i = 57 μM and K i = 54 μM for PMME and PDME, respectively) with respect to AdoMet. AdoHcy was a noncompetitive inhibitor of PEMT ( K) i , = 160 μM) and PLMT K i = 320 μM and K i = 120 μM) with respect to PE and PMME and PDME, respectively.

Modification of phospholipid polar head group with monomethylethanolamine and dimethylethanolamine decreases cholesteryl ester and triacylglycerol synthesis in cultured human fibroblasts

Modification of the phospholipid polar head group was achieved by supplementation of the growth medium of cultured human fibroblasts with the choline analogues monomethylethanolamine (ME) or dimethylethanolamine (DE) at a concentration of 80-200 micrograms/mL for 48 hr. The maximum concentration of phosphatidylmonomethylethanolamine (PME) or phosphatidyldimethylethanolamine (PDE) reached without affecting the phospholipid/protein ratio was about 45% of total phospholipids. Incorporation of oleic acid into cholesteryl esters and triacylglycerols was markedly inhibited after supplementation with ME or DE, and accounted for 60% and 40% of controls, respectively, at 200 micrograms/mL, whereas incorporation into phospholipids was not affected. AcylCoA:cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT) activities measured on cell-free extracts appeared to be decreased also by phospholipid polar head group modification, whereas the overall phospholipid acyltransferase activity remained unchanged. The intracellular content of cholesteryl esters and triacylglycerols, determined by the isotopic equilibrium method with radioactive cholesterol and glycerol, was found to be diminished to 50-60% and 40-50% of controls, respectively, after supplementation with the choline analogues. The study showed that modification of the phospholipid polar head group affects the activity of membrane-bound enzymes involved in the metabolism of neutral lipids.

Radial "high-performance" thin-layer chromatography used to assess fetal lung maturity

A radial "high-performance" thin-layer chromatographic (HPTLC) method is described by which the percentages and ratios of phosphatidylinositol, sphingomyelin, lecithin, phosphatidylethanolamine, phosphatidylglycerol, and dimethyl phosphatidylethanolamine may be determined simultaneously. An additional method for radial HPTLC determination of saturated phosphatidylcholine is described. We report results of application of these methods to greater than 2000 specimens of amniotic fluid from both diabetic and nondiabetic cases.

Phorbol 12-myristate 13-acetate, A23178 and l-adrenaline inhibit phospholipid methylation in human monocytes and lymphocytes. Inhibition is independent of oxyradical production and phospholipid hydrolysis

The Ca ++ ionophore A23187 and phorbol 12-myristate 13-myristate 13-acetate (PMA) caused dose-dependent inhibition of phospholipid (PL) methylation in unfractionated mononuclear cells (MNC), monocytes, and lymphocytes as measured by incorporation of 3- H-methyl-groups from [ 3 H-methyl]- L-methionine into phosphatidylcholine (PC), dimethyl phosphatidylethanolamine (PE), and monomethyl PE. This inhibitory effect did not correlate with monocyte superoxide release and was unaltered by the presence of either catalase and superoxide dismutase the NADPH osidase inhibitor, diphenylene iodonium (DPI), indicating that oxyradical-mediated oxidation of methionine was not the major cause of inbition of PL methylation. Furthermore L-adrenaline, which elevates cAMP and does not stimulate superoxide release, also inhibited PL methylation. Inhibition by PMA was not due to reduction in intracellular levels of methionine or S-adenosyl methionine. A23187 caused reduction of S-adenosyl methionine levels only at 1 μM, and had no effect at lower concentrations. Inhibition of PL methylation was shown not to be due to phospholipase A 2-dependent hydrolysis of newly methylated PL. Attmepts to reverse the inhibitory effect of either A23187 or PMA with the putative protein kinase inhibitors W-7 and H-7 were inconclusive. The mechanism of inhibition of PL methylation by A23187 and PMA remains ubclear, but does not appear to be due to oxidation of methionine or hydrolysis of newly methylated PL.

Head Group Specificity in the Requirement of Phosphatidylcholine Biosynthesis for Very Low Density Lipoprotein Secretion from Cultured Hepatocytes

We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.

Mutations in the Saccharomyces cerevisiae opi3 gene: effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis.

We report the isolation of two new opi3 mutants by EMS mutagenesis, and construction of an insertion allele in vitro using the cloned gene. We have demonstrated that the opi3 mutations cause a deficiency in the two terminal phospholipid N-methyltransferase (PLMT) activities required for the de novo synthesis of PC (phosphatidylcholine). The opi3 mutants, under certain growth conditions, produce membrane virtually devoid of PC although, surprisingly, none of the mutants displays a strict auxotrophic requirement for choline. Although the opi3 mutants grow without supplements, we have shown that the atypical membrane affects the ability of the mutant strains to initiate log phase growth and to sustain viability at stationary phase. The commencement of log phase growth is enhanced by addition of choline or to a lesser extent DME (dimethylethanolamine), and retarded by addition of MME (monomethylethanolamine). The mutant cells lose viability at the stationary phase of the cell cycle in the absence of DME or choline, and are also temperature sensitive for growth at 37 degrees especially in media containing MME. These growth defects have been correlated to the presence of specific phospholipids in the membrane. The opi3 growth defects are suppressed by an unusual mutation in the phospholipid methylation pathway that perturbs the N-methyltransferase (PEMT) activity immediately preceding the reactions affected by the opi3 lesion. We believe this mutation, cho2-S, alters the substrate specificity of the PEMT. A secondary effect of opi3 mutations is disruption of the cross pathway regulation of the synthesis of the PI (phosphatidylinositol) precursor inositol. Synthesis of inositol is controlled through regulation of the INO1 gene which encodes inositol-1-phosphate synthase. This highly regulated gene is expressed constitutively in opi3 mutants. We have used the opi3 strains to demonstrate that synthesis of either PC or PD (phosphatidyldimethylethanolamine) will restore normal regulation of the INO1 gene.

The incorporation of monomethylethanolamine and dimethylethanolamine in fetal brain aggregating cell culture.

Fetal rat brain aggregating cell cultures were exposed to varying concentrations of [3H]monomethylethanolamine (MME) and [3H] dimethylethanolamine (DME). The rate of labeling of water-soluble compounds was more rapid and the amount of radioactivity present was greater than in the lipids. After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases. Little label was associated with the free bases or their cytidyl derivative. In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of [3H]MME and [3H]DME respectively. The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed. There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline. The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated.

Modulation of phospholipid methylation in rabbit leukocytes by indomethacin.

Methylation of membrane phospholipids has been implicated as an early biochemical signal for the chemotactic migration of phagocytic cells into inflammatory sites. In this study, the ability of indomethacin to modulate phospholipid methylation as one of its mechanisms of antiinflammatory action was investigated. Nontoxic doses of indomethacin (10(-4) M and 10(-5) M) were found to retard the methylation of phosphatidylmonomethylethanolamine (PMME) to phosphatidyldimethylethanolamine (PDME) and phosphatidylcholine (PC) in rabbit leukocytes, suggesting inhibition of methyltransferase II activity. Indomethacin was, however, without effect on the uptake of L-[methyl 3H]methionine by rabbit leukocytes. It is suggested that the inhibition of membrane phospholipid methylation could result in the suppression of inflammatory responses such as prostaglandin and leukotriene synthesis, generation of reactive oxygen radicals, and chemotaxis.

The phospholipid- N-methyltransferase of rat brain microsomes

The phospholipid- N-methyltransferase activity of rat brain microsomes had an optimum pH of 11.0 in the absence or presence of phosphatidylethanolamine (PE) but pH 10.0 in the presence of phosphatidyl-monomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). An apparent K m for S-adenosyl methonine from 0.10 to 0.12 mM was observed with exogenous methylated phospholipids PMME or PDME. Methylated neutral lipid was the major lipid produced in the absence of the exogenous acceptors. Two exogenous phospholipids, PMME and PDME, significantly stimulated microsomal phospholipid- N-methyltransferase activity and the predicted methylated phospholipids were the major products. PE additions did not cause any stimulation of methylated lipid formation. Preincubation of particles at temperatures from 40 to 100° C resulted in a loss in the microsomal phospholipid- N-methyltransferase activity that was stimulated by PMME and PDME.

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg 2+ (10 m m) or Ca 2+ (1 m m), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 m m Mg 2+ and 1 m m Ca 2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent K m values for S-adenosyl- l-methionine were 20 and 400–500 μ m for the conversion of PE to PME and for the conversion of PME to PC they were 5 μ m and 40 μ m. S-Adenosyl- l-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [ 14C]methyl incorporation into total lipid. The apparent K i value for AdoHcy was 44.7 μ m. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approxiamtely 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A 2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A 2 in the thymocyte plasma membrane.

Norepinephrine stimulation of phospholipid methylation in rat cortical synaptosomes: Fact or artifact?

Synaptosomes from rat cerebral cortex incubated with 3H-S-adenosyl-L-methionine (3H-SAM) displayed an increase in chloroform- extractable tritium when norepinephrine was added to the reaction mixture. The products of this mixture were maximally generated from intact synaptosomes, only partially inhibited by propranolol, and not enhanced by exogenous phospholipids. Thin layer chromatographic analysis of these chloroform extracts in three solvent systems yielded large norepinephrine- stimulated peaks of radioactivity that did not consistently co-chromatograph with authentic methylated phospholipid standards: phosphatidylmonomethylethanolamine (PME), phosphatidyldimethylethanolamine (PDE), and phosphatidylcholine (PC). Further, attempts to identify these peaks of radio-activity using as standards several putative methylated products of varied chemical classes, failed to elucidate likely candidates. It appears that while norepinephrine markedly stimulates the amount of tritium extracted into the chloroform phase, careful and positive structural elucidation of formed products is required before it can be concluded that these are indeed methylated phospholipids.

Phospholipid methylation and human platelet function

Lysed human platelet preparations were shown to methylate phosphatidylethanolamine to the mono-, di- and trimethyl products. At a low concentration of S-adenosylmethionine (SAM). 2 μM, the major product formed was phosphatidylmonomethylethanolamine (PNE). There was a broad pH optimum from 9 to 11 for the formation of PNE. The combined formation of phosphatidyldimethylethanolamine (PNNE) and phosphatidylcholine (PC) showed a peak of activity at pH 8.2 followed by a broad shoulder up to pH 12. At a higher concentration of SAM (200 μM). the predominant product formed was PC; the pH dependence for formation of the combination of PNNE and PC under these conditions showed a broad peak from 9 to 11. Inhibitors of SAM-dependent methylations. S-adenosylhomocysteine (SAH) and 3-deazaadenosine (3-DZA), blocked phospholipid methylation in lysed platelets and whole cells respectively. 3-DZA, did not inhibit platelet responses to thrombin, ADP, epinephrine, collagen, arachidonic acid and the calcium ionophore A23187 as measured by serotonin release, platelet aggregation, malondialdehyde formation, clot retraction, or arachidonic acid release at high agonist concentrations, and appeared to potentiate responses at low concentrations. Prostaglandin-induced inhibition of platelet function was unaffected by 3-DZA. Thrombin stimulation of platelets failed to produce the changes in methylation of phospholipids that have been associated with receptor activation of other cell types. Phospholipid methylation is not required for intial activation of platelets but the inhibition of this methylation may activate platelet function at low levels of agonists.

Alteration of the membrane lipid composition and infectivity of vesicular stomatitis virus by growth in a Chinese hamster ovary cell sterol mutant and in lipid-supplemented baby hamster kidney clone 21 cells.

The cholesterol and phospholipid composition of the membrane of vesicular stomatitis (VS) virus was altered by growth in a sterol auxotroph Chinese hamster ovary (CHO MI) host cell and by infection of CHO MI and baby hamster kidney (BHK)-21 cells supplemented with fatty acids and dimethylethanolamine. VS virus released from infected CHO MI sterol auxotroph cells grown in delipidated serum had a 50% lower ratio of cholesterol to phospholipid and an 80% drop in infectivity measured by plaque formation on L-929 cells compared with VS virus released from infected CHO MI cells grown in fetal calf serum. When VS virus was harvested from infected BHK-21 cells fed the choline analogue dimethylethanolamine, 29% of the membrane phospholipids were phosphatidyldimethylethanolamine (PDME); 87% of the PDME was located in the external monolayer of the virus membrane as determined by phospholipase C hydrolysis. Exogenous fatty acids added to the medium of cells infected with VS virus comprised up to 30% of the fatty acyl chains of the viral glycerophospholipids. The presence of PDME or unusual fatty acyl chains in the viral membrane had no effect on viral infectivity. These data indicate that the lipid composition of the VS virus membrane is determined primarily by the lipids available in the host cell and that only cholesterol content affects the biological activity of the virus membrane.

The methylating system for 3-sn-phosphatidylcholine biosynthesis in Fusarium oxysporum

Cell extracts of hyphae of Fusarium oxysporum f. sp. lycopersici rapidly transferred the methyl group of S-[methyl-3 H]adenosyl-L-methionine (Ado-Met) to endogenous phosphatidylethanolamine (PE). About 80% of the radioactivity incorporated into the phospholipid fraction was found in phosphatidylcholine (PC) while the rest of the radioactivity was present in the intermediates monomethylphosphatidylethanolamine (MePE) and dimethylphosphatidylethanolamine (DiMePE). The phospholipid methylating system had a pH optimum of 8.5, a Km of 30 μm for Ado-Met, and a Vmax of 10 nmol/h per milligram protein. The specific activity of the methylating system was highest in early log phase and lowest in the late log phase of growth.The activity of the cell-free methylating system was reduced by incubation at temperatures above 25 °C, and at 37 °C about 50% of the initial methylating activity remained after incubation for 15 min. In contrast, the activity of the in vivo methylation system almost doubled when the incubation temperature was raised from 25 to 37 °C.

Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells.

We have cultured myogenic cells derived from primary explants and a cell line (L6) in a lipid-depleted medium (LDM) and produced large alterations of the fatty acyl and polar headgroup composition and of the cellular sterol levels. These alterations were produced by altering the composition of the media as follows: removing biotin and providing exogenous fatty acid; removing choline and providing exogenous ethanolamine or choline analogues; and by adding 25-OH cholesterol, an inhibitor of 3-hydroxy-3-methylglutarate (HMG)-CoA reductase. Relatively small, secondary alterations of other lipid classes accompany the large primary alteration. In general, they are not obviously compensatory for the primary alteration by retaining some physical property. We have explored the influence of these lipid alterations on myoblast proliferation and fusion into myotubes. In general, considerable variability appears tolerated, but there also appear to be limits. Long-term cultures grown in media containing a single fatty acid do not proliferate indefinitely, and the fatty acid does not become the sole fatty acyl component of the phospholipids. This phenomenon is also observed for cultures enriched in phosphatidylethanolamine (PE) or phosphatidyldimethylethanolamine (PDME). The influence of the lipid alterations on fusion is particularly interesting. The inclusion of 25-OH cholesterol inhibits fusion. Enrichment of the fatty acyl chains with elaidate or the polar headgroups with PE also inhibits fusion, but in contrast to that by 25-OH cholesterol, a significant fraction of the myoblasts are aligned and interacting with each other. Oleate enrichment enhances the rate of fusion.

Fatty acids of phospholipids in human neonatal lung surfactant.

The fatty acid chains of lecithin, phosphatidyl ethanolamine (PE), and phosphatidyl dimethylethanolamine (PDME) in human neonatal alveolar washes were analyzed. Both alpha- and beta-palmitic acid increased with gestational age on all phospholipids analyzed. No evidence for high concentrations of myristic acid was found on any phospholipid. Babies who were stillborn or who had died of hyaline membrane disease (HMD) had lower proportions of beta-lecithin palmitic acid when compared with babies who breathed normally before death. In the HMD group these levels were significantly lower.

Studies on the phospholipids in tracheal aspirate from normal full term newborn infants. Comparison with amniotic fluid

The phospholipids in tracheal aspirates from normal full term newborn infants were studied and compared to the phospholipids present in the corresponding amniotic fluids. The same classes of phospholipids were recovered from both fluids, i.e. lysophosphatidylcholine (LPC), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (S), phosphatidyldimethylethanolamine (PDME), phosphatidylethanolamine (PE), polyglycerophosphatides (PGP), phosphatidic acids (PA) and two different species of phosphatidylcholine (PC-1 and PC-2). Except for PA, the proportion as well as the fatty acid composition of each of these phospholipids was similar in tracheal aspirates and in amniotic fluids. The predominant fraction (PC-1) was the most saturated one. PI, PDME, LPC and S were, like PC-1, predominantly constituted by saturated fatty acids and accounted together for a concentration equivalent to that of PC-1. Since tracheal aspirate originates from the lung, these data suggest that the pulmonary surfactant activity in normal full term newborn infants results not only from the presence of PC-1, but also from that of PI, PDME, LPC and S.

Phospholipid composition and metabolism of Micrococcus denitrificans.

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of (32)P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of (32)P or acetate-1-(14)C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.

Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in the human fetus and newborn and etiology of the respiratory distress syndrome.

Extract: The surface-active complex lining alveoli in normal lung lowers surface tension on expiration, thus preventing alveolar collapse. Surface activity follows a developmental timetable. Infants with idiopathic respiratory distress syndrome (RDS) almost exclusively are prematurely born, and their lungs lack adequate surface activity and are deficient in the principal surface-active component, lecithin. This deficiency implies that RDS is a “disease of development,” with fetal and neonatal timetables for lecithin synthesis. The biosynthesis of lung lecithin in the living human infant was studied by examining phospholipids in lung effluent (pharyngeal aspirates, mucus), which have identical phospholipids to those in lung lavage (alveolar wash). The fatty acid esters of isolated lecithin and phosphatidyl dimethylethanolamine (PDME) were examined. The β-carbon fatty acids are indicators of the primary pathways of synthesis of lecithin: (1) a preponderance of palmitic acid signifying cytidine diphosphate choline (CDP-choline) + D-α,β-diglyceride → lecithin (choline incorporation pathway) and (2) a preponderance of myristic acid signifying phosphatidyl ethanolamine (PE) + 2 CH3 → PDME + CH3 → lecithin (methylation pathway). Fetal lung of 18 and 20 weeks showed slight incorporation by CDP-choline pathway, absence of PDME, and almost no methylation. Salivary lecithin had totally different fatty acids from lecithin in aspirates. Phosphatidyl dimethylethanolamine (PDME) (therefore methylation) was identified in aspirates as early as 22–24 week-gestation. Lecithin fatty acid esters in aspirates from premature infants after birth or those with no RDS closely resembled PDME fatty acids. With RDS, PDME disappears and β-carbon palmitic acid (therefore dipalmitoyl lecithin) increases. With recovery, PDME reappears, as does the premature infant's major lecithin, palmitoylmyristoyl. Full term infants are born with more β-carbon palmitic acid (20–40%) and by 12–18 hr have equal palmitic and myristic acids, indicating function of both lecithin synthesis pathways. Stress (hypoxia acidosis, hypothermia) cause disappearance of PDME and loss of β-carbon myristic acid. Similar changes in full term infants who do not get RDS are due to adequate lecithin synthesis by the GDP-choline pathway. The capacity to synthesize lecithin in the lung by methylation in the human fetus and newborn allows the human to be born prematurely; rabbits and sheep lack this capacity and cannot be prematures, but die from respiratory insufficiency if born too soon. Speculation: The diagnosis of RDS can be made objectively by looking for PDME in aspirates (lung effluent). Prognosis also can be established by lack or presence of PDME. The effects of various therapies also can be assessed this way. The techniques and consideration described herein can be employed to study biochemically the effects of other stresses to lung, including oxygen toxicity and anesthesia.

A two-dimensional thin-layer chromatographic system for phospholipid separation the analysis of yeast phospholipids

A two-dimensional thin-layer Chromatographie procedure is described for the separation of phospholipids. The first developing solvent was chloroform-methanol-acetic acid-water (52:20:7:3, by vol.). This was followed after the plates were washed with diethyl ether, by chloroform-methanol-40% aqueous methylamine-water (13:7:1:1, by vol.) in the second dimension. This procedure permitted the separation of each of the following phospholipids from one another: cardiolipin or diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidic acid, phosphatidyl ethanolamine, dimethylphosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, lysophosphatidyl ethanolamine, lysophosphatidyl choline and sphingomyelin. The method has been employed to study the phospholipid composition of log phase and stationary phase yeast. Both wild-type and respiratory-deficient yeast (cytoplasmic petite) have been studied after aerobic and anaerobic growth. Log phase yeast had more phospholipid than stationary phase yeast, particularly more phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol, which are the major yeast phospholipids. Cytoplasmic respiratory deficiency did not result in any differences in these major phospholipids. The phospholipid composition of log phase yeast cells was very similar irrespective of the strain and of the presence or absence of O 2 during growth. Stationary wild-type yeast, grown anaerobically, contained less phosphatidyl choline, phosphatidyl ethanolamine and cardiolipin, and more phosphatidyl inositol than aerobically grown cells.