Effect of a bis-thiazolium compound on the biosynthesis of Plasmodium falciparum phospholipids

In the eukaryotic cell, phospholipids can be biosynthesized by two pathways, one from choline and the other one from ethanolamine. The functional effectiveness of each pathway depends on the type of the cell. Thiazolium designed-drugs have shown, under in vivo conditions, antiplasmodial and antimalarial activities with inhibition of the phospholipids biosynthesis. This study aimed to discover the pathways involved in the biosynthesis of phospholipids in Plasmodium and deduce the biochemical steps inhibited by T4, a bis-thiazolium bromide drug. We compared the uptake of radiolabeled precursors and their selective incorporation in the phospholipids of cultured Plasmodium-infected and -uninfected erythrocytes which revealed that phosphatidylcholine of Plasmodium is synthesized both from choline and ethanolamine (4.7 vs 1.9 nmol/10¹⁰ cells.h^?1). T4 has no effect on the biosynthesis of phosphatidylethanolamine but T4 inhibited, in a selective way, the in vitro uptake of choline. However no enzymes in the biosynthesis of phospholipids seem to be inhibited by T4 but rather an inhibition of choline entry into the parasite.     

Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC₅₀ concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [¹⁴C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K⁺-efflux and greater leakage of intracellular [³²P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.     

Further studies on the metabolism of phospholipids inStreptomyces griseus: Turnover of phospholipid moieties

Phospholipid turnover inStreptomyces griseus was studied by pulse-chase techniques using 1-[¹⁴C]sodium acetate and [U-¹⁴C]glucose. Different phospholipids and their individual moieties were found to have different turnover rates. The moieties of inositol-containing phospholipids exhibited the fastest turnover rates among the major phospholipids, while only fatty acyl moieties of phosphatidylethanolamine showed rapid turnover. Cardiolipin did not show any significant turnover with both precursors.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

³²P-Labelled midguts (³²P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the ³²P-midguts were incubated in the absence of lipophorin, no ³²P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of ³²P-phospholipids derived from metabolically labelled ³²P-midgut or lipophorin particles after incubation with ³²P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of ³²P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of ³²P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A² was incubated with ³²P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.     

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine?phosphatidylethanolamine?0. Acidic phospholipids increased V_max-Ca²⁺ and lowered the value of K_0.5-Ca²⁺ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K_0.5-Ca²⁺ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1–116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Δ74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca²⁺-ATPase provides new evidence that type 2B Ca²⁺-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.     

THE EFFECT OF N-METHYL-4-PIPERIDYL-DIPHENYL GLYCOLATE ON THE INCORPORATION OF 32P INTO PHOSPHOLIPIDS FROM RAT BRAIN CORTEX SLICES AND ITS SUBCELLULAR LOCALIZATION

—N-Methy-4-piperidy1-diphenyl glycolate (N-methy1-4-piperidy1 benzilate), an anticholinergic drug, was shown to stimulate ³²P-incorporation into total phospholipids of rat brain cortex slices. Analysis of the total phospholipids showed stimulation in phosphatidic acid, phosphatidylinositol and phosphatidylethanolamine. Stimulated ³²P-incorpora-tion was accompanied by a decrease in the Qo₂ (μ1 O₂/mg dry tissue) value. The effects of the drug were compared with those of some other CNS-active drugs known to interfere with the ACh content of brain; tremorine, oxotremorine and atropine; and in the presence of eserine, with that of the neurotransmitter acetylcholine. Increase of the outer K+-concentration resulted in increased Qo₂ and ³²P-incorporation into the slices. The effect of the glycolate and perhaps that of atropine tended to increase in this medium. Subcellular fractionation of slices showed that the glycolate stimulated ³²P-incorporation occurred mainly in the nerve end fraction. The total amount of the individual phospholipids was not changed, but the specific activity had significantly increased in phosphatidic acid and phosphatidylinositol. The possibility that glycolate-induced stimulated ³²P-incorporation into nerve end phospholipids is due to increased glycolysis is discussed.     

Experimental pneumonitis: changes in the phospholipid metabolism of the lung of guinea pigs and rats

Changes of the metabolism of lung phospholipids and alveolar wash lipids were studied in guinea pigs and rats with experimental pneumonitis produced by the injection of complete Freund's adjuvant. The amount of total lipid obtained by lung lavage decreased significantly in contrast to a marked increase in the content of total protein in treated animals. Among the lipids in the pulmonary washings, only phospholipids were strikingly reduced, and free fatty acids were increased somewhat. However, the composition of phospholipids was not affected by the treatment. The in vitro incorporation of 1-¹⁴C-acetate into the total lipid and phospholipids in the lung with pneumonitis was slightly lower compared to controls, but the difference was not significant. The in vitro 1-¹⁴C-palmitic acid incorporation into phospholipids of the lesioned lung, was also similar to that in controls. Thus, phospholipid biosynthesis in the lung was not affected by pneumonitis. On the other hand, the in vitro activity of total phospholipase in rat lung with experimental pneumonitis was enhanced significantly. These results suggest that phospholipase activity is increased in the diseased lung and this may participate in the process of the inflammatory reaction. The possible role of activated macrophages in the inflammatory response of the lung is discussed.     

Heterogeneity of the apolipoprotein H *3 allele and its role in affecting the binding of apolipoprotein H (β2-glycoprotein I) to anionic phospholipids

Apolipoprotein H (apoH, protein; APOH, gene) has been implicated as a necessary cofactor for the binding of certain autoimmune antiphospholipid antibodies to anionic phospholipids. APOH exhibits genetically determined structural polymorphism with the occurrence of four alleles. Recently three IgG1_k monoclonal antibodies (mAb) to human apoH, designated 3G9, 1B4, and 3D11, have been produced. The mAb 3D11 does not recognize apoH bound to anionic phospholipids in contrast to mAb 3G9 and 1B4, which recognize free and phospholipid-bound apoH. In this investigation we have determined the reactivity of the three mAb with four APOH allele products and the binding ability of these allele products with anionic phospholipids. The mAb 3G9 and 1B4, like the polyclonal anti-apoH, were equally reactive with all four allelic products, but the 3D11 recognized only the APOH ^*3 allele product. In the 159 APOH ^*3 carriers tested from five ethnic groups, the reactivity of mAb 3D11 was observed with all the Chinese but none of the African blacks. For the U.S. whites and Polynesians 89% and 75%, respectively, of the APOH ^*3 allele products were recognized by 3D11, while 87% of the U.S. blacks with this allele had no 3D11 reactivity. These data show that the APOH ^*3 allele, originally identified as a single entity by the polyclonal anti-apoH, is heterogeneous with at least one distinct variation based on mAb 3D11 reactivity. Our data also demonstrate that the apoH from certain homozygous APOH ^*3 individuals is unable to bind to anionic phospholipids. Such ethnic-specific apoH variations could play a significant role in the binding properties of autoimmune antiphospholipid antibodies to anionic phospholipids.     

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane

Summary The role of phospholipids in the binding of [³H] tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A₂ (Crotalus adamanteus andNaja naja) or phospholipase C (Bacillus cereus andClostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A₂, and with phospholipase C the lipid hydrolysis was about 60–70% for a 70–80% reduction in the binding activity. Phospholipase C fromB. cereus andCl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A₂ but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A₂. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.     

Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.     

Incorporation of ω6 polyunsaturated fatty acids into phospholipids of the crab Carcinus maenas

The incorporation in vivo of ¹⁴C-18:2 ω6 and ³H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle.     

Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli

Synthesis of the high-affinity K⁺-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K⁺ limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K⁺ to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD. Received: 29 March 1999 / Accepted: 26 July 1999     

Mechanism of lipid activation of Na,K, Mg-activated adenosine triphosphatase and K,Mg-activated phosphatase of bovine cerebral cortex

Summary Na⁺, K⁺, Mg⁺⁺-activated adenosine triphosphatase and K⁺, Mg⁺⁺-activatedp-nitrophenyl phosphatase prepared from a membrane fraction of bovine cerebral cortex were studied with regard to the manner of their activation by phospholipids, using phosphatidyl serine, lysolecithin, monodecyl and didecyl phosphates. The kinetic and chromatographic studies suggested the following. (1) When the enzyme proteins bind the phospholipids in a proper ratio, they attain the optimum activation. (2) The binding causes a simple conversion of the enzymes from an inactive form to a fully activated form. (3) The lipids in both micellar form and molecular dispersion activate the enzymes. (4) Of the proteins contained in the enzyme preparation, only a group of proteins possessing the ATPase and the phosphatase activities bind phospholipids, and the amount of the bound lipids is limited.     

Studies on (Na + K)-activated ATPase XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na⁺ + K⁺)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na⁺ + K⁺)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na⁺ + K⁺)-ATPase activity. These results indicate that cholesterol is not essential for (Na⁺ + K⁺)-ATPase activity.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Phospholipid metabolism in Mycobacterium smegmatis ATCC 607 grown at 37° C and 27° C

The rate of synthesis and degradation of phospholipids in Mycobacterium smegmatis ATCC 607, grown at 27° C and 37° C was studied by incorporation of ³²P into phospholipids and chase of radioactivity of the pulse-labelled phospholipids. A relatively low rate of synthesis and degradation of phospholipids in cells growth at 27° C was observed as compared to those grown at 37° C. Phosphatidylethanolamine (PE) had the maximum turnover at 37° C. However, at 27° C, cardiolipin (CL) showed a turnover rate higher than PE. Phosphatidylinositol mannosides (PIMs) were metabolically more active at 37° C than at 27° C. The differences in metabolic activity of the phospholipids at the two temperatures have been discussed.     

Reduced Palmitate Turnover in Brain Phospholipids of Pentobarbital-Anesthetized Rats

Our laboratory has reported that pentobarbital-induced anesthesia reduced the incorporation of intravenously injected radiolabeled palmitic acid into brain phospholipids. To determine if this decrease reflected a pentobarbital-induced decrease in palmitate turnover in phospholipids, we applied our method and model to study net flux and turnover of palmitate in brain phospholipids (1). Awake, light and deep pentobarbital (25–70 mg/kg, iv) anesthetized rats were infused with [9,10-³H]palmitate over a 5 min period. Brain electrical activity was monitored by electroencephalography. An isoelectric electroencephalogram characterized deep pentobarbital anesthesia. Net incorporation rates (J _FA,i ) and turnover rates (F _i) of palmitate were calculated. J _FA,i for palmitate incorporated into phospholipids was dramatically reduced by pentobarbital treatment in a dose-dependent manner, by 70% and 90% respectively for lightly and deeply anesthetized animals, compared with awake controls. Turnover rates for palmitate in total phospholipid and individual phospholipid classes were decreased by nearly 70% and 90% for lightly and deeply anesthetized animals, respectively. Thus, pentobarbital decreases, in a dose-dependent manner, the turnover of palmitate in brain phospholipids. This suggests that palmitate turnover is closely coupled to brain functional activity.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180 000 sites/cell but this fell to 125 000 (p <0.001) in cells enriched in monounsaturated fatty acids and rose to 386 000 (p <0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants ( and ) of 0.03 and 0.01 nM⁻¹. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM⁻¹ whereas values of 0.01 and 0.005 nM⁻¹ were obtained in the cells enriched in polyunsaturated fatty acids (all comparison p <0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.     

Purification and characterization of a Mn2+/phospholipid-dependent protein phosphatase from pig brain membranes

A Mn²⁺/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase theV _max of the phosphatase but had no effect on theK _m value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn²⁺. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn²⁺ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn²⁺/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn²⁺ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn²⁺ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.     

THE INCORPORATION OF [1-14C]ACETATE INTO UNESTERIFIED FATTY ACIDS IN RAT CEREBRAL CORTEX IN VIVO

—The incorporation of [1-¹⁴C]acetate into unesterified fatty acids and into the fatty acids of neutral glycerides and of phospholipids has been measured in rat cerebral cortex in vivo. The most rapid incorporation is seen in the unesterified fatty acids which have a turnover time of 5-6 min. It is suggested that unesterified fatty acids are precursors to neutral glycerides and phospholipids rather than being derived from them by lipase activity.