The alkyl and alk-1-enyl types of ether-linked phospholipids are present in mammalian cells almost exclusively as structural analogues of phosphatidylcholine and phosphatidylethanolamine. Perhaps the most significant discovery and advancement in studies of the ether-linked analogues of phosphatidylcholine was the elucidation of the chemical structure of platelet activating factor which was identified in 1979 as 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast to the alkyl lipids that contain phosphocholine, only limited knowledge is available about the origin of the alk-1-enyl moiety of plasmenylcholine. Warner and Lands117 first demonstrated that lysoplasmenylcholine hydrolase in rat liver microsomes catalyzes the hydrolysis of lysoplasmenylcholine to produce free aldehydes and glycerophosphocholine. Platelet activating factor is inactivated by hydrolysis of the acetate moiety via a reaction catalyzed by an acetylhydrolase. Regulation of the enzymatic steps that catalyze the de novo synthesis of plasmanylcholines from 1-alkyl-2-lyso-GroP is poorly understood.

Considerable interest in plasmalogens has recently been rekindled because of their important linkage to the production of potent bioactive lipid mediators such as platelet-activating factor (PAF) and the various oxygenated metabolites of arachidonic acid. Moreover, much progress has been made towards attaining a better understanding of the metabolism and functional role of plasmalogens in mammalian cells, including the establishment of the direct metabolic relationship between the ethanolamine- and choline-containing plasmalogens. This chapter reviews the early studies that established the enzymatic reactions leading to the synthesis of the ethanolamine plasmalogens by a Δl-alkyl desaturase and discusses more recent results that have shown the O-alk-1-enyl linkage in choline plasmalogens can be derived from the ethanolamine plasmalogens by enzymatic reactions involving the remodeling of substituents at both the sn-2 and/or n-3 positions. Other topics covered include a critical discussion of criteria and methods required for establishing proof of the plasmalogenic structure isolated from biological materials and an updated view of how plasmalogens participate in the trafficking of arachidonate among membrane phospholipids. A number of studies suggest the ethanolamine plasmalogens serve as a repository for arachidonate and other polyunsaturates in that they appear to be the final destination in the movement of polyenoic acids through the choline and ethanolamine-containing subclasses of glycerophospholipids. Ethanolamine-containing lysoplasmalogens have been shown to be involved in the biosynthesis of PAF by serving as acyl acceptors for the arachidonoyl moiety from alkylarachidonoyl-glycerophosphocholine (a membrane precursor of PAF) in a reaction catalyzed by a CoA-independent transacylase. The lyso-PAF intermediate formed by the transacylase can then be acetylated to form PAF by an acetyl-CoA acetyltransferase. The coupled phospholipase A 2 /transacylase pathway also is closely associated with the production of eicosanoid mediators since arachidonic acid and other polyenoic fatty acids are released from the ethanolamine plasmalogens in the initial hydrolytic step that forms the lyso plasmalogen acceptor.

This study of sphingomyelin molecular species in undifferentiated and differentiated (granulocytic form) HL-60 cells demonstrated only minor differences in the distribution of species between the sphingomyelinase-sensitive and sphingomyelinase-resistant pools of sphingomyelin in these cells. The two most prominent species of sphingosine present in both the undifferentiated and differentiated cells were those containing 16:0 (slightly higher in the sphingomyelinase-resistant membranes) and 24:1 N-acyl moieties. Cell differentiation exerted little effect on the distribution of molecular species of sphingomyelin between the sphingomyelinase-sensitive and sphingomyelinase-resistant pools in HL-60 cells, although the levels of N-palmitoyl sphinganine were significantly lower and the N-nervonoyl sphingosine higher in both pools from the differentiated cells. Our results indicate the same species of sphingomyelin, available at both the outer layer of the plasma membrane and inner layer of the plasma membrane (plus intracellular membranes) of HL-60 cells, serve as precursors for generation of the ceramides that participate in signal transduction processes initiated by cell activation.

Microsomal membranes from six different rat tissues (spleen, lung, kidney, brain, testis, and liver) were found to possess CoA-independent transacylase activity that could both acylate lyso-[3H]PAF (1-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine) and then deacylate the 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine product via the transacylation of added exogenous 1-alk-1′-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Platelet-activating factor (1-[3H]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was produced when acetyl-CoA was added to the spleen microsomes during generation of lyso-[3H]PAF by the transacylases. More extensive studies with subcellular fractions from spleen revealed that, in addition to microsomes, the transacylase activities were also present in the 15 000 × g membrane fraction but not in the cytosol. Analysis of molecular species of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine before and after addition of 1-alk-1′-enyl-2-lyso-sn-glycero-3-phosphoethanolamine as the acyl acceptor demonstrated a high selectivity for polyunsaturated fatty acids (> 3 double bonds/acyl group) in both the acylation and deacylation processes that occurred in testicular microsomal membranes. The transfer of acyl groups by the transacylase appeared to be equally effective for either arachidonic or docosapentaenoic(n − 6) fatty acids, whereas linoleic and oleic fatty acids were not transferred from 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine following the addition of 1-alk-1′-enyl-2-yso-sn-glycero-3-phosphoethanolamine. Similar experiments with the membrane fraction of undifferentiated HL-60 cells showed that arachidonic acid supplementation of intact cells enhanced both the CoA-independent transacylation of lyso-[3H]PAF and the subsequent deacylation of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine caused by addition of 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. Differentiation of the HL-60 cells into a neutrophil-like form had no effect on the transacylase activity. Our results indicate the PAF-related transacylase is widely distributed among tissues and, although highly selective for polyunsaturated acyl groups, does not discriminate selectively among the polyunsaturates.

1-Alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) is a significant phospholipid component of many mammalian tissues. Plasmenylethanolamine contains relatively high concentrations of polyunsaturated fatty acids; and this phospholipid subclass has been proposed as a storage site and source of arachidonic acid. It has also been suggested that ethanolamine plasmalogens function as scavengers of oxidative free radicals in biological membranes. Plasmanylethanolamine desaturase is a membrane-bound enzyme that converts plasmanylethanolamine (1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine) to plasmenylethanolamine via an electron transport chain involving cytochrome b5, molecular oxygen, and either NADPH or NADH as cofactors. Tissues and cells used as enzyme sources to study plasmanylethanolamine desaturase include Ehrlich ascites cells, Fischer R-3259 sarcomas, rat brains, preputial gland tumors, Madin-Darby canine kidney cells, P388D1 cells, and hamster small intestines. The plasmanylethanolamine desaturase resides in the microsomal membrane fraction, although postmitochondrial supernatant fractions have also been used in some Δ1-desaturase studies.

Phosphatidylsulfocholine (PSC), the sulfonium analogue of phosphatidylcholine (PC), occurs naturally in some diatoms. The replacement of the −N+(CH3)3 group by a −S+(CH3)2 results in an increase in the polar head group size in PSC relative to that of PC, consistent with the observed increase in permeability of PSC bilayers towards urea. It was of interest to see whether replacement of the −N+(CH3)3 group in platelet activating factor (PAF) by an −S+(CH3)2 group leads to any change in platelet aggregation or other physiological activity. Synthesis of the sulfonium analogue of PAF was carried out by suitable modifications of known procedures. The PAF-sulfonium analogue was found to have almost the same platelet aggregating activity as PAF itself, in the concentration range 1–20 μM, but a much lower activity in the range 0.01–1 μM. The analogue had little or no effect on the platelet aggregation activity of PAF when added in the concentration range 0.01–1 μM and had about half the hypotensive activity of PAF towards hypertensive CDF male rats. The sulfonium analogue, however, was much more cytotoxic to HL-60 cells than PAF itself, in the concentration range 0–15 μM; replacement of the acetate group by a benzyl group increased the cytotoxicity to the level of that of the methoxy analogue of PAF. Thus, replacement of the −N+(CH3)3 group by a −S+(CH3)2 group in the polar head group region of PAF results in a relatively small change in its platelet aggregation activity and a decrease in its hypotensive activity, but greatly increases its antitumor activity.

Acylation of alkyllysophospholipids in most cells occurs by: (a) CoA-independent transacylation, (b) CoA-dependent transacylation, and (c) acyl-CoA-dependent acylation. Using a recently developed high-performance liquid chromatography method, we have investigated the factors that influence the molecular species composition of the acylated products formed via these pathways with 1-hexadecyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) or 1-hexadecyl-2-lyso-sn-glycero-3-phospho-ethanolamine (alkyllyso-GPE) as substrates for the enzymes in Fischer R-3259 sarcoma microsomes. We found that short incubation times and low substrate concentrations favored the formation of polyunsaturated molecular species, i.e., 16:0-22:6, 16:0-22:5 (n - 3), and 16:0-20:4. Also, in agreement with results from other systems, CoA-independent transacylation produced a high percentage of polyunsaturated molecular species; acyl-CoA-dependent acylations generated the least polyunsaturated molecular species and CoA-dependent transacylation gave intermediate values. Furthermore, no substrate selectivity occurred with respect to alkyl chain lengths of alkyllyso-GPE; similar molecular species composition was obtained with either hexadecyllyso-GPE or octadecyllyso-GPE as substrates. Responses to N-ethylmaleimide inhibition and heat inactivation as well as pH optima suggest the same enzyme catalyzes the CoA-independent transacylation of both alkyllyso-GPC and alkyllyso-GPE.

The goal of this investigation was to determine the effect of an alkylglycerol dietary supplement on the lipid composition of several major organs. Lipids from kidney, liver, and lung tissues of rats on a laboratory chow diet (controls) were compared to lipids from the same tissues of rats that had received oral supplements (300–600 mg/day) of 1-O-alkyl-2,3-diacetyl-sn-glycerol (alkyl groups were 65% 18∶1 and 17% 16∶1) for six days. Incorporation of the alkylglycerol into tissue lipids was indicated by both the presence of a neutral lipid in liver that had the same chromatographic migration as alkyldiacylglycerols and by a substantial increase (≈150% of controls) in the octadecenyl group of the alk-1-enyl- and alkyl-glycerol side chains derived from total phospholipids of all three tissues. Compared to controls, there was a significant increase in the amount of alkylacylglycerophosphocholine in all three tissues of the alkylglycerol supplemented group. Total lipids, total phospholipid phosphorus, or the distribution of phospholipid classes (except for small differences in lung tissue) were not affected by the dietary supplement. The increase in ether lipids was offset by a corresponding decrease in the diacyl subclass in tissues from animals on the alkyldiacetylglycerol supplement. Our results indicate that the amount of ether-linked glycerolipids in rat tissues can be easily increased with dietary supplements of alkylglycerols.

Ether-linked glycero-alpha- and beta-D-glucopyranosides and glycero-1-thio-alpha- and beta-D-glucopyranosides have been synthesized by modifications of the Königs-Knorr procedure, and their antitumor activities have been evaluated. The bioactivities of these compounds have been evaluated in five different cell lines (WEHI 3B, C653, X63/OMIL3, R6X-B15, and HL-60) and compared with the activities of 1-O-hexadecyl-2-O-methyl-sn-3-glycerophosphocholine (GPC) and its enantiomer, 3-O-hexadecyl-2-O-methyl-sn-1-GPC. The results indicate that a alpha-D-thioglucopyranoside [1-O-hexadecyl-2-O-methyl-3-S-(alpha-D-1'- thioglucopyranosyl-sn-glycerol)] is selective with respect to its action on target cells, with high activity for killing of WEHI 3B and C653 cells as determined by inhibition of [3H]thymidine incorporation into DNA and HL-60 cell cytotoxicity, but unable to induce aggregation of rabbit platelets at 10(-5) M. The corresponding beta-linked thioglycolipid was ineffective with respect to cytotoxicity against each cell line tested, indicating the importance of configuration at the anomeric position; the beta-thioglycoside was also ineffective with respect to inducing platelet aggregation. 1-O-Hexadecyl-2-O-methyl-sn-3-GPC and 3-O-hexadecyl-2-O-methyl-sn-1-GPC were potent inhibitors of growth of each cell line tested but also caused rabbit platelet aggregation at concentrations greater than or equal to 10(-7) M. Thus, 3-S-(alpha-thioglycopyranosyl)-sn- glycerols bearing a long-chain O-alkyl group at the sn-1 position and a methoxy group at the sn-2 position of glycerol appear to be a promising class of antineoplastic agents with lower risk of inducing thrombosis than the widely studied platelet activating factor analogue, 1-O-octadecyl-2-O-methyl-rac-3-GPC.

This chapter discusses the measurement of key enzyme activities involved in the metabolism of platelet activating factor (PAF). Evidence for the occurrence of PAF in mammalian cells is based on chemical, chromatographic and mass spectral studies, bioassays, and the detection of specific enzymes that synthesize PAF. 6 PAF can be biosynthesized through two independent pathways with the final steps catalyzed by either acetyl–coenzyme A (CoA): 1-alkyl-2-1yso- diacyl-glycerophosphocholines (GPC) acetyltransferase or cytidine 5'diphosphocholine (CDP)–choline: 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase. The composite picture of individual enzymatic steps that lead to the biosynthesis of PAF is illustrated in the chapter. The catabolic reactions involved in PAF metabolism are summarized. Detailed methods for the assay of the key enzymes in these PAF pathways under optimum conditions are also described.

l-~-Hexadecyl-2-acetyl-sn-glJ.cero-3-phosphocholine (l-hexadecyl2-acetyl-GPC, platelet activating factor, PAF) was previously shown to produce profound hypotension and sympathetic activation in conscious rats. To determine the role of the sympatho-adrenomedullary system in the cardiovascular responses elicited by 1-hexadecyl-2-acetyl-GPC, the vasoactive phospholipid was administered (1 nmol per 300 g) to a) intact, b) bilaterally demedullated, and c) propranolol- (a 8-adrenoceptor blocker) treated SHR and WKY rats. The hvpotensive response to 1-hexadecyl-2-acetyl-GPC was prolonged in demedullated or propranolol-pretreated WKY rats and in propranolol-treated SHR rats. The extreme tachycardia produced by 1-hexadecyl-2-acetyl-GPC in both the WKY and SHR rats was abolished by propranolol pretreatment. Pressor responses to norepinephrine during the 1-hexadecyl-2-acetyl-GPC-induced hypotension in propranolol-pretreated rats were suppressed in both the normotensive and SHR rats. Plasma acetylhydrolase activity, which inactivates PAF, was higher in hypertensive (SHR) rats or demedullated WKI rats than in the normotensive (wp;Y) rats. These results show that the tachycardia evoked by 1-hexadecyl-2-acetyl-GPC is mediated solely by sympathetic activation and the 0-adrenergic receptors and further indicate the major role of the sympathetic system and 0-adrenoceptors in recuperation from 1-hexadecyl-2-acetyl-GPC-induced shock. The data also suggest that acetylhydrolase in serum is an important regulatory enzyme for controlling PAF levels in the vascular compartment.