Zigrida L. Smith

Dietary Supplementation with Ether-Linked Lipids and Tissue Lipid Composition

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.

HL-60 cells become resistant towards antitumor ether-linked phospholipids following differentiation into a granulocytic form

1-0-Alkyl-2-0-methyl-sn-glycero-3-phosphocholine (alkylmethoxy-GPC) exerts a highly selective cytotoxic activity towards a variety of tumor cells that is not seen in normal cells. Human promyelocytic leukemia (HL-60) cells are particularly sensitive to this cytotoxic action. In this report we show that when HL-60 cells are differentiated into a granulocytic form by dimethylsulfoxide (Me2SO)they become resistant toward the cytotoxic effects of alkylmethoxy-GPC. Also, after short-term exposures of the HL-60 cells to alkylmethoxy-GPC, the uptake of [methyl-3H]choline is inhibited in the undifferentiated cells, but not in those differentiated with Me2SO. Thus, cellular choline uptake appears to be a useful index for assessing the susceptibility of cells to the cytotoxic effects of antitumor phospholipids. [3H]Alkylmethoxy-GPC is poorly metabolized by both cell populations as is evident by the trace quantities of labeled metabolites formed; also, alkylmethoxyglycerols do not exert any cytotoxic activity toward undifferentiated cells. These results demonstrate that differences in the cytotoxic response of sensitive (undifferentiated) and resistant (differentiated) cells to alkylmethoxy-GPC are not due to differences in their ability to metabolize alkylmethoxy-GPC or to a phospholipase C-generated toxic metabolite. Instead the data support our earlier hypothesis that the antitumor action of alkylmethoxy-GPC is, at least in part, caused by an impaired transport of small molecules across the membrane of sensitive cells.

Contributing factors in the trafficking of [3H]arachidonate between phospholipids

Cultured human promyelocytic leukemia cells (HL-60), depleted of arachidonic acid by continued growth in serum-free media, were used as a model system to examine various factors that control the incorporation and distribution of [3H]arachidonic acid into classes and subclasses of cellular lipids. Increasing the culture media concentration of [3H]arachidonic acid from 1 x 10(-8) M to 1 x 10(-5) M caused a greater percentage of the cellular tritium to be distributed into triacylglycerols (from less than 1% at 1 x 10(-8) M to 38% at 1 x 10(-5) M) with a corresponding decrease in cellular [3H]diradylglycerophosphoethanolamine (from 53% at 1 x 10(-8) M to 12% at 1 x 10(-5) M) during 2 h incubations. A greater proportion of the tritium present in diradylglycerophosphoethanolamine and diradylglycerophosphocholine, at the higher media concentration of [3H]arachidonic acid (1 x 10(-5) M), was found in the diacyl subclasses of these two lipids than was observed at the lower concentrations (less than 1 x 10(-6) M) of [3H]arachidonic acid. Significant amounts of diarachidonoyl molecular species were found in the phosphatidylethanolamine (10%) and phosphatidylcholine (15%) of HL-60 cells that were labeled for 2 h with 1 x 10(-5) M [3H]arachidonic acid. This was the only molecular species of phosphatidylcholine to completely disappear when prelabeled cells were placed in arachidonate-free media for 22 h. Prelabeling-chase experiments with 1 x 10(-5) M [3H]arachidonic acid were consistent with movement of [3H]arachidonate from triacylglycerols into diradylglycerophosphatides and from diacylphospholipids into ether-linked phospholipids. Increasing the concentration of HL-60 cells in the incubations influenced the distribution of [3H]arachidonic acid in cellular lipid classes in a manner analogous to decreasing the concentration of [3H]arachidonic acid in the media. Increasing the endogenous level of cellular arachidonate in phospholipid classes with supplements of unlabeled arachidonic acid changed the subsequent lipid class distribution of a low concentration (1 x 10(-8) M) of [3H]arachidonic acid to resemble results obtained with a much higher mass level of [3H]arachidonate in arachidonate depleted cells. HL-60 cells differentiated into granulocytes by treatment with dimethyl sulfoxide incorporated less [3H]arachidonic acid but had a greater proportion associated with alkylacylglycerophosphocholine and alk-1-enylacylglycerophosphoethanolamine than undifferentiated HL-60 cells.

Effects of eicosapentaenoic and docosahexaenoic acid supplements on phospholipid composition and plasmalogen biosynthesis in P388D1 cells

This investigation describes the influence of n-3 fatty acid supplements on the phospholipid composition and the metabolism of plasmalogens in P388D1 cells. The cellular content of phospholipid classes and subclasses was unchanged in P388D1 cells (a macrophage-like cell) grown for 24 h in media supplemented with 10 microM sodium eicosapentaenoate or sodium docosahexaenoate. However, phospholipids from these cells were highly enriched in acyl groups of the corresponding fatty acid supplement, with the largest increases occurring in the ethanolamine plasmalogens (e.g., 46% of the ethanolamine plasmalogens from cells supplemented with docosahexaenoate contained this acyl group at the sn-2 position). Eicosapentaenoate supplements lowered the levels of oleate in phosphatidylinositol/serine, diacyl-sn-glycero-3-phosphoethanolamine (GroPEtn), and alk-1-enylacyl-GroPEtn in the P388D1 cells but had little or no effect on the amounts of arachidonate in the cellular phospholipids. In contrast, supplementation of the cells with docosahexaenoic acid not only reduced the level of oleate but also decreased the amount of arachidonate by one-third in the alk-1-enylacyl-GroPEtn. When P388D1 cells were incubated for 1 h with [3H]alkyllyso-GroPEtn both [3H]alkylacyl-GroPEtn and [3H]alk-1-enylacyl-GroPEtn were formed. The sn-2 acyl composition of these two ether-containing GroPEtn lipids reflected the fatty acid supplement that the cells had received (e.g., 68% of the [3H]alk-1-enylacyl-GroPEtn from cells supplemented with docosahexaenoate contained this acyl group at the sn-2 position). Cells from both the controls and supplemented groups contained greater amounts of docosahexaenoate in the [3H]alk-1-enylacyl-GroPEtn (plasmalogen) than in the [3H]alkylacyl-GroPEtn subclass. Analysis of molecular species from pulse-chase experiments with intact cells and examination of the molecular species of [3H]alk-1-enylacyl-GroPEtn produced by the delta 1-desaturase system in cell-free membrane fractions suggest that the docosahexaenoate-containing species of [3H]alk-1-enylacyl-GroPEtn have a higher turnover rate than other molecular species. Possible biological implications of our findings are also discussed.

Arachidonate-containing triacylglycerols : biosynthesis and a lipolytic mechanism for the release and transfer of arachidonate to phospholipids in HL-60 cells

When HL-60 cells are incubated in media containing 10 microM [3H]arachidonic acid the label is immediately incorporated into both triacylglycerols and phospholipids. About one-half of the cellular tritium was associated with triacylglycerols after 2 h of incubation and this [3H]arachidonate was then transferred to phospholipids as soon as the labeled cells were placed in arachidonate-free media. A technique was devised to analyze the stereospecific distribution of [3H]arachidonate at the three sn-positions of glycerol in order to identify the mechanism(s) responsible for the biosynthesis of the labeled triacylglycerols. [3H]Arachidonate was found to be distributed in nearly equal amounts among all three glycerol positions of the triacylglycerols. In addition, analysis of intact triacylglycerols containing [3H]arachidonate revealed that 24% of the tritium eluted from reverse-phase HPLC with triarachidonoylglycerol. Both of these findings would be expected if a significant portion of the arachidonate-containing triacylglycerols were synthesized de novo. Homogenates prepared from [3H]arachidonate prelabeled HL-60 cells were capable of hydrolyzing the endogenous [3H]arachidonate-containing triacylglycerols to produce mainly free fatty acids and smaller amounts of monoacylglycerols. The relatively small amount of monoacyl- and diacylglycerols produced by the lipolytic activity of the homogenates indicated that [3H]arachidonate was hydrolyzed from all three sn-positions of the [3H]triacylglycerols. This lipase activity had a pH optimum of 4.5 and was associated to a greater extent with the soluble fraction than in the total membrane fraction. Although it is not known whether this lipolytic activity is the same as that expressed in the intact cells, the activity of the cell-free triacylglycerol lipase was of sufficient magnitude to have easily accounted for the decrease in [3H]triacylglycerols that was observed after transfer of the intact HL-60 cells (prelabeled with [3H]arachidonate) to fresh media. The data suggest that transfer of arachidonate from triacylglycerols to phospholipids probably occurs through an acyltransferase utilizing a lysophospholipid and arachidonoyl-CoA.

Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis

This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (greater than 90% of total activity); only a minimal level of activity (less than 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[3H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37 degrees C gave apparent values for Km and Vmax of 45 microM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[3H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca+2, Mg+2 or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[3H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[3H]hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.

O-alkyl-O-methylglycerophosphocholine, an antineoplastic lipid, undergoes spontaneous redistribution between biological membranes prepared from HL-60 cells

Cytotoxic actions of the unnatural phospholipid 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholine (alkylmethyl-GPC) appear to be targeted to the plasma membrane of sensitive cells. We analyzed the distribution of [3H]alkylmethoxy-GPC in subcellular membranes isolated from human promyelocytic leukemia (HL-60) cells. [3H]Alkylmethyl-GPC added to intact cells selectively labels plasma membrane-enriched fractions of the postnuclear supernatant, but the labeling profile is independent of the temperature and duration of the incubation, and concentration of the molecule. Also, an identical distribution pattern is obtained when [3H]alkylmethyl-GPC is directly added to postnuclear supernatants. Moreover, [3H]alkylmethyl-GPC translocates between subcellular membranes in a manner that does not depend on membrane-adsorbed or cytosolic transfer proteins. These results indicate that the subcellular localization studies reported for alkylmethyl-GPC and structurally-related molecules must be interpreted with caution.

Altered platelet-activating factor levels and acetylhydrolase activities are associated with increasing severity of bronchopulmonary dysplasia

Lipid inflammatory mediators are thought to play an important role in the pathogenesis of neonatal lung injury and bronchopulmonary dysplasia (BPD). Because preliminary studies from the intensive care nursery of the University of Tennessee Medical Center, Knoxville, revealed linear increased in blood platelet-activating factor (PAF) levels in very low birthweight infants developing chronic lung disease and lower cord blood PAF acetylhydrolase activities in premature infants, it was theorized that altered platelet-activating factor levels and PAF acetylhydrolase activities are associated with increasing severity of BPD. Platelet-activating factor levels (blood and tracheal lavage) and PAF acetylhydrolase activities (blood and tracheal lavage) were measured over days 1 to 2, 3 to 5 and 6 to 7 in 16 ventilated infants and weekly in 9 infants with bronchopulmonary dysplasia. Platelet-activating factor values were normalized per nanogram of lavage blood urea nitrogen. Severity of bronchopulmonary dysplasia was estimated using the scoring system developed by Toce. Mean blood and lavage PAF levels and PAF acetylhydrolase activities were compared in infants developing bronchopulmonary dysplasia with those without the disease over the first seven days of life. Infants developing chronic lung disease were significantly smaller and of younger gestational age. In infants with bronchopulmonary dysplasia, higher PAF levels in blood were seen on days 3 to 5, along with increased lavage acetylhydrolase activities on days 1 to 2. Increased levels of PAF in lavage on days 3 to 5 were associated with increasing severity of bronchopulmonary dysplasia. Altered blood and lavage platelet-activating factor levels and PAF acetylhydrolase activities appear to be associated with the pathogenesis and severity of bronchopulmonary dysplasia.

Membrane-Targeted Biochemical Effects and the Role of Cellular Differentiation in the Selective Antitumor Actions of Alkylmethoxyglycerophosphocholine

Various studies of the selective antitumor activity of unnatural derivatives of platelet-activating factor (PAF), including clinical trials in Germany, have been reviewed by Berdel and Munder (1) and Berdel et al. (2). The close structural relationship between the antitumor agent, 1-alkyl-2-methoxy-sn-glycero-3-phosphocholine (alkylmethoxy-GPC) and PAF is illustrated in Figure 1. It appears that there is an inverse relationship between phospholipids of similar structure (i.e., those with a short chain aliphatic group at the sn-2 position) that possess PAF biological activities versus those that exhibit selective cytotoxic properties toward sensitive tumor cells. The natural enantiomer of PAF (L form) exhibits no cytotoxicity toward undifferentiated cells, whereas in stark contrast, D-PAF is highly toxic under the same conditions (3).