Claude Piantadosi

Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols

Alkylacylglycerols are synthesized when choline-phospholipids are degraded by a phospholipase C. This class of compounds has been shown to have biological activities; however, the mechanism of action is unknown. A series of alkyl-linked diglycerides were synthesized and tested for activity in an in vitro assay for protein kinase C. When protein kinase C activity was stimulated with the synthetic diacylglyceride analog 1-oleoyl-2-acetyl-sn-glycerol, the addition of alkyl glycerides caused a concentration-dependent inhibition of protein kinase C activity. Comparison of the protein kinase C inhibition by this series of 1-O-alkyl-2-acyl analogs revealed that both saturated and unsaturated long-chain groups in position 1 were effective and that dietherglycerols with short-chain moieties in position 2 were also effective. It is concluded from these studies that the biological activity of alkyl-linked glycerides may be expressed through protein kinase C inhibition.

Quantitative analysis of ether-linked lipids as alkyl- and alk-1-enyl-glycerol benzoates by high-performance liquid chromatography

Methodology for the quantitative and qualitative analyses of alkyl- and alk-l-enyl-glycerols derived by Vitride reduction of ether-linked glycerolipids in the presence of an internal standard was developed. The procedure involved preparation of benzoate derivatives that were subsequently analyzed by high-performance liquid chromatography with detection at 228 nm. Separation of the glycerol ether dibenzoates on a C18 reverse-phase column allowed for the simultaneous quantitation and the determination of chain length of both alkyl- and alk-l-enyl-glycerols in a single chromatographic run of less than 15 min. The method was accurate (less than 10% error), reproducible, and sensitive (less than 1 microgram per component). Application of the method to the analysis of phospholipids from L-M cells grown in the presence and absence of elaidic acid demonstrated that the cells incorporated a portion of the trans acid supplement (presumably via the fatty alcohol) into the side chains of both alkyl- and alk-l-enyl-glycerol-containing phosphatides.

A method for the quantitative determination of glycerolipids containing O-alkyl and O-alk-1-enyl moieties

We have developed a spectrophotometric procedure, based on a combination of established methods, for the quantitative determination of aklyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids. It depends upon the release of alkylglycerols and alk-1-enylglycerols from phospholipids by phosphlipase C (Bacillus cereus) followed by saponification or by Vitride reduction the phospholipids; aldehydes are subsequently formed and measured colorimetrically after reacting them with a fuchsin reagent. The total alkyl and alk-1-enyl content of glycerolipids is determined oxidation of the sample withperiodate to form aldehydes and alkylglycolic aldehydes. The O-alk-1-enyl lipid content is determined on a separate sample by measuring the aldehydes produced after acid hydrolysis. The quantity of O-alkyl lipids is calculated from the difference between the values obtained for the total ether-lipid content and that of the O-alk-1enyl lipid content. Alternately, direct determination of alk-1-enylglycerols and alkylglycerols can be made if these hydrolytic products are first separated by thin-layer chromatography.

Neoplastic cell inhibition with new ether lipid analogs

Bioactive phospholipid analogs of platelet-activating factor (PAF) represent a new approach to cancer chemotherapy. Various modifications of the basic structure of PAF lead to different ether lipid (EL) analogs. Data from the evaluation of thioalkyl and amidoalkyl glycerophosphocholine and of glycerophosphoinositol EL analogs against different experimental tumors in vitro (HL60 and K562 human leukemia cells, BG1 and BG3 ovarian adenocarcinomas) are presented. Exclusion of trypan blue after short exposure to the drugs determined cytotoxicity, and a soft agarose clonogenic assay measured the ability of the analogs to inhibit tumor cell proliferation. The thioalkyl EL are very active against the cell lines using both end points, and the amidoalkyl EL showed efficacy against the leukemic cell lines, whereas the phosphoinositol EL are active only at high concentrations. Combined use of EL analogs, which are membrane-interactive, with classical DNA-interactive chemotherapeutic drugs revealed that the combinations have additive antiproliferative effects. These results are promising leads in the development of the anticancer potential of ether lipid analogs.

Tetrahydropteridine-dependent cleavage enzyme for O-alkyl lipids: substrate specificity

The tetrahydropteridine-requiring enzyme in rat liver microsomes that oxidatively cleaved the O-alkyl moiety of glycerolipids does not utilize alkyl glycerolipid substrates that contain carbonyl, acyl, or phosphate groups on the glycerol portion. However, the ether linkage in rac and 1-, 2-, or 3-isomeric forms of alkylglycerols and 1-alkyl-sn-glycero-3-phosphorylethanolamine is readily cleaved by the pteridine-requiring enzyme. Therefore, it appears that hydrolytic enzymes play an essential role in conjunction with the cleavage enzyme in the regulation of cellular levels of O-alkyl moieties in glycerolipids.

Hypolipidemic activity of in vitro inhibitors of hepatic and intestinal sn-glycerol-3-phosphate acyltransferase and phosphatidate phosphohydrolase

A number of agents including a series of 1,3-bis (substituted phenoxy)-2-propanones were screened in vitro for their ability to inhibit hepatic and intestinal microsomal sn-glycerol-3-phosphate acyltransferase and phosphatidate phosphohydrolase. Effective inhibitors reduced in vivo hepatic and intestinal glycerolipid production and with one exception also lowered serum triglyceride levels, suggesting that agents which inhibit potential rate-limiting steps of glycerolipid biosynthesis may be effective hypolipidemic agents. Two compounds, 1-methyl-4-piperidyl bis (p-chlorophenoxy) acetate (Sah 42-348) and 1,3-bis (p-methylphenoxy)-2-propanone were the best inhibitors of glycerolipid biosynthesis and lipid-lowering agents. The lipid-altering effects of both drugs were compared to chlorophenoxyisobutyrate during high fructose intake in rats. Each agent reduced fructose induced glycerolipid biosynthesis and serum triglyceride levels to similar degrees.

Modification of glycerolipid metabolism in L-M fibroblasts by an unnatural amino-alcohol, N-isopropylethanolamine

Abstract The unnatural amino-alcohol, N-isopropylethanolamine, is incorporated into a phospholipid by monolayers of L-M fibroblasts. This phospholipid was identified as 1,2-diacyl- sn -glycero-3-phosphoisopropylethanolamine by using chemical and enzymatic procedures combined with thin-layer and gas-liquid chromatography. Since the phospho-N-isopropylethanolamine moiety is removed by phospholipase C, the stereochemistry of the phospholipid analog is identical to naturally occurring phosphoglycerides. Incubation of cells in 10 mM N-isopropylethanolamine inhibited the incorporation of [14C]choline and [14C]ethanolamine into phospholipids and stimulated the incorporation of [1-14C]palmitic acid and [1-14C]hexadecanol into triacylglycerols and alkyldiacylglycerols. These results indicate that N-isopropylethanolamine affects glycerolipid synthesis at the diradylglycerol branch point.

Membrane lipid modification and stearoyl-coenzyme A desaturase activity in L-M cells.

Abstract The stearoyl-coenzyme A desaturase system of L-M cells, grown as monolayers, was examined in microsomal membranes that contained 8.2% phosphatidylisopropylethanolamine, an unnatural phospholipid analog. Desaturation of both [1- 14 C]stearic acid by whole cells and [1- 14 C]stearoyl-coenzyme A by cell-free homogenates, or microsomes, was decreased to about 40% of control levels in cells that had been grown for 24 h in the presence of 10 m m N -isopropylethanolamine. No decrease in microsomal NADH- or NADPH-dependent cytochrome c reductase activities or the level of cytochrome b 5 was found in the L-M cells that had been treated for 24 h with N -isopropylethanolamine. Although amino acid transport into L-M cells was not affected by treatment with N -isopropylethanolamine, protein synthesis was decreased by about 30%. These results indicate that the decrease in stearoyl-coenzyme A desaturation in the modified membranes is specifically associated with the terminal oxidase activity (cyanide-sensitive factor) of the desaturase enzyme complex.

The synthesis of14C- and3H-labeled glycerol ethers

The racemic14C-and3H-labeled alpha and beta derivatives of octadecyl glycerol ether (batyl alcohol) and of hexadecyl glycerol ether (chimyl alcohol) of high specific activity were synthesized by treating the appropriate alkyl halides with a large excess of the potassium salts of isopropylidene or benzylidene glycerol. By use of the trifluoroacetic anhydride esterification procedure, the labeled diesters of alpha and beta octadecyl and hexadecyl glycerol ethers were prepared. The labeled monoesters of beta octadecyl and of beta hexadecyl glycerol ethers were isolated from the reaction mixtures by silicic acid column chromatography.

An alternate enzymic route for the synthesis of the alkyl analog of phosphatidic acid involving alkylglycerol

A key intermediate in the biosynthesis of complex ether lipids is 1-alkyl-2-acyl-sn-glycero-3-phosphate. This lipid is known to be formed by enzymic reduction of alkyldihydroxyacetone phosphate and subsequent acylation. We have now obtained evidence for an alternate pathway for its formation from hexadecylglycerol. The reaction is catalyzed by a mitochondrial supernatant fraction prepared from mouse preputial gland tumors; CoA, ATP, and Mg++ are required as cofactors.