The hydroxy fatty acids: Isolation, structure determination, quantitation

This review and commentary on quantitative methodology of the hydroxy fatty acids covers methods of isolation, structure determination, and quantitative analysis. Individual topics are: methods of liberation from bound form, separation by partition, separation by partition columns, separation by precipitation, separation by adsorption columns, separation by thin‐layer chromatography, separation of homologous fatty acids, locating the hydroxyl group, characterization of the optically active hydroxyl group, locating the double bonds, gravimetric determination, colorimetric determination, gas chromatographic determination, infrared absorption, titrimetric determination, and radiometric determination.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141899/1/aocs0569.pd

Killing cancer cells by poly-drug elevation of ceramide levels

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66186/1/j.1432-1033.2001.01845.x.pd

Drug design: hiding in full view

Compounds that can produce potent biological effects in cells encompass a variety of structural motifs. Many of these compounds share a structural feature that has rarely been noted. It is an allylic cluster of atoms, a 3-carbon chain with a double bond between two of the atoms and an oxygen atom at the other end. The oxygen can be in a hydroxyl group, or in an ether or ketal or ester linkage, or simply a carbonyl form. In the latter case, the linkage is an allylic ketone (ene-one) structure. Nitrogen is often seen in equivalent forms. Inclusion of at least one allylic moiety appears to be able to turn a modestly active or inert compound into an effective drug or toxin. Some compounds lack the allylic moiety but develop one by enzymatic action, usually via cytochrome P-450 enzymes. These metabolites probably represent the active drug forms. The above concepts seem to be radically simplistic and improbable, but the evidence supporting them and the explanations for the biological activities are hidden “in plain view.” Comparisons with the pleiotropic activities of the allylic sphingolipid, ceramide, indicate that many allylic drugs operate by controlling the state of protein phosphorylation, by activating proteases, by generating reactive oxygen species, by slowing mitochondrial electron transport, or by lowering cellular glutathione concentrations. Drug Dev Res 69:15–25, 2008 © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58639/1/20223_ftp.pd

Preparation of psychosines (1‐O‐hexosyl sphingosine) from cerebrosides

A convenient method for large or small scale preparation of psychosine from cerebroside has been developed by adaptation of published procedures. Cerebroside is refluxed with butanol and aqueous KOH, then the KOH is removed with perchloric acid. The fatty acids are removed by extraction with hexane and the excess perchloric acid is removed by partitioning between chloroform, ethanol, and water.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142232/1/lipd0358.pd

Uptake of cerebroside, cholesterol and lecithin by brain myelin and mitochondria

The uptake of emulsified labeled lipids by rat brain myelin and mitochondria was studied. Cerebroside and lecithin uptakes were greatly stimulated by addition of salts, particularly those containing divalent cations. Cholesterol uptake was not influenced by salts. Increasing concentrations of detergent (nonâ ionic) were inhibitory. Delipidated membranes took up much less lipid, but pretreatment with lecithin partially restored the ability to take up cerebroside and cholesterol. The lipid uptake appears to be nonenzymatic and appears to depend on the size of the emulsified particles. The possible role of such a phenomenon in membrane formation and maintenance is discussed.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141986/1/lipd0439.pd

Metabolism of brain glycolipid fatty acids

The metabolism of the fatty acid moieties of brain cerebrosides, sulfatides, and gangliosides is reviewed and discussed. The methodology involved in the isolation of the fatty acids is described briefly. It seems clear now that most of these acids are made by chain elongation of intermediate length fatty acids by addition of acetate residues. The unsaturated acids are made by desaturation of the intermediate length acids (palmitic, heptadecanoic, stearic) followed by chain elongation. The hydroxy acids are made directly from the corresponding nonhydroxy acids, saturated, unsaturated, and odd‐numbered. All the hydroxy acids undergo oxidative decarboxylation to yield fatty acids containing one less carbon atom. The odd‐numbered acids are also made from propionate, which is elongated to intermediate length acids and then to longer acids. The major intermediate length “primer” acid seems to be palmitate, but there is evidence that the stearate used for cerebroside synthesis is also madede novo from acetate. The ganglioside fatty acids were found to turn over somewhat faster than the other fatty acids. Two metabolic pools for the cerebroside acids were found, one with a very high turnover rate, the other with a very low turnover rate.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141473/1/lipd0047.pd

Preparation of 6‐ 3 H glucocerebroside

Glucocerebroside (1–0‐β‐glucosyl ceramide) can be labeled with 3 H‐borohydride at the 6‐position of the glucose moiety. The 6‐trityl ether of cerebroside is formed first, the remaining hydroxyl groups are acetylated, the trityl group is removed, and the free 6‐hydroxyl group is oxidized to an aldehyde. The carbonyl group is then reduced with borohydride and the acetyl groups are removed, regenerating the original glycolipid.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90217/1/2580130309_ftp.pd

Stimulation of liver growth and DNA synthesis by glucosylceramide

The nature of the growth‐stimulating effect of glucosylceramide was studied. Mice were injected intraperitoneally with emulsified glucosylceramide and conduritol B epoxide, an inhibitor of cerebroside glucosidase. Within one or two days, the liver grew 18–24%, as reported. Two enzymes involved in DNA synthesis also increased more than the weight. The total liver activity of thymidine kinase increased 46–73%, and the total activity of ornithine decarboxylase increased as much as 101%. It is suggested that elevated liver levels of glucocerebroside stimulate cell proliferation through a relatively direct mechanism.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141613/1/lipd0508.pd

Alternate pathways of cerebroside catabolism

A search was made for new degradative pathways for glucosyl and galactosyl ceramides in an effort to explain the failure of these lipids to accumulate in the brains of children with Krabbe’s or Gaucher’s disease. Using various buffers and incubation conditions, we tested brain homogenates from 12 day old rats with the stearateâ labeled and galactoseâ labeled lipids. No evidence for direct deacylation (and formation of psychosines) could be obtained, nor was there any evidence for transacylation of sphingocyl phosphoryl choline or oxidation of the 6 position of the galactose moiety. Two new derivatives of galactosyl ceramide were observed, possibly fatty acid esters of unknown polar compounds. It is tentatively proposed that the etiology of infantile Krabbe’s and Gaucher’s diseases involves, not an accumulation of galactosyl and glucosyl ceramides, with consequent formation of toxic products, but rather malfunction of some other role of the corresponding glycosidases.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141643/1/lipd0732.pd

A 2‐phase liquid scintillation assay for glycolipid synthetases

Glycolipid synthetases can be assayed conveniently by incubating the lipid substrate with the radiosugar‐labeled nucleotide in a small plastic scintillation vial. At the end of the incubation period, water and perchloric acid are added, thenn‐butanol, then a toluene‐based scintillation cocktail. The radioactive lipid partitions into the scintillation fluid, leaving excess sugar nucleotide in the aqueous phase. Only a small fraction of the total radioactivity in the aqueous layer is detectable. This method is illustrated for ceramide: UDP‐glucose glucosyltransferase. The approach should be applicable to other lipid synthetases that can be assayed with a radioactive hydrophilic substrate.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141182/1/lipd0764.pd