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

Changes in liver lipids after administration of 2‐decanoylamino‐3‐morpholinopropiophenone and chlorpromazine

The enzyme which forms glucocerebroside, ceramide: UDP‐glucose glucosyltransferase, is inactivated in vitro by a cationic analog of cerebroside, 2‐decanoylamino‐3‐morpholinopropiophenone. A study of the inhibitor using intraperitoneal injection into young mice showed that the level of the enzyme activity in liver was appreciably lowered between 3 and 6 hr after injection. The activity increased subsequently, overshooting the normal level within 24 hr by about 20%, then returning to normal within the next 24 hr. Additional effects observed in liver were an increase in lipid content (primarily in the triglyceride fraction and ceramides) and a decrease in the glucocerebroside level. Body temperature dropped rapidly. Markedly similar effects were produced by injecting chlorpromazine, which was tried in order to reduce the hyperirritability and inhibitory effects on monoamine oxidase previously demonstrated by the glucosyltransferase inhibitor. Chlorpromazine did indeed block the hyperirritability and resulted in enhancement of the keto amine’s effects on the enzyme and lipids. It is possible that the two drugs in combination would be helpful in ameliorating the symptoms due to the cerebroside accumulation that occurs in Gaucher disease. Diazepam also produced a reduced level of glucosyltransferase. A color reaction for chlorpromazine, possibly suitable for quantitative determination in tissues, was accidentally discovered.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142035/1/lipd0538.pd

Determination of glucocerebroside, sphingomyelin, free fatty acid and total lipid by thin-layer chromatography and charring-scintillation quenching

A previously described method has been extended to various specific lipids of liver and brain. The basic method involves thin-layer chromatography followed by charring to reveal the bands. The intensity of each band is determined by suspending the silica gel in a radioactive scintillation gel and measuring the optically quenched activities. The lipids are extracted with hexane/isopropanol and, in the case of total lipid determinations, the extract is simply applied to a silica gel plate and charred without use of a development step. For brain cerebroside, the extract is applied to the plate and developed in the usual way. For liver cerebroside, the dried lipid extract is fractionated with a silica gel column to purify the glycolipid, which is then purified further by development with a plate. For sphingomyelin the ester type lipids in the extract are cleaved by alkali for 1 min and the resultant lipids are applied directly to the thin-layer plate. Free fatty acids are chromatographed and measured after a preliminary solvent partitioning to remove most lipids. The method is useful for samples of 5-40 [mu]g. Methods for quantitative application of samples to plates are described. A modification of the Camag sample streaker is described which yields precise 1-cm streaks.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23807/1/0000045.pd

Treatment of Gaucher disease with an enzyme inhibitor

The hypothesis is offered predicting that Caucher patients could be treated with a drug that slows the synthesis of glucosylceramide, the lipid that accumulates in this disorder. The present therapeutic approach involves augmenting the defective enzyme, glucosylceramide β-glucosidase, with exogenous β-glucosidase isolated from human tissue. This spectacularly expensive mode of treatment should be replaceable with a suitable enzyme inhibitor that simply slows formation of the lipid and matches the rate of synthesis with the rate of the defective, slowly working β-glucosidase. Several drugs that possess this ability are available, the best known of which is 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a designer inhibitor that resembles the synthase's substrate and product. PDMP has been found to be effective in mice, rats, fish, and a wide variety of cultured cells. Its use, at suitable dosages, seems to be harmless, although long-term tests have not been made. The lack of suitable animal models of Gaucher disease has made it difficult to test the hypothesis adequately, but PDMP does rapidly lower the levels of glucosylceramide in normal animal tissues and the animals evidently do well with the lowered levels of glucosylceramide and its more complex glycolipid metabolites.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45689/1/10719_2004_Article_BF00731489.pd