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This study describes the effects of the glucolipid synthase inhibitor P4, (DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol), on various functional and phenotypic parameters of 5T33 murine myeloma cells. Cell recovery was reduced by >85% following incubation of the cells for 3 days in the presence of 4 μM P4 (the IC50 concentration). Both cytostatic and cytotoxic inhibition was observed with tumour cell metabolic activity and clonogenic potential reduced to 42% and 14% of controls, respectively, and viability reduced to 52%. A dose-dependent increase in cells undergoing apoptosis (from 7% to 26%) was also found. P4 induced a decrease in the number of cells expressing H-2 Class I and CD44, and a large increase in cells expressing H-2 Class II and the IgG2b paraprotein. It did not affect surface expression of CD45 or CD54 (ICAM-1). Based on these alterations in tumour cell growth, adhesion molecule expression and potential immunogenicity, it is anticipated that P4 will provide a novel therapeutic approach for the treatment of multiple myeloma. In addition, given that essentially all tumours rely heavily on overexpressed or abnormal glucosphingolipids for growth, development and metastasis, glucolipid synthase inhibitors may prove to be universally effective anti-cancer agents. © 1999 Cancer Research Campaign

A recent case report in the journal described a Gaucher’s disease patient, dangerously infected with salmonella that responded poorly to antibiotic treatment.1 However, use of enzyme replacement with glucosylceramide glucosidase lowered tissue levels of glucosylceramide (GlcCer), and produced a slow but dramatic improvement. The article points out that infection is an important aspect of the genetic disorder. I would like to mention important observations that explain this unfortunate complication and their significance for many patients with infections. Many research studies have shown that a wide variety of bacterial and viral infections involve binding of the organism to a glucosphingolipid in the cell surface. Patients …

The hypothesis offered here is that many different illnesses could be treated by slowing the synthesis of glucosphingolipids (GSLs) with a suitable inhibitor. In people with inadequate hydrolases for the GSLs (e.g. Gaucher's disease), the lipids accumulate to a pathological degree. It should be possible to eliminate the accumulation by slowing the synthesis of the GSLs to match the ability of the patient to degrade them. In people with cancer, the tumors secrete excessive amounts of GSLs, which block the ability of the immune system to attack the tumor cells. By blocking the synthesis of tumor GSLs, it should be possible to enable the patient to generate antibodies and activated T cells that can destroy the tumor. Tumors exhibiting multidrug resistance may do so by synthesizing GSLs even faster than usual. It should be possible to restore the sensitivity of the tumor to anti-cancer drugs by inhibiting their synthesis of GSLs. Metastasis of tumors also appears to require the formation of GSLs, so an inhibitor should help block tumor dissemination. Diabetics tend to have high levels of blood glucose, which acts to stimulate kidney growth via more rapid synthesis of GSLs. This pathological growth can be blocked by inhibiting the formation of kidney GSLs. Viruses, bacteria, and bacterial toxins have been found to bind to specific GSLs of human and animal cells. Presumably, this binding leads to the damaging process of infection. It should be possible to treat such infections by depleting the host's body of its GSLs.

A ceramide glycanase (CGase activity has been characterized from lactating rat mammary tissue which cleaves the glycosidic bond between sphingosine and the glucose chain of a glycosphingolipid (GSL) thus liberating the intact oligosaccharide chain from a GSL. The majority (65%) of the hydrolase activity was detected in the supernatant fraction when the rat mammary tissue homogenate was centrifuged at 100,000 x g. Attempts to purify the enzyme indicated that the CGase protein is of hydrophobic nature as it binds to hydrophobic columns. The enzyme has been partially purified using hydrophobic columns in tandem. The partially purified protein was found to be immunoreactive to the antibody raised against the purified clam CGase. The immunostained band corresponded to a 64 kDa protein as also found with the clam enzyme. This immuno cross-reactivity indicated probable structural similarities between CGase proteins isolated from widely separated species in the evolutionary tree. The rat CGase was found to have a specific detergent requirement for optimal activity, and the pH optimum was found to be between 5 and 6. The enzyme activity is partially heat stable. It is not a divalent cation requiring enzyme; however, the activity is totally inhibited in the presence of mercury, indicative of a sulfhydryl group in the active site of the enzyme. The rat mammary CGase activity is inhibited in the presence of both D- and L-PPMP (1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol. HCl), homologs of PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol. HCl), a well-known inhibitor of GlcT-1 (Ceramide: UDP-Glc Glucosyltransferase), an enzyme in the glycolipid synthetic pathway. The inhibition seems to be of a competitive nature and the same type of inhibition is also observed with clam CGase. The CGase activity was found to be highest in lactating tissue compared to the activity found in either pregnant or post-lactating rat mammary tissues. Tissue survey indicated the presence of high levels of CGase in lactating rat liver, uterus, and ovary; moderate activity was detected in kidney and spleen. Both virgin and male rat mammary tissue also indicated a basic level of CGase activity. However, newborn spleen and mammary tissue showed a comparable level of activity to that found in lactating rat tissues. This report is mainly concerned with the characterization of CGase activity from a mammalian source and its importance in cellular processes.

A unique transacylase that catalyzes esterification of a short chain ceramide, N-acetylsphingosine, was found in Madin-Darby canine kidney cell and mouse tissue homogenates. It esterified the hydroxyl group at the carbon-1 position of the ceramide. The enzyme has a pH optimum of 4.2 and a Km of 9.4 microM for N-acetylsphingosine at pH 4.5. The transacylase activity is independent of free fatty acid or acyl-CoA and instead uses the 2-acyl group of phosphatidylethanolamine or phosphatidylcholine. The transacylase activity in the homogenate was present in the 100,000 x g supernatant, and the lipid extracted from the membranous fraction could function as a donor of the acyl group. When liposomes consisting of dioleoylphosphatidylcholine:1-palmitoyl-2-[14C]arachidonoyl-phosphati dylethanolamine:sulfatide (70:0.2:30) were incubated with the supernatant and N-acetylsphingosine, the formation of free arachidonic acid and O-arachidonoyl-N-acetylsphingosine was observed. The ratio of the two products depended on the concentration of ceramide; only the free acid was formed if the truncated ceramide was absent. Both deacylase and transacylase activities were inhibited 50-60% by 20 microM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of several glucosphingolipid synthases. Neither activity was inhibited by nonadecyltetraenyl trifluoromethyl ketone, a potent inhibitor of cytosolic phospholipase A2. N-Acetyldihydrosphingosine and N-octanoylsphingosine were only 55 and 10%, respectively, as effective as N-acetylsphingosine as acyl acceptors. Oleoylsphingosine was only slightly reactive. An esterase that releases the truncated ceramide from its ester linkage appears to be membrane bound. Lecithin was less effective than phosphatidylethanolamine as an acyl donor in the transacylation. Madin-Darby canine kidney cell cultures treated with N-acetyl-[3-3H]sphingosine formed radioactive polar sphingolipids, long chain ceramide, free sphingosine, and O-acyl-N-acetylsphingosine. This suggests that the deacylation and transacylation reactions observed in vitro occur in growing cells as well.

In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptor. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2'-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G1/S and G2/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G1/S and G2/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34cdc2 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is a structural analog of ceramide that inhibits glucosylation of this molecule and thus of glucosphingolipid (GSL) expression by living cells. In this study, we used PDMP to slow the synthesis of the globoseries of GSLs (globo-GSLs) (derived from the precursor Gal alpha 1-4Gal beta 1-4Glc-ceramide) by cultured human kidney and large intestinal epithelial cells. The aim was to deplete the cells of receptors for P-fimbriated Escherichia coli and to examine the effects on the bacterially induced cytokine response. The mammalian cells (A498, HT-29, and Caco2) were cultured in the presence of PDMP in order to deplete them of GSLs. The cells were then subjected to GSL analysis or used to test bacterial adherence and cytokine production. The globo-GSLs were identified by thin-layer chromatography. Bacterial adherence was quantitated by microscopy, and interleukin-6 secretion was quantitated by the B9 bioassay. The interaction of bacteria with the globo-GSLs was studied by using E. coli strains and recombinant clones expressing P fimbriae. E. coli strains expressing type 1 fimbriae binding to mannose-containing glycoproteins were used as controls. PDMP treatment was found to reduce the content of the globo-GSLs in mammalian cells and the adherence of P-fimbriated E. coli to these cells. In contrast, PDMP treatment had no effect on the adherence of type 1-fimbriated E. coli or their activation of cytokine production by A498 cells. P-fimbriated E. coli elicited an interleukin-6 response in the A498 cells; this response was reduced after treatment with PDMP. The results emphasize the role of GSLs as receptors for P-fimbriated E. coli and for the cytokine response elicited by attaching bacteria.

Glucosylceramide (GlcCer) and related glycosphingolipids have been implicated as causal elements in both the growth of cells and in the regulation of hormonal signaling. We therefore studied whether the renal hypertrophy induced by diabetes was associated with enhanced synthesis of glycosphingolipids. 16 d after the induction of diabetes, increases in renal size and concentration of glucocerebroside and ganglioside GM3 were observed paralleling an increase in UDP-Glc concentration. GlcCer synthase and beta-glucosidase-specific activities were no different between control and diabetic kidneys. The apparent Km of the GlcCer synthase with respect to UDP-Glc was 250 microM and was unchanged in the diabetic kidneys. The observed concentrations of UDP-Glc were 149 and 237 microM in control and diabetic kidneys, respectively. The UDP-Glc level is thus rate limiting with regard to GlcCer synthesis. To determine whether the changes in glycolipid content were functionally significant, diabetic and control groups were treated with the GlcCer synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1- propanol, 2 wk after the induction of diabetes. Kidney weights in the diabetic rats treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol were no different than the control groups. Morphometric analysis of glomerular volumes paralleled changes in renal growth. Glycosphingolipid formation may therefore represent a significant pathway for glucose utilization in early diabetic nephropathy.

An inhibitor of glucosylceramide (GlcCer) synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), has been reported to deplete cells and mice of their glucosphingolipids. This inhibitor has proved useful for the elucidation of the many functions of this lipid family [reviewed by Radin, N.S.Inokuchi, J. (1991) Trends Glycosci. Glycotechnol. 3, 200-213]. In the present study, we have synthesized homologs of PDMP having different acyl chains (C6-C18) and compared their effectiveness for the inhibition of GlcCer synthase in vitro and their inhibition of GlcCer, protein, and DNA synthesis in cultured MDCK (Madin-Darby canine kidney) cells. Using MDCK homogenates and mouse brain and liver microsomes, we found that the C6 compound was relatively inactive and that the longer chain compounds did not differ much in inhibitory power. However, the use of intact MDCK cells showed that the longer chain homologs were much more effective in inhibiting GlcCer synthesis, cell growth, and incorporation of [3H]thymidine. Tests with two radioactive homologs showed that the inhibitor with a longer acyl chain was taken up much more effectively by MDCK cells and that this difference explains the much greater effectiveness of this homolog in intact cells. The inhibitors were effective when solubilized either with a nonionic detergent or with bovine serum albumin. The extent of decrease in DNA synthesis was not directly proportional to the decrease in cellular glucosylceramide, possibly because only a low level of the glycolipid is needed for DNA synthesis.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is an effective inhibitor of the glucosyltransferase that makes glucosylceramide. Virtually all of the hundreds of naturally occurring glycolipids are formed from this primary glycolipid, so the inhibitor acts to lower their concentrations by the process of attrition (hydrolytic catabolism). Trials with mice carrying ascites carcinoma cells showed that PDMP could produce a permanent cure in some of the animals and marked prolongation of life in the others (Inokuchi, J., I. Mason, and N.S. Radin. 1987. Cancer Lett. 38: 23-30). In order to maximize the effect, we studied the metabolism of PDMP by labeling it with [3H] on carbon one, using a labeling method that discriminated against the unwanted erythro-isomer. The active enantiomer of the inhibitor (D-) was isolated by chromatography of the camphanate esters, followed by methanolytic cleavage. Examination of the fate of the labeled drug after a single injection showed that it was very rapidly converted to several polar products that were rapidly excreted. The drug penetrated all of the organs readily and a small portion was oxidized at the C-1 position to yield 3H2O. From these findings it appeared likely that the amine is attacked by a mixed function oxidase based on cytochrome P450. This conclusion was confirmed by showing that the tissue levels of PDMP could be greatly elevated, for a much longer time, when the mice were pretreated with piperonyl butoxide or cimetidine. The amount of conversion to polar metabolites was substantially reduced and tissue levels of PDMP were maintained much longer. Analysis of mice injected with one or both drugs showed that piperonyl butoxide augmented the effects of PDMP on ceramide, glucosylceramide, and dihexosylceramide levels, as well as on the activity of glucosylceramide synthase. It is suggested that piperonyl butoxide be used as an adjuvant for the many useful drugs that are inactivated by the P450 system.

Article de synthese traitant de la structure et des fonctions des glycosphingolipides dans le rein des mammiferes et de leur association fonctionnelle avec des evenements cellulaires comme la croissance, la differenciation, la generation de messagers secondaires et le transport d'ion.

Incubation of rat red cells with liposomes containing radioactive glucosylceramide, together with unlabeled phosphatidylcholine and cholesterol, produced cells labeled with the glycolipid. Part of the bound cerebroside could be removed by further incubation with unlabeled liposomes. Both processes, uptake and release, were stimulated by adding a preparation obtained from bovine spleen. The active material was purified partially by ammonium sulfate precipitation and gel permeation with a Sephadex G-75 column. The latter step indicated a molecular weight of about 22,000. The material lost much of its activity when treated with trypsin or N-ethylmaleimide, indicating that it is a protein. The "cerebroside uptake protein" was quite stable if stored in dithioerythritol at 4 degrees C. Tests with liposomes containing labeled phosphatidylcholine or cholesterol showed that the red cells took up these lipids as well, but the transfer protein exerted little effect on this. Ghosts prepared from rat red cells also took up the three lipids from the liposomes and here, as well, the uptake protein showed high specificity in facilitating the uptake of cerebroside. This appears to be the first demonstration of the existence of a glycolipid transfer protein. Possible physiological roles for the uptake protein in red cell metabolism and in the modulation of the glycosphingolipid composition of cell membranes are discussed.

Galactose oxidase can be used to oxidize the terminal carbon atom of lipids containing galactose or N-acetylgalactosamine, and the resultant aldehyde group can be reduced back to the original carbinol with radioactive borohydride. The efficiency of the first reaction has been investigated systematically by using [6-3H]galactosyl ceramide as substrate and measuring the amount of radioactive water formed. This enabled us to establish that the addition of catalase and peroxidase greatly speeded the oxidation, that phosphate and PIPES buffers were the best among those tested, that the reaction continued for 24 hr without a second addition of galactose oxidase, and that the optimum concentration of organic solvent (tetrahydrofuran) was 50%. The suggestion if made that a similar set of variables be studied for each lipid or nonlipid by the same basic technique: labeling by the oxidase/borohydride method and use of the resultant compound as substrate.

A method is described for the determination of sulfur-35 in large amounts of sulfuric acid using a liquid scintillation counter. The acid is converted to a neutral salt of a high molecular weight aliphatic primary amine and is dissolved in toluene-ethyl alcohol. The efficiency of counting is almost independent of the amount of sulfate, but does depend on the amount of amine. Other acids, such as radioactive citric, phosphoric, and hydrochloric acids can be counted by this technique. Finally, preliminary findings on the counting of monosaccharides are presented also.

Incubation of rat red cells with liposomes containing radioactive glucosylceramide, together with unlabeled phosphatidylcholine and cholesterol, produced cells labeled with the glycolipid. Part of the bound cerebroside could be removed by further incubation with unlabeled liposomes. Both processes, uptake and release, were stimulated by adding a preparation obtained from bovine spleen. The active material was purified partially by ammonium sulfate precipitation and gel permeation with a Sephadex G-75 column. The latter step indicated a molecular weight of about 22,000. The material lost much of its activity when treated with trypsin or N-ethylmaleimide, indicating that it is a protein. The "cerebroside uptake protein" was quite stable if stored in dithioerythritol at 4 degrees C. Tests with liposomes containing labeled phosphatidylcholine or cholesterol showed that the red cells took up these lipids as well, but the transfer protein exerted little effect on this. Ghosts prepared from rat red cells also took up the three lipids from the liposomes and here, as well, the uptake protein showed high specificity in facilitating the uptake of cerebroside. This appears to be the first demonstration of the existence of a glycolipid transfer protein. Possible physiological roles for the uptake protein in red cell metabolism and in the modulation of the glycosphingolipid composition of cell membranes are discussed.

beta-Glucosidase activator (SAP-2) is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. In this study, we improved the purification method and found that SAP-2 is highly heterogeneous. A hot water extract of frozen guinea pig liver was fractionated by ammonium sulfate sedimentation, then chromatographed with DEAE-Sephacel, Sephadex G-75, and concanavalin A-Sepharose. A fraction binding to concanavalin A-Sepharose was purified further with a C4 high performance liquid chromatography reverse phase column. This yielded several peaks, the main one of which was studied. The specific activity of the purified SAP-2 was 35 units/micrograms (1 unit produces 50% stimulation of a basal glucosidase preparation). N-terminal amino acid sequencing showed that this preparation is a mixture of polypeptides differing in the presence or absence of one or two of the end amino acids. The complete amino acid sequence of the 81 residues in SAP-2 has been determined. Comparison of the sequence of guinea pig SAP-2 with the sequence of human sphingomyelinase activator revealed 58% homology and quite similar hydropathy profiles. Both proteins possess a highly hydrophilic region around Asn-22, which is glycosylated, and 6 cysteine residues, in oxidized form, located in the same positions. Comparison with the published nucleotide sequence for the precursor form of the human activator protein for sulfatide sulfatase (SAP-1) suggested that this activator also has a possibly glycosylated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat different. Examination of another region of the precursor's nucleotide sequence, assuming a few changes in the identifications, revealed the presence of the sphingomyelinase activator. It appears that two or more activators are derived from a single precursor protein. Marked homologies were seen also with a lung surfactant protein and a sulfated glycoprotein from Sertoli cells.

Eicosapentaenoic acid prevents platelet aggregation and inhibits arachidonate conversion into thromboxane A2 and prostaglandins. Consequently eicosapentaenoic acid might protect the brain from the ischemia that follows cerebral arterial occlusion. We studied the effect of eicosapentaenoic acid on cerebral ischemia in anesthetized gerbils. Ischemia was produced by bilateral carotid occlusion for 10 min, followed by reperfusion for 60 min, in gerbils fed either a standard diet (control) or a diet supplemented with menhaden fish oil for 2 months. The menhaden fish oil contained 17 mole % eicosapentaenoic acid. Regional cerebral blood flow was measured by the hydrogen clearance method and brain water by the specific gravity technique. In control animals cerebral blood flow was decreased 30 and 60 min after reperfusion (p less than .001) and brain water was increased (p less than .001). In the experimental group cerebral blood flow did not fall during reperfusion and edema did not appear. Brain prostaglandins and thromboxane were measured by radioimmunoassay. PGF2 alpha, PGE2, 6-keto PGF1 alpha and TXB2 increased after severe ischemia and reperfusion. The synthesis of brain diene prostaglandins was not altered by eicosapentaenoic acid. Our study indicates that eicosapentaenoic acid prevented post-ischemic cerebral edema and hypoperfusion, without affecting the levels of brain diene prostaglandin and thromboxane.

Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.

SummaryGlucose utilization, serum insulin, human growth hormone, and free fatty acids were studied in 18 unipolar depressed patients and 14 normal controls, using the intravenous glucose tolerance test. Patients were kept drug-free for at least two weeks before testing, and physical activity and diet were carefully controlled. Previously described abnormalities in the balance between glucose and free fatty adds were not seen, but endogenous depression was associated with lowered glucose utilization rate with insulin resistance. Changes in kinetics of membrane transport in endogenous depression which could account for this finding are discussed.