Published by Springer Verlag
Online ISSN: 1558-9307
Print ISSN: 0024-4201
Dietary energy density (ED) appears to have a major influence on the regulation of food intake and body weight. If people consume a fixed weight of food each day, then high-ED diets should be associated with high energy intakes and with overweight. In contrast, low-ED diets should result in lower daily energy intakes and therefore weight loss. For this approach to work, low-ED foods must be as palatable as high-ED foods and, calorie for calorie, have a greater satiating power. Each of those assumptions is debatable. Dietary ED depends chiefly on the water content of foods. As a rule, high-ED foods are more palatable but less satiating, whereas low-ED foods are more satiating but less palatable. Consumer preferences for high-ED foods can be explained in terms of good taste, low cost, and convenience. Low-ED foods, such as fresh produce, provide less energy per unit cost than do high-ED foods, which often contain added sugars and fats. Poverty and obesity may well be linked through the habitual consumption of a low-cost, high-ED diet.
Chemical examination of the ethyl acetate extract from the fermentation broth of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new glycolipids, ieodoglucomide C (1) and ieodoglycolipid (2). The structural characterization of 1 and 2 was achieved by extensive spectroscopic evidence, including 2D NMR experiments. A combination of chemical derivatization techniques followed by NMR studies, LC-MS data analysis and a literature review was deployed for the establishment of the stereo-configurations of 1 and 2. Compounds 1 and 2 exhibited good antibiotic properties against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa with MICs ranging from 0.01 to 0.05 μM. Furthermore, the antifungal activity of 1 and 2 was evaluated against plant pathogenic fungi Aspergillus niger, Rhizoctonia solani, Botrytis cinerea and Colletotrichum acutatum as well as the human pathogen Candida albicans. Compounds 1 and 2 inhibited the mycelial growth of these pathogens with MIC values of 0.03-0.05 μM, revealing that these compounds are good candidates for the development of new fungicides.
The seed oil ofEuonymus verrucosus Scop., family Celastraceae, contains more than 90% 1,2-diacyl-3-acetins (monoacetotriglycerides). The constituent triglyceride acids, other than acetic, are the usual long-chain fatty acids. Thin-layer chromatography (TLC), infrared (IR) and hydrolysis with pancreatic lipase indicate that the acetic acid is esterified exclusively on the outer glycerol carbon atoms. The isolated mixed monoacetotriglycerides exhibit optical rotation caused by asymmetry of the central glycerol carbon atom. Comparison with synthetic products of known configuration shows that the natural material is essentially all (S)-1,2-diacyl-3-acetin. IR, TLC and GLC analyses indicate the presence of monoacetotriglycerides in seven other species of Celastraceae and five species in three other plant families in amounts from 13 to 98%.
Five 1,2-cyclic acetal-type sn-glycero-3-phosphoethanolamines (CGPE) were isolated in a pure state from the sea anemone, Actiniogeton sp. (Coelenterata). Their structures, including the absolute configurations, have been determined on the basis of chemical and spectral data to be so-called Feulgen's acetalphosphatides, which have been regarded as artifacts derived from original plasmalogens. We examined whether these CGPE are intact constituents in the animal tissues and obtained reliable confirmation that CGPE are normally present in the sea anemone.
The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100, n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100-130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.
Reaction of methyl 9,10-dioxostearate (1) and 9,12-dioxostearate (2) with 1,2-diaminoethane under concomitant ultrasonic irradiation (10-15 min, 60 degrees C) in water furnished the corresponding 2,3-dihydropyrazine (4, 79%) and 1,2,3,4-tetrahydro-1,4-diazocine (5, 70%) derivatives, respectively. Reaction of methyl 10,12-dioxostearate (3) with 1,2-diaminoethane was successful only when glacial acetic acid was used instead of water under ultrasonic irradiation (4 x 10 min, 70 degrees C) to give a 2,3-dihydro-1H-1,4-diazepine (6, 95%) derivative. The structures of these novel six-membered (4), seven-membered (6), and eight-membered (5) N-heterocyclic fatty ester derivatives were confirmed by a combination of infrared, nuclear magnetic resonance spectroscopic and mass spectral analyses.
(A) General reaction scheme for lipase-catalyzed hydrolysis of TAG to glycerol and FFA. (B) Reaction scheme for lipase-catalyzed hydrolysis/methanolysis of triricinolein to 1,2(2,3)-diricinolein and FFA/FAME.
RP-HPLC analysis of a fraction from the Penicillium roquefortii lipase (PRL)-catalyzed reaction in diisopropyl ether (DIPE) at water activity (a w ) of 0.11. An ODS column (Beckman Instruments Inc., Fullerton, CA) was used, and the mobile phase was based on methanol and water (see the Experimental Procedures section). The peaks were identified as ricinoleic acid, BHT, methyl ricinoleate, diricinolein (DAG), and triricinolein (TAG).
(A) Mass spectrogram of diricinolein generated in the single MS mode, and the chemical structure of 1,2(2,3)-diricinolein. (B) Mass spectrogram of the parent ion of diricinolein generated in the tandem MS-MS mode, and the chemical structure of 1,2(2,3)-diricinolein minus one of its FA chains. R = ricinoleic acid, as illustrated in Figure 1.
Normal-phase HPLC analysis of a fraction from the PRL-catalyzed reaction in DIPE at an a w of 0.53. A CN column (Phenomenex, Torrance, CA) and a mobile phase consisting of hexane and methyl tert-butyl ether (see the Experimental Procedures section) were used. The peaks were identified as methyl ricinoleate, ricinoleic acid, triricinolein (TAG), 1,3-diricinolein (1,3-DAG) and 1,2(2,3)-diricinolein [1,2(2,3)-DAG]. For other abbreviations see Figure 2.
The objective of this study was to find the optimal parameters for lipase-catalyzed methanolysis of triricinolein to produce 1,2(2,3)-diricinolein. Four different immobilized lipases were tested, Candida antarctica type B (CALB), Rhizomucor miehei (RML), Pseudomonas cepacia (PCL), and Penicillium roquefortii (PRL). n-Hexane and diisopropyl ether (DIPE) were examined as reaction media at three different water activities (a w), 0.11, 0.53, and 0.97. The consumption of triricinolein and the formation of 1,2(2,3)-diricinolein, methyl ricinoleate, and ricinoleic acid were followed for up to 48 h. PRL gave the highest yield of 1,2(2,3)-diricinolein. Moreover, this lipase showed the highest specificity for the studied reaction, i.e., high selectivity for the reaction with triricinolein but low for 1,2(2,3)-diricinolein. Recoveries of 93 and 88% DAG were obtained using PRL in DIPE at a w of 0.11 and 0.53, respectively. Further, NMR studies showed that a higher purity of the 1,2(2,3)-isomer vs. the 1,3-isomer was achieved at higher a w (88% at a w=0.53), compared to lower a w (71% at a w=0.11). The DAG obtained was acylated by the DAG acyltransferase from Arabidopsis thaliana. Therefore, this enzymatic product is a useful enzyme substrate for lipid biosynthesis. Accordingly, the use of PRL in DIPE at a w 0.53 is considered optimal for the synthesis of 1,2(2,3)-diricinolein from triricinolein.
Oral administration of 1,2-propanediol to rats in a daily dose of 1 ml of 28.4% aqueous solution per 100 g body weight for 30 days caused a significant decrease in the total lipids, fatty acids, phospholipids, and triglycerides of plasma, liver, and heart. The cholesterol content in plasma decreased while that in the tissues increased significantly. The accumulation of cholesterol in tissues tends to discourage long term use of 1,2-propanediol even by the oral route.
Alfalfa leaflet tissue incorporated choline-1,2-14C into at least 6 molecular speies of phosphatidylcholine. After 7 hr incubation the species containing palmitic and linoleic acids (16∶0–18∶2) and the species containing linolenic and linoleic acids (18∶3–18∶2) had the highest specific activities. After 24 hr incubation, the species containing palmitic and linolenic acids (16∶0–18∶3) and the species containing 2 linolenic acids (18∶3–18∶3) had the highest specific activities. The most likely interpretation of these data is that choline is accepted by diglycerides containing one linoleic acid residue and that this residue is then desaturated within the phosphatidylcholine molecule to linolenic acid.
Long chain 1,2-alkanediol diesters comprise about 15-20% of the acetone soluble skin surface wax of golden Syrian hamsters. The constituent 1,2-alkanediols, obtained through acidic methanolysis, were fractionated by preparative gas liquid chromatography of their isopropylidene derivatives. The major component (57%) was identified by nuclear magnetic resonance and mass spectrometry as 15-methyl-1,2-hexadecanediol.
Thirteen-day-old rats were divided into two groups; one group received 1,2-(1-14C) dipalmitoyl phosphatidylcholine and the other 1-14C palmitic acid in the form of an intraperitoneal injection. One half of the total number of rats in each group was sacrificed 24 hr after injection, and the other half was allowed to survive for 17 days after the injection. Radioactivity incorporated into brain and liver total lipids and into individiual polar lipid components of the brain was determined at both intervals. Phosphatidylcholine was isolated and partially deacylated with phospholipase A2 fromCrotalus Admanteus venom. The ratio of radioactivity FA 2/FA 1 (fatty acid attached to 2 and 1 carbon of the glycerol moiety) 24 hr after the injection was 8.3, when the tracer was radioactive phosphatidylcholine, compared to only 0.7 when radioactive palmitate was injected. From this different labeling ratio and different pattern of labeling the polar lipid components, it was concluded that the radioactive phosphatidylcholine was not deacylated completely before being taken up directly into the brain. Possibilities are discussed to show that the observed radioactive ratio could result from direct uptake of intact phosphatidylcholine, with little or no restriction from the blood brain barrier system, followed by partial degradation by phospholipase A1 in the brain itself.
Unsaturated long-chain fatty acids are oxidized more rapidly than are saturated fatty acids of similar chain length in intact animals and isolated mitochondria. Gamma-oxidation of the 3-dodecenoic acid intermediate in beta-oxidation of oleate would yield propionate which is metabolized via methylmalonate.14C-labeled fatty acids were administered to intact rats, muscle homogenates and lysed mitochondria. Methylmalonate, succinate and CO2 were isolated and14C determined. Incorporation of U-14C-linoleate into methylmalonate in vitro was 20 times greater than from U-14C-palmitate. Rats fed 20% corn oil grew more slowly on B12 deficient than B12 sufficient diets. Biotin and vitamin B12 deficiencies were found to decrease the in vivo metabolism of linoleate. These data suggest that one pathway of linoleate oxidation has methylmalonate as an intermediate.
The initial incorporation of glycerol-3H into the molecular species of liver 1,2-diacyl-sn-glycerols and phosphatidylcholines was studied in vivo using goldfish acclimated to 10 C and 30 C. A 1.5- and 2.2-fold higher proportion of the total radioactivity in the diacylglycerols from cold acclimated fish was found to be associated with the trienoic and pentaenoic species, respectively, when compared to warm acclimated fish. In the phosphatidylcholines, 1.9- and 1.3-fold greater percentages of the newly-incorporated radioactivity were found in tetraenoic and pentaenoic molecules, respectively, from cold relative to warm acclimated fish which suggests a preferential synthesis of these molecules relative to other molecular species in response to a lowering of environmental temperature. The present results indicate, therefore, that environmental temperature influences the complement of molecular species of diacylglycerols an phosphatidylcholines which fish produce by way of de novo biosynthesis in vivo.
A novel phosphonium salt methodology was utilized for the first time to synthesize 1,3-, and 1,2-diphosphatidylglycerol. Optically active 1,2-di-O-acyl-sn-glyceryl phosphate was coupled with unprotected glycerol in the presence of pyridiniumbromide perbromide and triethylamine to yield, after final removal of phosphate protecting group, the title compounds. The 1,2-diphosphatidylglycerol (1,2-isomer of cardiolipin) may be a member of a new class of phospholipids for industrial applications similar to other phosphocholines.
A simple chemical method for the synthesis of 1,2-diacyl-sn-glycerophosphatidylserine (PS), with the same fatty acid composition in the sn-1 and sn-2 glycerol positions as egg phosphatidylcholine (PC), is described. PS synthesis was carried out by a phosphite-triester approach, using 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (phosphoramiditate) as the phosphorylating agent, for the formation of phosphate linkage between serine and diacylglycerol. 1,2-Diacylglycerol, obtained from PC hydrolysis by phospholipase C, was coupled with N-t-BOC-L-serinebenzhydryl ester phosphoramidite with tetrazole as catalyst. Phosphite-triester was oxidized to the corresponding phosphate-triester with 30% H2O2 in CH2Cl2. The cyanoethyl group was removed by addition of an Et3N/CH3 CN/pyridine mixture, and trifluoroacetic acid was used to eliminate the protecting groups of O-(1,2-diacylglycero-3-phospho)-N-t-BOC-serinebenzhydryl ester. Purified PS was identified by thin-layer chromatography, infrared, and 1H nuclear magnetic resonance.
1,2-Dimethylhydrazine is a procarcinogen with selectivity for the colon and proximal small intestine. In weekly subcutaneous (s.c.) doses of 20 mg/kg body weight, this agent produces colonic and proximal small intestinal tumors in a high percentage of rodents with a latency period of approximately six months. To determine whether alterations in the glycosphingolipid content of rat proximal and/or distal small intestinal mucose existed before the development of dimethylhydrazine-induced cancer, rat were given s.c. injections of this agent (20 mg/kg body weight per wk) or diluent for five wk. Animals were killed at this time, and mucosa was isolated from each small intestinal segment of both groups. Glycosphingolipids then were extracted from these tissues and analyzed by high performance thin layer chromatography and gas liquid chromatography. The results of these studies demonstrated that (1) the content of neutral and acidic glycosphingolipids was significantly decreased (approximately 20%) in the proximal small intestine of treated rats compared with their control counterparts; (2) no significant difference in the glycosphingolipid content was seen, however, in the distal small intestinal mucosa of control and treated rats; and (3) while significant differences were noted in the majority of fatty acids of GM3, glucosyl- and globotriaosylceramide in the proximal small intestine of control and treated animals, differences in the fatty acids of these glycosphingolipids in the distal segment of these groups were confined to stearic (18∶0) acid and/or arachidic (20∶0) acid.
The influence of dietary cholesterol on the relative rates of synthesis of hepatic lipids in the male Mongolian gerbil, Meriones unguiculatus, was studied. The semi-purified starch-based diet used lard as the dietary fat and was fed with or without a 0.5% (by wt.) cholesterol supplement. Each animal received 300 microCi [2-3H]-glycerol i.p. after 3 or 7 days on the dietary regimens. Relative rates of [2-3H]-glycerol incorporation into the major hepatic glycerides in vivo was not affected significantly by dietary cholesterol (0.5% level), suggesting that alteration in the relative biosynthesis of these lipids could not readily account for the higher triglyceride (TG) to phospholipid (PL) mass ratio in liver with cholesterol feeding. However, there was evidence for an increased formation of 1,2-diglyceride (1,2-DG). The complement of molecular species of hepatic 1,2-DG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) formed de novo, as measured using isotopic glycerol, was not influenced greatly by dietary cholesterol, although lower mean rates of synthesis of tetraenoic relative to dienoic species of phospholipids were indicated in cholesterol-fed gerbils.
(A) Reaction scheme for lipase-catalyzed hydrolysis/methanolysis of triricinolein to 2-monoricinolein. (B) Reaction scheme for lipasecatalyzed esterification of 2-monoricinolein and ricinoleic acid to 1,2(2,3)-diricinolein.
Time course of the ROL-catalyzed methanolysis reaction of triricinolein in (A) DIPE and (B) toluene. Data points represent the means of two experiments. For abbreviations see Figure 2.
The purpose of this investigation was to develop conditions for producing 2-monoricinoleoyl DAG. We used lipase-catalyzed hydrolysis of triricinolein to obtain 2-monoricinolein and thereafter synthesized 1,2(2,3)-diricinolein through esterification of 2-monoricinolein, using ricinoleic acid as the acyl donor. Five different 1,3-specific immobilized lipases were tested for the initial methanolysis reaction: Candida antarctica type B, Rhizomucor miehei, Rhizopus oryzae (ROL), Thermomyces lanuginosus, and Aspergillus niger. For the second esterification reaction, we investigated these five lipases plus Pseudomonas cepacia, Penicillium roquefortii, Candida rugosa, and Pseudomonas fluorescence. Toluene and diisopropyl ether (DIPE) were examined as reaction media at a water activity of 0.11. ROL in DIPE gave the highest yield of 2-monoricinolein from triricinolein, 78% after 3 h of reaction. The isolated 2-monoricinolein was esterified with ricinoleic acid for synthesis of 1,2(2,3)-diricinolein. ROL in DIPE gave the highest yield of 1,2(2,3)-diricinolein, 58% after 1 h of reaction, and NMR analysis showed that the purity was 97.2%. This methodology can be used for synthesizing radiolabeled 1,2(2,3)-diricinolein to study lipid biosynthesis in castor and other oilseeds.
The incorporation of 1,2-14C-ethanolamine into the liver phosphatidylethanolamines (PE) and phosphatidylcholines (PC) in female rats was studied. These phosphatides were fractionated according to their degrees of unsaturation and the specific activities of monoenoic, dienoic, tetraenoic and hexaenoic fractions were measured at intervals of 1, 20, 60 and 300 min after injection of the labeled precursor. Hexaenoic and dienoic PE incorporated and lost the label rapidly. Although the labeled precursor was incorporated into tetraene PE at a similar rate, this fraction attained a relatively low specific activity that remained essentially constant between 10 and 300 min after injection of the label. Hexaenoic PC had the highest specific activity among the PC fractions at all time periods. Estimations of the rate of loss of radioactivity in the hexaenoic PE fraction and its appearance in hexaenoic PC indicate that the docosahexaenoic acid is conserved, possibly by being reincorporated into PE after becoming a part of the hexaenoic PC species. The high rate of turnover of the hexaenoic PE also suggests that this fraction might have some special role in endogenous choline synthesis.
Reaction of C60 fullerene with dialkyl bromomalonate (where the alkyl groups consist of short-, medium-, and long-saturated chains or unsaturated long chains) in the presence of sodium hydride gives [6,6]-bridged mono-adducts of methanofullerene. The spectroscopic properties of such fullerenoid lipids are reported.
Some properties of the pure, soluble phospholipase D (phosphatidycholine phosphatido hydrolase, EC interactions with phosphatidyl choline (1,2 diacyl-sn-glycerol-3-phosphoryl choline) in a system also containing dodecylsulfate and Ca2+ ions were studied. Concentrations of Ca2+ greater than 50 mM were necessary both for activity and adsorption of the enzyme to the "supersubstrate." Ethylenediamine tetraacetic acid caused inhibition of activity, greater than one would expect from its chelating capacity. A nonlinear increase in activity with the increase of enzyme protein was observed, suggesting a subunit aggregation into a higher mol wt protein, catalytically more active. Upon centrifugation of the supersubstrate-enzyme complex at 4.5 X 10(5) g-min at 30 C, most of the substrate molecules sedimented regardless of the pH. The reverse was true when centrifugation was done at 1 C. Phospholipase D hydrolyzed phosphatidylcholine molecules present in the supersubstrate at temperatures around 0 C at a rate 1/5 that of a maximal value measured at 30 C. The Arrhenius plot was linear in the range from 0 to 30 C, and at that temperature the curve broke with a smaller slope. Activation energy of 9.1 Kcal/mol, below 30 C, was calculated. Adsorption of the enzyme to the sedimentable supersubstrate occurred at pH 8.0, regardless of temperature. At pH 5.6, a considerable portion of phosphatidylcholine was degraded at 30 C, thus minimizing the capacity of the supersubstrate to adsorb the enzyme. Although Mg2+ could replace Ca2+ in the formation of sedimentable supersubstrate, it neither assists in adsorption of the enzyme nor in activation of the phosphatidylcholine hydrolysis.
A detailed procedure for quantitative determinations of molecular species of lecithins is described and applied to several lecithins isolated from natural sources. The method is based on the conversion of lecithin to acetylated 1,2-diglycerides and analysis of these compounds by methodology used for the determination of triglyceride structure. The preparation of the acetylated 1,2-diglycerides was carried out via hydrolysis with phospholipase C and acetylation of the resultant, 1,2-diglycerides with pyridine-acetic anhydride. Preparation of acetylated 1,2-diglycerides from lecithin by acetolysis with acetic acid-acetic anhydride was shown to be accompanied by intermolecular as well as intramolecular rearrangement of the fatty acids. The structure of the acetylated 1,2-diglycerides was determined by a combination of argentation-TLC and pancreatic lipase hydrolysis using internal standards for quantification. The method was applied to lecithins isolated from milk serum, egg, soybean, safflower seed and wheat germ lipids.
The synthesis of rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline of high specific activity was carried out on a microscale by making 7 mumol of rac-1-palmitoyl-2-oleoylglycerol react first with an equimolar amount of POCl3 and then of [3H]choline. After purification by thin-layer chromatography and normal-phase high-performance liquid chromatography and normal-phase high-performance liquid chromatography (HPLC), the yield of the synthesis of [3H]phosphatidylcholine (120 microCi/mumol) was 22%. rac-1-Palmitoyl-2-oleoylglycerol was purified before use by reversed-phase HPLC under conditions which were nonisomerizing and allowed the separation of 1,2- and 1,3-isomers of diacylglycerol. Ethanol, but not benzene, was shown to cause isomerization of long-chain diacylglycerol and, therefore, was not used for drying the substrate before reaction. A rapid and complete separation of 1,2- and 1,3-isomers of long-chain phosphatidylcholine was obtained by reversed-phase HPLC using 20 mM choline chloride in methanol/acetonitrile/water (50:50:1, by vol) isocratically as the mobile phase. Under these conditions, analysis of the synthesized rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline showed a total absence of 1,3-isomer.
A spray reagent of 4-amino-5-hydrazino-1,2,4-triazole-3-thiol, 2% in aqueous 1N NaOH, was used for the detection of aldehydes on chromatograms of lipids. Purple spots are produced with aldehydes, and 1 μg of palmitaldehyde can be detected easily on thin layer chromatograms. The reagent is specific for aldehydes and does not react with ketones. The aldehyde moiety from plasmalogens is detected only after acidic hydrolysis. Purple or red stains arise also with ozonides. Simple precautions against autoxidation of the lipids should be taken to prevent formation of aldehydes which could lead to misinterpretations.
The activity of the intestinal phosphatidylcholine deacylation-reacylation cycle has been found to be stimulated by 1,25-dihydroxy-vitamin D3. The stimulation of this cycle thus provides a possible mechanism for the reported retailoring of the fatty acid composition of phosphatidylcholine in intestinal cell membranes by 1,25-dihydroxy-vitamin D3 and its analogue, 1alpha-hydroxyvitamin D3.
Vitamin D3 and its metabolites comprise an endocrine system which plays a critical role in calcium homeostasis. The active form of vitamin D3 is 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Chromatin localization of 1,25(OH)2D3 and sucrose density gradient centrifutation have demonstrated the presence of an intestinal mucosa cytosol receptor which specifically binds 1,25(OH)2D3. The kinetic parameters of 1,25(OH)2D3 binding to its receptor have been determined by hydroxylapatite and reconstituted chromatin cytosol assays. Utilization of these assays has also permitted a determination of the precise structural requirements of the vitamin D ligand for the intestinal receptor. Furthermore, it has been possible to propose two receptor-ligand models which are capable of accommodating the conformationaly modile A ring of the vitamin D seco-steroids.
The triacylglycerol (TG) analog 1,3-dioctadecenoyl-2-hexadecyl glycerol was used in the study of the transport of dietary lipids by lipoprotein fractions of rat intestinal lymph. 1,3-Diacyl-2-alkyl glycerols (DAG) are hydrolyzed by pancreatic lipase to form 2-alkyl glycerols and free fatty acids. These hydrolysis products are then absorbed, and DAG are resynthesized within the intestinal mucosa. Intestinal lymph of rats was collected following intragastric administration of 1,3-dioctadecenoyl-2-hexadecyl glycerol. The DAG to TG ratios in very low density lipoprotein (VLDL) and chylomicron fractions were determined as a measure of the incorporation of lipid of dietary origin. The ratio of DAG to TG in the VLDL-2 (Sf 12–100) fraction ranged from0.06 to 0.56 indicating a significant amount of DAG transported relative to TG. The glyceryl ether to TG ratio increased with mean lipoprotein volume from the VLDL-2 fraction to the chylomicron (Sf>400) fraction. The correlation between glyceryl ether to TG ratio and average volume and between the amount of DAG per ml of original lymph and average volume within the chylomicron fraction was 0.99. Thus, the amount of dietary fat transported was correlated with the size of the chylomicrons produced. The glyceryl ether to TG ratio was positively correlated with the average volume of the lipoprotein fractions isolated (chylomicrons, chylomicron rich (Sf>100), VLDL-1 (Sf 100–400) and VLDL-2) (r=0.87). These results suggest that the size of the lipoproteins produced by the intestine is determined by the amount of fat available for transport and that particles of larger diameter are formed by the addition of lipid of dietary origin to existing VLDL.
It has been reported that, compared with TAG, DAG suppresses postprandial hypertriacylglycerolemia and reduces visceral fat levels in experimental animals and humans. To clarify the mechanism responsible for these beneficial effects, we compared the lymphatic transport of 1,3-DAG, a major isomer of DAG, and TAG in rats. Male SD rats, after insertion of a cannula into the thoracic duct, were given 1,3-di[1-14C]oleoylglycerol or tri[1-14C]oleoylglycerol via a stomach tube. The 24-h recovery of the radioactivity from 1,3-di[14C]oleoylglycerol in the lymph was slightly but significantly lower than that from tri[14C]oleoylglycerol (81.3+/-1.0 vs. 86.5+/-1.2%, respectively). However, in the first 1-h interval after administration, the recovery of radioactivity from 1,3-dioleoylglycerol was almost half of that from trioleoylglycerol (17.5+/-2.0 vs. 31.1+/-1.4%). The amount of TAG and phospholipids secreted into the lymph was significantly lower 1 h after the administration of 1,3-dioleoylglycerol compared with that after the administration of trioleoylglycerol. More than 90% of the radioactivity recovered in the lymph in the first 3 h was distributed in the TAG fraction for both 1,3-dioleoylglycerol and trioleoylglycerol. These results suggest that slower lymphatic transport of 1,3-DAG compared with TAG could be a factor in the suppression of postprandial hypertriacylglycerolemia. The possibility that the slower lymphatic transport of DAG contributes to the anti-obesity action observed in the feeding of 1,3-DAG cannot be excluded.
Diacylglycerol (DAG) is a component of various vegetable oils. Approximately 70% of the DAG in edible oils are in the configuration of 1,3-DAG. We recently showed that long-term ingestion of dietary oil containing mainly 1,3-DAG reduces body fat accumulation in humans as compared to triacylglycerol (TAG) oil with a similar fatty acid composition. As the first step to elucidate the mechanism for this result, we examined the difference in the bioavailabilities of both oils by measuring food energy values and digestibilities in rats. Energy values of the DAG oil and the TAG oil, measured by bomb calorimeter, were 38.9 and 39.6 kJ/g, respectively. Apparent digestibility expressed according to the formula: (absorbed) x (ingested)(-1) x 100 = (ingested - excreted in feces) x (ingested)(-1) x 100 for the DAG oil and the TAG oil were 96.3+/-0.4 and 96.3+/-0.3% (mean +/- SEM), respectively. The similarity in the bioavailabilities of both oils supports the hypothesis that the reduced fat accumulation by dietary DAG is caused by the different metabolic fates after the absorption into the gastrointestinal epithelial cells.
The dietary effect of 1,3-biseicosapentaenoyl-2-γ-linolenoyl glycerol (STG) on the fatty acid composition of guinea pigs was examined and compared with that of an eicosapentaenoic acid ethyl ester (EPA-F) and of a soybean oil (SBO) diet. In terms of content of plasma lipid, EPA-E had a greater hypolipidemic effect than STG. On the other hand, in terms of EPA incorporation, contents of EPA in liver lipid were almost the same in the STG and EPA-E groups. Considering that the amount of EPA administered in the EPA-E group was almost 1.5 times that of the STG group, FPA may be absorbed more effectively as the glycerol ester than as the ethyl ester in guinea pigs. In all the tissue lipids, the STG group had a higher unsaturation index (UI) than the EPA-E group even though there is a lower UI in the STG diet than the EPA-E diet. These results suggest that greater amounts of desaturase products as a whole were synthesized in the STG group than in the other two groups. The dihomo-γ-linolenic acid/arachidonic acid (DGLA/AA) ratio in plasma total lipids in the STG group was 3.5 times that of SBO group, and the DGLA/AA ratio in the EPA-E group was half that of the SBO group. In liver lipid, the ratios of DGLA/AA and EPA/AA in the STG group were 0.687 and 0.488 (phosphatidylcholine fraction) and 0.237 and 0.752 (phosphatidylethanolamine fraction), respectively. The ratio of DGLA/AA as well as the high EPA/AA ratio obtained in the present study with the STG diet may lead to physiological alterations, including enhanced synthesis of 1-and 3-series eicosanoids.
In investigating the preparation of 2-acylglycerol by debenzylation of 1,3-di-O-benzyl-2-O-acylglycerol, twelve 1,3-di-O-benzyl-2-O-acylglycerols were synthesized. 2-O-Acyl moieties with one or more protons at the carbon atom adjacent to carbonyl group were found to migrate from C-2 to C-1 of the glycerol moiety during hydrogenation under Pd/C at room temperature.
2-(2,4-Dimethylphenyl)indan-1,3-dione was shown to be a potent hypolipidemic agent in rodents, lowering significantly both serum cholesterol and triglyceride levels at 20 mg/kg/day. The agent in vivo inhibited the enzymatic activities of ATP-dependent citrate lyase, acetyl-CoA synthetase, cholesterol-7-alpha-hydroxylase, acyl-CoA cholesterol acyl transferase, sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase. Tissue lipid levels of liver and small intestine also were reduced by the agent. The rat serum lipoprotein lipid content was modulated by the drug, which should be favorable for the removable of cholesterol from peripheral tissue for conduction to the liver for clearance from the body. Low density lipoprotein (LDL) cholesterol levels were reduced after treatment, which suggests that the agent potentially reduces deposition of cholesterol in plaques. If chemotherapy for atherosclerosis is to be successful, then the high density lipoprotein (HDL) cholesterol level needs to be elevated more than 16% to 25%, the level produced by current hypolipidemic agents. 2-(2,4-Dimethylphenyl)indan-1,3-dione offers a 75% increase in HDL cholesterol levels and a 30% reduction of LDL cholesterol levels with a suppression of de novo synthesis of lipids and a reduction of tissue cholesterol deposition.
Significant hypolipidemic activity was demonstrated by 6-ethoxycarbonyl-1-3-phenyl-1,3,5-triazabicyclo[3.1.0]hexane-2,4-dione, 2-ethoxycarbonyl-5-phenyl-1,3,5-triazine-4,6(1H,5H)-dione and 2-ethoxycarbonyl-5-(4-chlorophenyl)-1,3,5-triazine-4,6(1H,5H)-dione in rodents at 20 mg/kg/day. These agents lowered serum cholesterol and triglyceride levels by approximately 40% in mice after 16 d. Tissue lipids in rat liver, small intestinal mucosa, aortic wall and feces were reduced by treatment with the agents. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol levels were reduced in the rat; high density lipoprotein (HDL) cholesterol levels were elevated after 14 d of treatment. The activities of regulatory enzymes, e.g., acetyl-CoA synthetase, acyl-CoA:cholesterol acyltransferase, cholesterol 7 alpha-hydroxylase, sn-glycerol-3-phosphate acyltransferase, phosphatidylate phosphohydrolase and heparin-induced lipoprotein lipase, involved in de novo synthesis of hepatic lipids were affected by the agents. The new compounds may represent another class of potentially useful hypolipidemic agents for the treatment of atherosclerosis since HDL cholesterol levels were increased and VLDL and LDL cholesterol levels were lowered by some of the agents.
The platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) antagonistic activity of thienotriazolodiazepines has recently been described. The lead compound in this series was brotizolam, which also exhibits sedative and hypnotic effects. By combination of brotizolam with the benzodiazepine receptor antagonist RO 15-1788, PAF antagonistic and central nervous system (CNS) activities could be segregated. Systematic structure variation has led to potent and selective PAF antagonists without CNS effects. WEB 2086 and its analogues WEB 2170 and STY 2108 are representative examples of this structural type and have shown a high potency and selectivity in PAF-induced and PAF-dependent in vitro tests and in experimental models. Studies in healthy volunteers have demonstrated potent pharmacological activity and good safety and tolerance of oral, intravenous or inhaled WEB 2086 in man. These agents should therefore prove useful for the further elucidation of the pathophysiological role of PAF and provide an opportunity for therapeutic applications in diseases in which the involvement of PAF has been implicated.
Racemic 1,4-dihydroxynonane mercapturic acid (DHN-MA) and 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d 3-DHN-MA) are synthesized on a 400-mg scale (over-all yield ∼40%) by a two-step sequence involving Michael addition of N-acetyl-l-cysteine to methyl-4-hydroxynon-2(E)-enoate or methyl 9,9,9-trideutero-4-hydroxynon-2(E)-enoate, followed by reduction of the intermediate adducts with lithium borohydride. The requisite starting methyl esters are obtained, respectively, from heptanal or 7,7,7-trideuteroheptanal and methyl 4-chlorophenyl-sulfinylacetate via a sulfoxide piperidine and carbonyl reaction described in the literature. The 7,7,7-trideuteroheptanal is easily prepared by classical methods in four steps from 6-bromo-1-hexanol. 13C NMR data indicate that DHN-MA as well as d 3-DHN-MA are obtained as mixtures of four diastereomers. Preliminary results show that d 3-DHN-MA could be used as an internal standard for mass spectrometric quantification of DHN-MA in human urine.
The mechanism of action of lithium as a drug for patients with affective disorders was investigated. Three-week-old male rats were orally administered 2.7 mEq Li2CO3/kg/d for 1 or 3 wk, and phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), inositol phosphate (IP), inositol diphosphate (IP2) and inositol triphosphate (IP3) levels in brain were measured. The levels of IP were increased 1.7 and 2.4 times after 1 wk and 3 wk of lithium administration, respectively, while PI, PIP, PIP2, IP2 and IP3 levels were not altered. IP3 was further fractionated by high-performance liquid chromatography into I-1,3,4-P3 and I-1,4,5-P3. In the control rat brain, the relative percentages of I-1,3,4-P3 and I-1,4,5-P3 were 95.8 and 4.2, respectively. However, after 3 wk of lithium administration, the values were changed to 69.6 and 30.3%, respectively. This increase in the neurotransducer I-1,4,5-P3 in the brain may be relevant to the mechanism of action in the lithium treatment of patients with manic-depressive disorders.
Platelet-activating factor (PAF) activates human platelets by binding to a putative PAF receptor which evokes the rapid formation of inositol-1,4,5-trisphosphate (IP3) by phospholipase C mediated phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis. Stimulation of [3H]inositol-labeled human platelets by PAF (1 nM-1 microM) resulted in a concentration-dependent increase of intracellular IP3, IP2 and inositolmonophosphate (IP1). IP1 levels increased up to three-fold upon maximum stimulation by 100 nM PAF. The EC50 concentration for PAF was 1.2 +/- 0.3 nM. Addition of the hetrazepinoic PAF antagonist, WEB 2086, inhibited PAF stimulated hydrolysis of PIP2 in a dose-dependent manner. WEB 2086 (100 microM) blocked inositol-1,4,5-trisphosphate formation down to baseline levels (IC50 = 33 +/- 12 microM WEB 2086). In thrombin and ADP stimulated platelets, inositol phosphate (IP) generation was not influenced by WEB 2086. It is concluded that WEB 2086 selectively antagonizes PAF-induced increases in IP and does not interfere directly with intracellular signal transduction. Instead, WEB 2086, which has been shown to bind specifically and with high affinity (Ki 15 nM) to human platelets, acts as a competitive antagonist at the PAF receptor level.
Linoleic acid was heated at 200 degrees C under helium. Analysis of degradation products by GC on a long polar open tubular capillary column showed the presence of CLA isomers. The identified mono trans CLA isomers were cis-9,trans-11, trans-9,cis-11, trans-10,cis-12, cis-10,trans-1 2, trans-8,cis-10, and cis-11,trans-13 18:2 acids. Oils containing different levels of linoleic acid (peanut, sesame seed, and safflower seed oils) were also heat treated, resulting in similar CLA distributions. Elution order was confirmed using cis-9,trans-11 and trans-10,cis-1 2 acid methyl esters standards and their respective configuration isomers (trans-9,cis-11, cis-10,trans-12), obtained after mild selenium-catalyzed isomerization. These results indicated that two conjugated mono trans isomers of 18:2 acid, cis-8,trans-10 and trans-11,cis-13 18:2 were absent from the series, thus strongly suggesting that some constraints were preventing their formation. By heating pure methyl rumenate (cis-9,trans-11 18:2) under similar conditions, isomerization resulted principally in a nearly equimolar mixture of methyl rumenate and trans-8,cis-10 18:2. Similarly, the methyl ester of trans-10,cis-12 18:2 acid was partially transformed into cis-11 ,trans-13 18:2 acid. Respective geometrical isomers were also formed in trace amounts. A concerted pericyclic isomerization mechanism, a [1,5] sigmatropic rearrangement, is proposed that limits the conjugated system to isomerization from a cis-trans acid to a trans-cis acid, and vice versa. This mechanism is consistent with undetected cis-8,trans-10 and trans-11,cis-13 18:2 isomers in heated oils containing linoleic acid.
Fructose-1,6-bisphosphate (FBP) is a high-energy glycolytic intermediate that decreases the effects of ischemia; it has been used successfully in organ perfusion and preservation. How the cells utilize external FBP to increase energy production and the mechanism by which the molecule crosses the membrane bilayer are unclear. This study examined the effects ofFBP on membrane bilayer permeability, membrane fluidity, phospholipid packing, and membrane potential to determine how FBP crosses the membrane bilayer. Large unilamellar vesicles composed of egg phosphatidylcholine (Egg PC) were made and incubated with 50 mM FBP spiked with 14C-FBP at 30 degrees C. Uptake of FBP was significant (P < 0.05) and dependent on the lipid concentration, suggesting that FBP affects membrane bilayer permeability. With added calcium (10 mM), FBP uptake by lipid vesicles decreased significantly (P < 0.05). Addition of either 5 or 50 mM FBP led to a significant increase (P < 0.05) in Egg PC carboxyfluorescein leakage. We hypothesized that the membrane-permeabilizing effects of FBP may be due to a destabilization of the membrane bilayer. Small unilamellar vesicles composed of dipalmitoyl pC (DPPC) were made containing either diphenyl-1,3,5-hexatriene (DPH) or trimethylammmonia-DPH (TMA-DPH) and the effects of FBP on the fluorescence anisotropy (FA) of the fluorescent labels examined. FBP caused a significant decrease in the FA of DPH in the liquid crystalline state of DPPC (P < 0.05), had no effect on FA of TMA-DPH in the liquid crystalline state of DPPC, but increased the FA of TMA-DPH in the gel state of DPPC. From phase transition measurements with DPPC/DPH or TMA-DPH, we calculated the slope of the phase transition as an indicator of the cooperativity of the DPPC molecules. FBP significantly decreased the slope, suggesting a decrease in fatty acyl chain interaction (P< 0.05). The addition of 50 mM FBP caused a significant decrease (P< 0.05) in the liquid crystalline/gel state fluorescence ratio of merocyanine 540, indicating increased head-group packing. To determine what effects these changes would have on cellular membranes, we labeled human endothelial cells with the membrane potential probe 3,3'-dipropylthiacarbocyanine iodide (DiSC3) and then added FBP. FBP caused a significant, dose-dependent decrease in DiSC3 fluorescence, indicating membrane depolarization. We suggest that FBP destabilizes membrane bilayers by decreasing fatty acyl chain interaction, leading to significant increases in membrane permeability that allow FBP to diffuse into the cell where it can be used as a glycolytic intermediate.
Incubation of a major subfraction, HDL/sub 2b/(d 1.063-1.100 g/ml), with single-bilayer liposomes of dimyristoylphosphatidylcholine (DMPC) resulted in uptake of DMPC by the HDL/sub 2b/ and dissociation of lipid-free apolipoprotein A-I (apoA-I). In the presence of excess DMPC, the dissociated apoA-I was also incorporated with DMPC into discoidal complexes. Preliminary studies with model apoA-I-DMPC ciomplexes indicated that they also can interact with native HDL/sub 2b/ with the resultant transfer of their DMPC to HDL/sub 2b/ and the concomitant release of their apoA-I. After interaction of HDL/sub 2b/ with DMPC lipsomes, the DMPC-enriched HDL/sub 2b/ product showed a lower hydrated density and a larger particle size than the control HDL/sub 2b/. The molecular properties of the lipoprotein product suggest that stabilization of the apoA-I-depleted HDL/sub 2b/ probably occurred via substitution of DMPC for the apoA-I at the HDL/sub 2b) surface rather than by fusion of the apoA-I-depleted HDL/sub 2b/. The above interactions of HDL/sub 2b/ with single-bilayer liposomes and discoidal cpomplexes indicate pathways of phospholipid transfer relevant to the possible role of HDL in the metabolism of lipoprotein surface components in vivo.
The antitumor activity of alkyl lysophospholipids has raised some questions concerning the degradation of O-alkyl bonds in naturally occurring ether lipids. In this report, we describe the first continuous assay for O-alkylglycerol monooxygenase (AGMO), the only enzyme known to cleave the O-alkyl bond in saturated ether lipids and ether phospholipids. AGMO activity was monitored at 340 nm by coupling the NADH redox reaction to the tetrahydropteridine cofactor of the rat liver microsomal enzyme. Turnover rates as low as 0.6 nmol/min could be measured. Using radiolabeled substrates, the products were identified with a TLC-Linear-Analyzer. The only interference with this assay can arise from other reducing agents, e.g. dithiothreitol. The assay was used to develop protocols for the solubilization of AGMO from membrane preparations in the presence of detergents.
Synthetic alkyl lysophospholipids (ALP) show antineoplastic activity. In the present discussion, the cytotoxicity of ALP is attributed to their accumulation in tumor cells. Some neoplastic cell species, in contrast to normal cells, cannot metabolize ALP because of a lack of O-alkylglycerol monooxygenase (AGMO) activity. To understand the metabolic fate of ether lipids and ether-linked phospholipids, AGMO substrate specificity studies were undertaken. Thirty-five different natural and synthetic ether lipids and their metabolites (including a thioether) were tested as substrates for AGMO. The study revealed that the potent cytostatic substance, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine is not a substrate for AGMO. Therefore, its selective toxicity to tumor cells cannot be based on the differences in direct detoxification of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine by AGMO in normal and malignant cells. However, 1-O-octadecyl-2-O-methyl-rac-glycerol, which can be formed by phospholipase C hydrolysis, is a good substrate for AGMO.
The previously unknown linoleic acid peroxidation product 9,12-dioxo-10(Z)-decenoic acid (Z5) was detected in lentil seed flour (Lens culinaris Medik.) by electron impact mass spectrometry (EI-MS) after derivatization with pentafluorobenzyl-hydroxylamine-hydrochloride, methylation of acidic groups with diazomethane, and protection of hydroxylic groups with N-methyl-N-trimethylsilyl-trifluoroacetamide. The structure of the natural product was confirmed by synthesis of Z5, 9,12-dioxo-l0(E)-decenoic acid, and derivatives. EI-MS, nuclear magnetic resonance and gas chromatographic data of these compounds and synthetic intermediates are discussed.
FA Composition of CLA Supplements 
Partial silver ion-high performance liquid chromatograms indicating the separation of cis-trans and trans-cis 9,11 and 10,12 isomers of CLA methyl esters prepared from treatment T3 attained using four silver impregnated silica columns coupled in series and a mobile phase containing (A) 0.1% (vol/vol) acetonitrile in heptane (22), or (B) 2.0% (vol/vol) acetic acid (23) under isocratic conditions at 22°C. Peak identification: (1) trans-10,cis-12 CLA, (2) cis-10,trans-12 CLA, (3) cis9,trans-11 CLA, and (4) trans-9,cis-11 CLA
Temporal changes in milk fat content and milk fat yield during abomasal infusion of mixtures of CLA supplements. Infusions were continuously infused during a 4-d period. Values represent means from four animals. SEM for milk fat content and milk fat yield was 1.66 g/kg and 53.8 g/d, respectively. The mean amounts of CLA isomers infused for each treatment are listed in Table 3. (+) Control, (G G) T1, (L) T2, (I I) T3. For treatment descriptions see Figure 1.
The trans-10,cis-12 isomer of conjugated linoleic acid (CLA) decreases TAG accumulation in 3T3-L1 adipocytes, reduces lipid accretion in growing animals, and inhibits milk fat synthesis in lactating mammals. However, there is evidence to suggest that other FA may also exert antilipogenic effects. In the current experiment, the effects of geometric isomers of 10,12 CLA on milk fat synthesis were examined using four Holstein-British Friesian cows in a 4 x 4 Latin Square experiment with 14-d periods. Treatments consisted of abomasal infusions of skim milk, or skim milk containing trans-10,cis-12 CLA (T1), trans-10,trans-12 CLA (T2), or a mixture of predominantly 10,12 isomers containing (g/l00 g) trans-10,cis-12 (35.0), cis-10,trans-12 (23.2), trans-10,trans-12 (14.9), and cis-10,cis-12 (5.1). CLA supplements were prepared from purified ethyl linoleate and infused as nonesterified FA. Infusions were conducted over a 4-d period with a 10-d interval between treatments and targeted to deliver 4.5 g/d of 10,12 CLA isomers. Compared with the control, trans-10, trans-12 CLA had no effect (P> 0.05) on milk fat yield, whereas treatments T1 and T3 depressed (P < 0.05) milk fat content (19.8 and 22.9%, respectively) and decreased milk fat output (20.8 and 21.3%, respectively). Comparable reductions in milk fat synthesis to 4.14 and 1.80 g trans-10,cis-12/d supplied by treatments T1 and T3 indicate that other 10,12 geometric isomers of CLA have the potential to exert antilipogenic effects. The relative abundance of cis-10,trans-12 CLA in treatment T3 and the low transfer efficiency of this isomer into milk suggest that cis-10,trans-12 CLA was the active component..
Potato tuber lipoxygenase (ptLOX) has been shown to catalyze the aerobic formation of at least four major oxygenated derivatives of DHA. Two of the products—7,17(S)- and 10,17(S)-dihydro(pero)xy-DHA [7,17- and 10,17-diH(P)DHA]— were formed from soybean 15-LOX-derived 17(S)-hydro(pero)xy-DHA [17(S)-H(P)DHA], whereas two novel oxylipin compounds— 10(S)-hydro(pero)xy-DHA and 10,20-dihydro(pero)xy-DHA [10(S)-H(P)DHA and 10,20-diH(P)DHA, respectively]—were the major direct products of DHA oxidation by ptLOX. The reactions proceeded relatively slowly but could be stimulated by catalytic amounts of SDS. Micromolar concentrations of 10(S)-HPDHA effectively abolished the kinetic lag period of ptLOX activation. Enzymatic activity with DHA or 17(S)-HPDHA as substrate was about 8% of that with linoleic acid—a standard natural ptLOX substrate—whereas 17(S)-HDHA was converted at a rate of ∼1%. The enzyme was relatively unstable and quickly inactivated during the reaction with DHA on with 17(S)-HPDHA (first-order kinetic constant of inactivation k in=1.5±0.3 min−1), but not with 17(S)-HDHA. Both 7,17- and 10,20-diH(P)DHA were clearly products of double oxygenation catalyzed by soybean 15-LOX and/or ptLOX. Our observation that ptLOX could convert 17-HDHA to 10,17-diH(P)DHA indicates that this dihydroxylated derivative of DHA also can be formed via a double lipoxygenation mechanism.
Administration of 2,2'''-([1-methyl-4, 4-diphenylbutylidene] bis(p-phenyl-eneoxy]) bistriethylamine oxalate (SQ 10,591) at 20 mg/Kg daily for 5 days to cholesterol-fed rabbits resulted in no change in aortic microsomal cholesterol esterification with a palmitoyl coenzyme A substrate or of aortic mitochondrial cholesterol esterification with a palmitate substrate. Esterification by both reactions in the adrenal was much higher than in either aorta or testes. Adrenal and testicular mitochondrial esterification and testicular microsomal esterification were inhibited significantly after SQ 10,591 administration. In vitro addition of 0.0001 M SQ 10,591 significantly inhibited both microsomal and mitochondrial cholesterol esterification in aorta, adrenal, and testes.
The binding of squalene, lanosterol, desmosterol, and cholesterol to proteins in 105,000 g supernatant fraction (S105) from brain and liver of rats was investigated. The S105 fractions from both tissues contain specific binding sites for sterols, which are sensitive to trypsin. The dissociation constants for squalene and sterol protein complexes were in the range of 10(-6) M and were not appreciably different for proteins in brain and liver S105. Competition studies revealed that both brain and liver S105 contain one receptor protein which binds lanosterol and is specific for methyl sterols, and a second receptor which binds both desmosterol and cholesterol. Binding of 7-dehydrocholesterol reported by others must occur at a third independent site since this compound does not interfere with the binding of lanosterol, desmosterol, or cholesterol. Although binding of squalene to proteins in brain and liver S105 does occur, we were unable to show the specificity of squalene binding. The concentration of desmosterol and cholesterol binding sites, which ranged from 6 to 10 nmol/mg protein, was 3- to 5-fold higher than the concentration of squalene and lanosterol binding sites (1.6-2.3 nmol/mg protein). The brain S105 from suckling rats contained fewer binding sites for desmosterol and cholesterol than the brain S105 from weaned rats. However, the concentration of lanosterol binding sites in brain S105 did not show an age-dependent change. The receptor proteins in brain and liver appear to be identical.
Intravenous infusion of endotoxin (0.25 mg/kg/hr for 4 hr) was shown to induce disseminated intravascular coagulation (DIC) in rats, which resulted in hypofibrinogenemia, prolongation of prothrombin (PT) and partial thromboplastin time (PTT), thrombocytopenia, and elevated levels of fibrinogen/fibrin degradation products (FDP). Oral administration (100 mg/kg) of the selective PAF antagonist, SM-10661 ((+/-)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl), counteracted the changes caused by the endotoxin. Intravenous infusion of SM-10661 (6mg/kg bolus 2 min before endotoxin infusion + 6 mg/kg/hr for 4 hr infusion) also counteracted DIC. When suboptimal doses of gabexate mesilate, a synthetic protease inhibitor (3 mg/kg i.p.), and SM-10661 (2 mg/kg bolus + 2 mg/kg/hr for 4 hr infusion) were administered concomitantly, hematological parameters improved. The results suggest that PAF may play a role in the pathogenesis of DIC, and that together with the results already reported for other PAF antagonists, SM-10661 may be useful in the treatment of DIC.
Top-cited authors
Douglas R Tocher
  • University of Stirling
Peter JH Jones
  • Nutritional Fundamentals for Health Inc
J.G.Bell Gordon Bell
  • University of Stirling
Junji Terao
  • The University of Tokushima
Patricio Peirano
  • University of Chile