Intravascular metabolism of the cholesteryl ester moiety of rat plasma lipoproteins

The intravascular metabolism of the cholesteryl ester moiety of rat plasma LDL, HDL1, and HDL2 was determined in intact male rats. Biosynthetically labeled lipoproteins were prepared by zonal ultracentrifugation from the plasma of rats injected with [3H]cholesterol. The lipoproteins were concentrated by vacuum ultrafiltration as other procedures were found to alter the biological properties of the lipoproteins. After injection of labeled LDL, [3H]cholesteryl esters remained with the injected lipoprotein and decayed from plasma with a t1/2 of 7-8 hours. [3H]Cholesteryl esters in HDL1 behaved similarly and decayed with a t1/2 of 10.5 hours. With HDL2, however, a different metabolic pattern was observed with intraplasma conversion of some [3H]cholesteryl ester HDL2 particles to HDL1. Since such conversion of HDL2 to HDL1 was not observed after in vitro incubations of rat plasma, this process seems to depend on metabolic events that occur in vivo. [3H]Cholesteryl esters disappeared from HDL2 with a t1/2 of 6-7 hours, while the esters that were transferred to HDL1 decayed with a t1/2 of 10-11 hours, similar to labeled cholesteryl esters injected with HDL1. The study demonstrated that the high apoE content of rat plasma HDL1 is not associated with rapid catabolism of the lipoprotein and that a major source of HDL1 in the rat is the intraplasma conversion of HDL2 particles to HDL1.     

Practical enantioresolution of alcohols with 2-methoxy-2-(1-naphthyl)propionic acid and determination of their absolute configurations by the (1)H NMR anisotropy method.

The enantioresolution of racemic alcohols as esters of 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1) and the determination of their absolute configurations on the basis of (1)H NMR anisotropy effect are described. The enantiopure MalphaNP acid (S)-(+)-1 was allowed to react with racemic 2-alkanols and 1-octyn-3-ol, yielding diastereomeric mixtures of esters, which were easily separated by HPLC on silica gel. To determine the absolute configurations of the first-eluted diastereomeric esters by the (1)H NMR anisotropy method, the general scheme was proposed. Separated esters were reduced with LiAlH(4) or hydrolyzed with KOH/EtOH to recover enantiopure alcohols.     

Selective incorporation of n-3 and n-6 fatty acids in essential fatty acid deficient rats in response to short-term oil feeding

Essential fatty acid deficient male Sprague Dawley rats were fed for 7 days a fat-free semi-synthetic diet supplemented with 10% by weight of different oil supplements. The oil supplement was a mixture of olive, safflower and linseed oils prepared at different proportions so the dietary n-9/n-6/n-3 ratios were approximate 2/1/1, 1/2/1, 1/1/2, and 1/1/1. The fatty acid compositions of plasma and liver lipids were then examined. Our results show polyunsaturated n-6 and n-3 fatty acids were selectively incorporated into plasma and liver phospholipids, and also into plasma cholesteryl esters. A preferential incorporation of n-6 over n-3 fatty acids into plasma cholesteryl esters and phospholipids was also observed.     

Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharide.

1. Cell walls from rapidly growing cell suspension cultures of Spinacia oleracea L. contained ferulic acid and p-coumaric acid esterified with a water-insoluble polymer. 2. Prolonged treatment with trypsin did not release may feruloyl esters from dearabinofuranosylated cell walls, and the polymer was also insoluble in phenol/acetic acid/water (2:1:1, w/v/v). 3. Treatment of the cell walls with the fungal hydrolase preparation "Driselase' did liberate low-Mr feruloyl esters. The major esters were 4-O-(6-O-feruloyl-beta-D-galactopyranosyl)-D-galactose and 3?-O-feruloyl-alpha-L-arabinopyranosyl)-L-arabinose. These two esters accounted for about 60% of the cell-wall ferulate. 4. It is concluded that the feruloylation of cell-wall polymers is not a random process, but occurs at very specific sites, probably on the arabinogalactan component of pectin. 5. The possible role of such phenolic substituents in cell-wall architecture and growth is discussed.     

Very long-chain phenylpropyl and phenylbutyl esters from Taxus baccata needle cuticular waxes

The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified by various chemical transformations with product assignment employing GC-MS. Homologous series of (1) 3-(4'-hydroxyphenyl)-propyl esters of C(20)-C(36) fatty acids, (2) 4-(4'-hydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids, (3) 3-(3',4'-dihydroxyphenyl)-propyl esters of C(20)-C(32) fatty acids, and (4) 4-(3',4'-dihydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids were identified. The four compound classes amounted to 0.1-3.6 micro g/cm(2) of needle surface area, corresponding to 0.2-7.6% of the wax mixture, respectively. While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters homologs containing eicosanoic and docosanoic acids predominated.     

Estimation of plasma free fatty acids as the trimethylsilyl (TMS) esters

Quantitative estimates of free fatty acids in total lipid extracts of plasma were obtained by glc on nonpolar columns following trimethylsilylation. The presence of other lipid esters in the reaction mixture had no effect upon the yield of the trimethylsilyl (TMS) esters or upon their resolution on the glc column. Routine quantitations by gas-liquid chromatography (glc) were obtained on 2 ft × 1/8 in. o.d. stainless steel columns packed with 3% OV-1 on 100–120 mesh Gas Chrom Q by means of temperature programming in the range 175–350°C with tridecanoin as internal standard. High resolution glc of the TMS esters of fatty acids was done on a 6 ft × 1/8 in. o.d. glass column packed with 1% SE-30 on Gas Chrom Q. In both instances the fatty acids were resolved on the basis of carbon number and by the presence or absence of double bonds. On gas chromatography/mass spectrometry (GC/MS), TMS esters of fatty acids were shown to yield proportionally greater amounts of high mass fragments, including the parent ions, than their methyl or ethyl esters, which has special advantages for the detection and characterization of polyunsaturated fatty acids.     

Plasma transport and tissue distribution of β-carotene, vitamin A and retinol-binding protein in domestic cats

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240±64 ng/ml, mean±S.D.) represented one-third of total retinyl esters (736±460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61±6, 36±13 and 5±3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22±21 ng/ml) were found in the urine. Vitamin A in the livers (4317±1956 μg/g) was significantly higher than in the kidney cortex and medulla (14.16±8.92 and 7.59±4.52 μg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. β-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of β-carotene and the excretion of vitamin A in the urine.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Inhibitors of sterol synthesis. Metabolism of [2,4-3H]5 alpha-cholest-8(14-)-en-3 beta-ol-15-one after intravenous administration to a nonhuman primate

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol synthesis with marked hypocholesterolemic activity, has been studied after the intravenous administration of a mixture of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one and [4-14C] cholesterol to a baboon. The levels of 3H in plasma which was associated with the free 15-ketosterol decreased very rapidly (T1/2 approximately 9 min) after injection of the labeled sterol. By 4 h, the level of the [3H]15-ketosterol in plasma was negligible. The rapid decrease in the levels of the free 15-ketosterol was associated with rapid formation of fatty acid esters of the 15-ketosterol. The maximum level of 3H-labeled 15-ketosteryl esters was observed at 20 min after the injection of the 15-ketosterol. Thereafter, the levels of the 15-ketosteryl esters decreased rapidly with an apparent T1/2 of approximately 3.5-4.0 h. The results also indicated rapid formation of 3H-labeled cholesterol and cholesteryl esters. Substantial formation of [3H]cholesterol was observed at 20 min after the injection of the 15-ketosterol and reached a maximum level in plasma at 2 h. The maximum levels of [3H]cholesteryl esters in plasma were observed much later. These and other findings indicated that the observed slow clearance of total 3H from plasma is a consequence of metabolism of the 15-ketosterol to cholesterol and cholesteryl esters, normal constituents of plasma whose turnover in the whole animal is known to be relatively slow.     

De novo biosynthesis of arachidonic acid and 5,11,14-eicosatrienoic acid in the cricket Teleogryllus commodus

The de novo biosynthesis of 5,11,14-eicosatrienoic acid (5,11,14-20:3), arachidonic acid (20:4(n - 6] and eicosadienoic acid (20:2(n - 6] and the elongation/desaturation of linoleic acid (18:2(n - 6] to 20:4(n - 6) and alpha-linolenic acid (18:3(n - 3] to eicosapentaenoic acid (20:5(n - 3] were demonstrated in adult males of the field cricket Teleogryllus commodus. Sodium [1-14C]acetate, [1-14C]18:2(n - 6) and [1-14C]18:3(n - 3) were injected into adult male crickets and after an incubation period, the testes and remaining tissues were extracted and the methyl esters obtained from the phospholipid and triacylglycerol fractions were analyzed. After 5 days of daily injections of [1-14C]acetate, the methyl esters of the triene and tetraene fatty acids from the testicular phospholipid fraction were purified by AgNO3-TLC and HPLC and analyzed by GLC, radio-HPLC, and radio-GLC of ozonolysis products. The results demonstrate the de novo biosynthesis of 20:2(n - 6), 20:4(n - 6) and an isomer of 20:3(n - 6) with double bonds in the 5,11,14 positions. the elongation/desaturation of 18:2(n - 6) to 20:4(n - 6) and 18:3(n - 3) to 20:5(n - 3) was demonstrated by analysis of the methyl esters derived from the testicular phospholipid fraction by radio-HPLC after injecting crickets with radiolabeled substrates.     

Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.     

Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica)

Three olean-12-ene type triterpenoid saponins, named TR-saponins A, B and C, were isolated as methyl esters from tea roots (Camellia sinesis var. assamica) after treatment with diazomethane. Their structures were established as the methyl esters of 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21, 22-di-O-angeloyl-R1-barrigenol-23-oic acid, 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-me thylbutanoyl-R1- barrigenol-23-oic acid and 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-16 alpha-O-acetyl-21-O-angeloyl-22-O-2-methylbutanoyl-R1-bar rigenol-23-oic acid, by extensive 1D and 2D-NMR as well as FABMS and HR-MS analyses.     

The hydrolysis of esters of N-hippurylglycine and N-pivaloylglycine by carboxypeptidase A

The kinetics of the hydrolysis of five esters of N-hippurylglycine (C6H5CONHCH2CONHCH2CO2CRR1CO2H (2 approximately) and seven esters of N-pivaloylglycine ((CH3)3CCONHCH2CRR1CO2H (3 approximately)) by bovine pancreatic carboxypeptidase A (Peptidyl-L-amino-acidhydrolase, EC 3.4.12.2) have been studied at pH 7.5, 25 degrees C and ionic strength 0.5. All N-hippurylglycine esters (2: R=H, R1=H, C2H5, 4-ClC6H4, C6H5CH2) display Michaelis-Menten kinetics up to at least 0.1 M substrate. The N-pivaloylglycine esters display either Michaelis-Menten kinetics (3 approximately: R=H, R1=H, C2H5 C6H5), substrate activation (3 approximately: R=H, R1=4-ClC6H4; R=R1=CH3) or substrate inhibition (3 approximately: R=H, R1=(CH3)2CHCH2, C6H5CH2). Kinetic parameters have been evaluated for each ester and compared with those for the corresponding hippuric acid esters (1 approximately). The enzymic specificity is shown to be identical for the alcohol moieties of the esters 1 approximately, 2 approximately and 3 approximately and unrelated to the occurrence of substrate activation or inhibition phenomena. These latter phenomena are shown to be characteristic of the enzymic hydrolysis of N-acyl amino acid esters but unimportant for N-acyl dipeptide ester substrates.     

蝉虫草样品的分级萃取与化学成分分析

利用索氏萃取技术,依次采用石油醚、乙酸乙酯、丙酮、无水乙醇和甲醇等5种溶剂对蝉虫草纯粉进行分级萃取,运用傅里叶变换红外光谱分析法和气相色谱-质谱联用技术对5级萃取物进行分析鉴定。傅里叶变换红外光谱分析显示萃取物中含有与烯烃类、羧酸类、酯类、醇类和酮类等化合物相关的C-H、C=O、C-O和C=C等官能团。气相色谱-质谱联用技术共鉴定出有机小分子化合物34种,以酯类和脂肪酸类为主,多为碳链长度为15-20的长链脂肪酸及对应的酯,其中十八碳不饱和脂肪酸相对含量高达28.95%;分别存在于两种或以上萃取物中的有机化合物共有11种;仅存于石油醚萃取物中的化合物6种,乙酸乙酯萃取物中3种,丙酮萃取物中2种,无水乙醇萃取物中6种,甲醇萃取物中6种。在一定极性范围内,利用溶剂的极性梯度变化,可实现蝉虫草中活性物质的按极性梯度分离;采用分级萃取技术可有效分离蝉虫草中部分化学成分。鉴定结果充实了蝉虫草中化合物的种类资源,为蝉虫草中活性物质谱图库的完善、构效关系的建立及蝉虫草产品的利用开发提供支撑。     

The oxidation products of crude mesobilirubinogen

Bile pigment esters were separated by ascending t.l.c. Apparently pure pigments, obtained by ferric chloride oxidation of crude mesobilirubinogen, derived from commercial bilirubin by reduction with sodium amalgam, were shown to be complex mixtures. Successive chromatography of their dimethyl esters on silica gel in methyl acetate-methyl propionate-dichloromethane-carbon tetrachloride (1:1:1:1, by vol.), ethyl methyl ketone-1,2-dichloroethane (1:2, v/v) and benzene-ethanol (100:3, v/v) revealed two major blue pigments (verdins), six major violet pigments (violins) and a red pigment (rhodin) together with numerous minor components. i-Urobilin dimethyl ester, prepared from mesobilirubinogen by dehydrogenation with aqueous iodine, was resolved into three major and at least four minor components on silica gel-kieselguhr (3:1, w/w) in benzene-ethanol (25:2, v/v). The chemical nature of these pigments was investigated by oxidation, by visible and u.v. spectroscopy, by mass spectrometry and by n.m.r. spectrometry. The evidence suggests unusual rearrangement of bilirubin during reduction leading to the formation of IIIalpha and XIIIalpha isomers. Isomeric forms of mesobiliviolin IXalpha and of i-urobilin IXalpha may also be formed.     

Binding rates, O--S substitution effects, and the pH dependence of chymotrypsin reactions.

The pH dependence for acylation of alpha-chymotrypsin by N-acetyltryptophan p-nitrophenyl-, p-nitrothiophenyl-, ethyl-, and thiolethyl esters has been studied by the stopped-flow technique. Values for the acylation rate constant, k2, and the binding constant, KS, were obtained by using measurements of phenolate release, for the p-nitrophenyl esters, and proflavin displacement, for the ethyl esters. The oxygen esters tested have slightly higher k2 values, and substantially higher KS values relative to the analogous thiol esters. Whereas k2/KS for the thiolethyl ester is higher than that for the analogous oxygen ester, the k2/KS values for oxy- and thio-p-nitrophenyl esters are nearly identical. These data are interpreted to indicate rate-determining formation of a tetrahedral intermediate in acylation of alpha-chymotrypsin by p-nitrophenyl esters, and rate-determining breakdown of such an intermediate in the case of the ethyl esters. It is also concluded that the oxygen to sulfur substitution causes a substantial increase in the proportion of nonproductive binding in these substrates. pH dependent k2 and KS values were used to calculate values for k1 and k-1, the binding and debinding rate constants for the two p-nitrophenyl compounds. This is the first such calculation based on experimentally determined acylation rate constants.     

Reversal of dietary-induced hyperlipidemia in Biomphalaria glabrata (Gastropoda).

1. To determine if dietary induced hyperlipidemia in snails could be reversed, thin-layer chromatography studies were done on lipids in the digestive gland-gonad (DGG) complex of Biomphalaria glabrata fed either hen's egg yolk for 2 weeks (Group A), yolk for 1 week followed by leaf lettuce for 1 week (Group B), or lettuce for 2 weeks (Group C). 2. The major lipid fractions in Group A were triacylglycerols, phosphatidylethanolamine, and phosphatidylcholine, along with lesser amounts of sterol esters, methyl esters, free fatty acids, free sterols and unidentified phospholipids. 3. Groups B and C had about one-half the amount of triacylglycerols than Group A and did not show sterol esters or methyl esters. 4. The triacylglycerol increase of the DGG resulting from the hen's egg diet could be reversed by returning the snails to the lettuce diet.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.     

Natural anti-HIV agents-part I: (+)-demethoxyepiexcelsin and verticillatol from Litsea verticillata

The eudesmane sesquiterpenoid, verticillatol (1), as well as the lignan, (+)-5'-demethoxyepiexcelsin (2), and a known lignan, (+)-epiexcelsin (3), were isolated from Litsea verticillata Hance. Lignan 2 showed moderate anti-HIV activity with an IC(50) value of 16.4 microg/ml (42.7 microM), while the known lignan 3 was inactive up to a concentration of 20 microg/ml (48.3 microM). Compound 1 demonstrated weak activity with an IC(50) value of 34.5 microg/ml (144.7 microM) while being devoid of cytotoxicity at 20 microg/ml. The structures were elucidated by 1D and 2D NMR spectroscopy, and the absolute configuration of the new sesquiterpenoid was determined by the generation of Mosher esters.