Skin surface lipids of the mink

Skin surface lipids from mink (Mustela vison) were collected in acetone and analyzed by thin-layer chromatography and gas chromatography. The principal components were wax monoesters (92%), cholesteryl esters (5%), free fatty acids (1%), fatty alcohols (1%) and cholesterol (1%). The fatty acids and alcohols contained in these lipids were composed principally of homologous series of straight chained omega 7-unsaturated structures (C16-C24), accompanied by lesser proportions of homologous series of saturated (C14-C22) and omega 9-unsaturated (C18-C22) structures.     

Novel anti-inflammatory omega-3 PUFAs from the New Zealand green-lipped mussel, Perna canaliculus

The present study has identified in the marine mollusc, Perna canaliculus, an homologous series of novel omega 3 polyunsaturated fatty acids (ω-3 PUFA) with significant anti-inflammatory (AI) activity. The free fatty acid (FFA) class was isolated from a supercritical-CO₂ lipid extract of the tartaric acid-stabilised freeze-dried mussel powder by normal phase chromatography, followed by reversed-phase high performance liquid chromatography (RP–HPLC). The RP–HPLC involved separation based on carbon numbers, followed by argentation–HPLC (Ag–HPLC) of the methyl esters based on degree of unsaturation. Identification of the FFA components was performed using gas chromatography (GC) with flame ionisation detection, and individual structures were assigned by GC-mass spectroscopy (GC-MS). Inhibition of leukotriene production by stimulated human neutrophils was used as an in vitro screening method to test the AI activity of the purified PUFAs. A structurally related family of ω-3 PUFAs was identified in the most bioactive fractions, which included C18:4, C19:4, C20:4, and C21:5 PUFA. The C20:4 was the predominant PUFA in the extract, and was a structural isomer of arachidonic acid (AA). The novel compounds may be biologically significant as AI agents, as a result of their in vitro inhibition of lipoxygenase products of the AA pathway.     

我国健康成人红细胞膜脂类的脂酸组成

 本文采用双向簿层层析分离红细胞膜脂类,继以毛细管气相色谱法分析其脂酸含量,检测了15名我国健康成人红细胞膜脂类的脂酸摩尔百分组成。结果表明:各脂类中,脂酸的类别基本相同,但其含量组成相差甚远。如磷脂酰乙醇胺(PE)富含C_(20:4);磷脂酰胆碱(PC)富含C_(18:2);神经鞘磷脂(SM)主要含C_(16:0);磷脂酰丝氨酸(PS)主要含C_(18:0);而以红细胞糖苷脂(GL)中脂酸含量最少。膜总脂中饱和脂酸与不饱和脂酸的含量大致相等,胆碱磷脂(PC+SM)的脂酸饱和度则明显高于氨基磷脂(PE+PS)。     

毛细管气相色谱细胞脂肪酸测定法鉴定28种分枝杆菌的研究

用毛细管气相色谱对分枝杆菌属28种参考株的细胞挥发性脂肪酸进行测定,发现均含有较多量的脂肪酸C18:1、C18:0及本属特异性组分TBSA。利用色谱图中这三种脂肪酸峰高的比例关系,可将所测菌株分应8群。再借助具有种间特异性的脂肪酸C19:0、C21:0和C26:0将其中的1 7株(60％)鉴定到种,11株鉴定到含有2或3个种的群。本鉴定程序区分多数临床常见的致病菌(如人型、牛型结核杆菌及一些分枝杆菌复合体),经计算机对细胞脂肪酸模式的聚类分析,表明了这一程序的客观合理性。     

柱前衍生反相高效液相色谱法测定绞股蓝茶叶中17种游离氨基酸含量

建立2,4-二硝基氟苯柱前衍生化-反相高效液相色谱法测定绞股蓝茶叶中17种游离氨基酸的含量。以Phenomenex Gemini NX C18(4.6mm×250mm,5μm)为分析柱,采用梯度洗脱,流动相A为0.05mol·L-1乙酸钠(pH=6.4,含0.1%N,N-二甲基甲酰胺),流动相B为乙腈-水(1∶1,v/v),检测波长为360nm,柱温35℃;经方法学考察,该方法具有良好的稳定性和重现性。测定结果表明,绞股蓝茶叶中17种游离氨基酸总量为39.79mg·g-1,其中人体必需氨基酸占游离氨基酸总量的36.57%。从氨基酸含量考虑,绞股蓝茶叶具备一定的开发利用价值。     

Analysis of Glucocerebrosides of Rye (Secale cereale L. cv Puma) Leaf and Plasma Membrane

Glucocerebrosides of whole rye (Secale cerale L. cv Puma) leaf and plasma membrane were analyzed using gas chromatography-mass spectrometry and gas chromatography following hydrolysis or as intact molecules purified by reverse-phase high performance liquid chromatography. Fatty acids of acid-hydrolyzed leaf and plasma membrane glucocerebrosides consisted of >98 weight percent saturated and monounsaturated 2-hydroxy fatty acids which contained 16 to 26 carbon atoms. The major fatty acids detected were 2-hydroxynervonic acid (24:1h), 2-hydroxylignoceric acid (24:0h), 2-hydroxyerucic acid (22:1h), and 2-hydroxybehenic acid (22:0h). Long-chain bases of alkaline-hydrolyzed glucocerebrosides consisted primarily of cis-trans isomers of the trihydroxy base 4-hydroxysphingenine (t18:1) and the dihydroxy base sphingadienine (d18:2) with lesser amounts of 4-hydroxysphinganine (t18:0) and isomers of sphingenine (d18:1). Intact, underivatized glucocerebroside molecular species of rye leaf and plasma membrane were separated into more than 30 molecular species using reverse-phase HPLC. The molecular species composition of leaf and plasma membrane were quantitatively and qualitatively similar. The major molecular species was 24:1h-t18:1 which constituted nearly 40 weight percent of leaf and plasma membrane extracts. Several other species including 22:1h-t18:1, 24:1h-t18:1 (isomer), 22:0h-t18:1, 24:1h-d18:2, and 24:0h-t18:1 each comprised 4 to 8% of the total. It is anticipated that the high performance liquid chromatography procedure developed in this study to separate intact, underivatized lipid molecular species will be useful in future studies of the physical properties and biosynthesis of plant glucocerebrosides.     

Xterra RP18柱高效液相色谱法快速分离测定氨基酸

建立了一种用XterraRP1 8色谱柱快速分离测定水解氨基酸的方法。所采用的色谱条件是 :WatersAlliance系统 ,柱温 5 6℃ ,流速 1 .8ml/min ,检测波长 2 4 8nm ,梯度分离 ,运行周期 2 5min,柱反压低于 2 0 0 0Psi。在 1 7.5min内分离了包括AMQ、NH3 和牛磺酸在内的 2 1种氨基化合物 ,适应于复合氨基酸注射液、含牛磺酸的氨基酸口服液及水解氨基酸样品的分析测定     

Serum fatty acids analysis by high performance liquid chromatography, after enzymatic hydrolysis and isolation by Sep-Pak C18 minicolumn

A simple method of high performance liquid chromatography for the determination of fatty acid composition of free fatty acid, triglyceride, cholesterol ester and phospholipid in 0.4 ml of human serum is described. The procedure includes the enzymatic hydrolysis of serum lipoproteins, the isolation of fatty acids using Sep-Pak C18 minicolumn, the p-bromo-phenacylester formation and the high performance liquid chromatography. Thirteen fatty acids including the internal standard separated into 10 peaks within a 30-min run. The recovery rates of Sep-Pak treatment were satisfactory. The coefficients of variation were 2-27%. The present method showed a limit of detection of 0.02-4 nEq fatty acid.     

Characterization of the phospholipid and fatty acid composition of Sendai virus

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.     

Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC

Arachidonic acid (AA) was separated and purified from microbial lipids by the combined method of urea inclusion reaction and reversed-phase high performance liquid chromatography. At first, AA was concentrated from free fatty acids made from microbial lipids by a urea inclusion reaction. The optimum conditions were as follows: methanol was the suitable solvent, the ratio of free fatty acids to urea to methanol was 1:2:8 (wt/wt), and the temperature of the urea inclusion reaction was -10 degrees C. The AA content was increased from 38% to 79%, and then AA was purified on a C(18) preparative column (300 mm x 30 mm I.D., d(p)=15 microm), using methanol-water (95:5, v/v) as the mobile phase, at a flow rate of 5 mL/min. The purity of AA after two steps purification reached 99%. This result indicates that the combined method of the urea inclusion reaction and reversed-phase high performance liquid chromatography is a promising technique for purification of AA.     

Sphingolipids of influenza viruses.

Total lipid of four egg grown influenza viruses (A2-Asia, A2-England, A2-Taiwan and fowl plague virus) were extracted with chloroform-methanol. After mild alkali treatment of the extracts, glycosphingolipids and sphingomyelin were separated by a silicic acid column, and finally purified by thin layer chromatography. Fatty acid, sphingosine and carbohydrate components of individual lipid classes were then analysed by gas-liquid chromatography. Nearly identical results were obtained with all viruses investigated. Approximately 20% of the total lipid was monohexosylceramide, distributed equally between glucosyl- and galactosyl- analogues. Lactosylceramide and oligohexosylceramides were found in much smaller concentrations (approx. 2%). About 15% of the total lipid was attributed to sphingomyelin. A large proportion of fatty acids (around 25% in sphingomyelin and 60% in glycolipids) belonged to the long chain (C19-C26) normal- and 2-hydroxy series. C18-sphingosine was found to be the only base present in all lipid classes investigated.     

Detection of plasmalogen from plasma low density lipoprotein and high density lipoprotein in carp, Cyprinus carpio, and rainbow trout, Oncorhynchus mykiss

The study revealed the presence of plasmalogens in the low density lipoprotein (LDL) and high density lipoprotein (HDL) of the fish. The composition of the plasmalogen in the carp plasma LDL phospholipids was 0.94 and 0.23% in the HDL; the LDL phospholipids in the rainbow trout were 0.44% and the HDL was 0.18%. Aldehydes from the plasmalogen were derivatized with dansylhydrazides and separated by high performance liquid chromatography (HPLC). Their presence was detected using a fluorescence detector. Hexadecanal (C16: 0), octadecanal (C18: 0) and octadecenal (C18: 1) were determined to be the major components in the carp and rainbow trout.     

Analysis of steroidal car☐ylic acids by high pressure liquid chromatography

A procedure utilizing high pressure liquid chromatography has been developed for the analysis of steroids containing 17β-car?ylic acid or 20-hydroxy-21-oic acid substituents. Acids were converted to thep-bromophenacyl esters and were separated on octadecylsilyl columns with methanol: water mixtures as mobile phase. Structurally similar steroids, including the 20α- and 20β- epimers of the 21-oic acids were well separated. The lower limits of detection ranged from 0.25 to 0.5 nm. The method is simple, rapid, quantitative, sensitive, and specific.     

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.     

Structures of Dimers Produced from Methyl Linoleate during Initial Stage of Autoxidation

A structural investigation on the main fraction of dimers formed during the induction period of autoxidation in methyl linoleate (ML) was carried out. The dimeric fraction (A₂), which was isolated from the autoxidized ML (POV= 18) by various Chromatographic techniques and gave a single spot on TLC, was further separated into four major components (components 1–4) by high performance liquid chromatography (HPLC). The mean molecular weights of these components were found to be 643–655 and component 4 gave the parent peak 652 on an FD-mass spectrum which corresponded to 2 × ML + 4O. The reduced products of each component with stannous chloride were identified in common as methyl 9- or 13-hydroxy octadecadienoate and methyl 9,13-dihydroxy octadecenoate by GC-MS. These results show that all of these dimers contained a peroxide bridge linking between a pair of MLs across C-9 or C-13 on one of the MLs and C-9 or C-13 on the other, with a hydroperoxy group.     

Oligosaccharide structures of human colonic mucin

Purified human colonic mucin was separated into six distinct components by DEAE-cellulose chromatography, and the structures of oligosaccharide side chains from the three most abundant species were determined. Oligosaccharide side chains were isolated from colonic mucin species III, IV, and V after alkaline borohydride reductive cleavage in the presence of sodium borotritide. After initial separation of acidic and neutral oligosaccharides by ion exchange chromatography, individual oligosaccharides were isolated by sequential chromatography on Bio-Gel P-4 and Bio-Gel P-2 resins followed by preparative normal phase high performance liquid chromatography. Composition and structure of individual oligosaccharides were determined by combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Collectively, 21 discrete oligosaccharide structures were identified in the major human colonic mucin species including 10 acidic oligosaccharides and 11 neutral structures which ranged in size from 2 to 12 sugar residues. Although detailed structures were defined for each oligosaccharide, the majority of the structures identified were variations of a relatively small number of "basic" structures, and several generalizations pertained. First, many oligosaccharides represented variations of a biantennary structure in which branch chains arise in N-acetylglucosaminyl residues linked to C3 and C6 of a galactosyl residue linked in turn to a GlcNAc beta (1-3)GalNAc core; second, non-branched oligosaccharides appeared to be linear chain derivatives of the same core structure; third, all acidic oligosaccharides could be derived from neutral structures present in the mucin species; fourth, sialic acid substitution was limited to few sites and always included substitution in alpha 2-6 linkage to the reducing terminal N-acetylgalactosamine, and finally several structures contained both sialic acid and fucose residues. Individually, mucin species III, IV, and V were found to contain unique mixtures of 13, 14, and 10 oligosaccharide structures, respectively. These data demonstrate that human colonic mucin contain a wide range of oligosaccharides reflecting variations of common core oligosaccharide structures. The major chromatographically defined constituents of normal colonic mucin appear to possess characteristic and distinguishable combinations of oligosaccharide structures. These findings support the concept that colonic mucin contains structurally and functionally distinct subpopulations.     

Characterization of Alkylgalactolipids from Calf Brain by High Performance Liquid Chromatography

Minor nonpolar galactolipids were isolated from the total lipids of calf brain stem by column chromatography and were separated by preparative thin-layer chromatography into four groups. The material recovered from the bottom band of the thin-layer chromatography consisted of monogalactosyl diglyceride and its 1-0-alkyl isomer, alkylgalactolipid, present in a molar ratio of 11 :9. After perbenzoylation. they were separated by preparative thin-layer chromatography and characterized. The fatty acid compositions of these lipids were similar to each other and to those of the ester-linked fatty acids of cerebroside esters. The major alkyl group of alkylgalactolipid was palmityl, and the other, minor components were oleyl. myristyl, and stearyl ethers. Perbenzoylated derivatives of these lipids were further separated by reverse-phase high performance liquid chromatography. The chromatograms from these two lipids were similar; however, most of the peaks were still mixtures of homologs containing different fatty acids or an alkyl group.     

Novel fucolipids accumulating in human adenocarcinoma. I. Glycolipids with di- or trifucosylated type 2 chain

Among a series of fucolipids accumulating in human colonic and liver adenocarcinoma, two neutral fucolipids have been isolated and their carbohydrate structures have been characterized by methylation analysis, mass spectrometry, enzymatic degradation, and proton NMR spectrometry as shown in Structures 1 and 2 below. These glycolipids are either absent or present in very small quantity in normal colonic mucosa and normal liver tissue. (formular; see text) Difucosyl neolactonorhexaosylceramide (Structure 1) gives a doublet or a triplet on high performance thin layer chromatography and can be separated into four to five fractions on high pressure high performance liquid chromatography. These components have the same carbohydrate structure, but have different fatty acid composition. Fractions are characterized by a predominance of either alpha-hydroxy C16 fatty acid or alpha-hydroxy C24 fatty acid. Trifucosyl neolactonoroctaosylceramide (structure 2) gives two discrete bands, which are designated Z3 and Z4, on high performance thin layer chromatography. Methylation analysis and nuclear magnetic resonance spectra show that the Z3 and Z4 have identical carbohydrate structures. All the cases of cancer tissue that accumulate di- or trifucosyl structure (1 or 2 above) also accumulated lactofucopentaose(III)ceramide. A possible enhancement in a specific synthetic pathway including type 2 chain elongation coupled with alpha 1----3 fucosylation in human cancer is discussed.