Source of lung surfactant phospholipids: Comparison of palmitate and acetate as precursors

The phospholipids and the fatty acid compositions of major phospholipids in rat lung parenchyma, microsomes, lamellar bodies and alveolar wash were quantified. Adult rats were injected simultaneously with [³H] palmitate and [¹⁴C] acetate into the femoral vein. The appearance of labeled phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) in each lung fraction was measured during short periods of time (5 min to 2 hr) after isotope administration. Relatively more PC, DSPC and PG labeled with acetate radioactivity in lung microsomes entered lamellar body and alveolar wash fractions than those labeled with palmitate radioactivity. However, there was no difference between palmitate and acetate labeled phospholipids in the transport from microsomes to lamellar bodies by phospholipid exchange proteins. On the other hand, prior injection of colchicine resulted in decrease in the transport of PC from microsomes to alveolar space to a relatively greater extent in the acetate radioactivity than in the palmitate radioactivity.     

Reversibility of cholinephosphotransferase in lung microsomes

The effect of cytidine 5′-monophosphate (CMP) on the incorporation of cytidine 5′-diphosphate (CDP) [methyl-¹⁴C]choline or [1-¹⁴C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDP choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[¹⁴C]choline into PC was markedly stimulated, but the incorporation of [¹⁴C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool.     

Molecular species analysis of phosphoglycerides from the ripe roes of cod (Gadus morhua)

Molecular species of the 3,5-dinitrobenzoyl derivatives of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were quantitated by UV detection at 254 nm after reversed-phase HPLC using solvent systems modified from Takamuraet al. (Lipids 21, 356–361, 1986). Three isocratic solvent systems were used and a total of 39 different molecular species detected. Four species, 16∶0/20∶5, 18∶1/20∶5, 16∶0/22∶6 and 18∶1/22∶6 contributed 67.2% and 61.8% of PC and PE respectively but only 23.0% of PI. In PI the most important species was 18∶0/20∶4 at 36.7% but this species only constituted 0.7% in each of PC and PE. Small amounts of dipolyunsaturated species were also found in PC and PE. Molecular species are abbreviated as follows: e.g., 16∶0/20∶5 PC is 1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine.     

Identification of compounds in an HPLC fraction from female extracts that elicit mating responses in male screwworm flies,Cochliomyia hominivorax

When hexane extracts of mature screwworm females were chromatographed on a silica gel column, mating stimulant activity was concentrated in a fraction that eluted with hexane-ether (946, v/v). Separation of this fraction with HPLC (acetonitrile-acetone; 6040, isocratic) resulted in a chromatogram of some 20 peaks. Only peaks 4–11 elicited mating responses. Peaks 5–10 had most of the activity, with peak 8 producing the highest response. Sixteen compounds were characterized from peak 8 by gas chromatography-mass spectrometry: six unbranched secondary acetates (C₃₁H₆₂O₂); seven previously unreported methyl-branched secondary acetates (C₃₂H₆₄O₂); one unbranched ketone (C₃₁H₆₂O); and one methyl-branched ketone (C₃₂H₆₄O). The isomeric acetates were not completely resolved from each other by capillary gas chromatography (CGC) on methyl silicone columns. The sixteenth compound was an aldehyde (C₃₀H₆₀O) that was present only in occasional peak 8 preparations. These compounds and several derivatives were characterized by capillary gas chromatography-mass spectrometry (CGC-MS). The position of the acetate group was ascertained by conversion to a keto group or by replacement of the acetate with a methyl group. Pheromone activity was not observed in peaks trapped either from CGC or by recombination of the trapped CGC peaks from HPLC peak 8. This apparent loss of activity from CGC peaks or from TLC cannot currently be explained.Mention of a proprietary or commercial product does not constitute an endorsement by the U.S. Department of Agriculture.     

Microscale synthesis of phosphatidyl-[3H]choline from 1,2-diacylglycerol. Assessment of isomerization by reversed-phase high-performance liquid chromatography

The synthesis ofrac-1-palmitoyl-2-oleoylglycero-3-phospho-[³H]choline of high specific activity was carried out on a microscale by making 7 μmol ofrac-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 (HPLC), the yield of the synthesis of [³H]phosphatidylcholine (120 μCi/μmol) 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 synthetizedrac-1-palmitoyl-2-oleoylglycero-3-phospho-[³H]choline showed a total absence of 1,3-isomer.     

HBIW的纯度分析方法

研究了流动相、流速、柱温等因素对HBIW纯度分析的影响.建立了HBIW的高效液相色谱分析条件:反相色谱柱,紫外检测波长241nm,二元流动相四氢呋喃-水体积比90:10,流速1.000mL/min,柱温25℃.采用归一化法、外标法和内标法对HBIW进行了纯度分析.结果表明,3种方法标准偏差均小于1%,归一化法准确度受样...     

Determination of phenolic antioxidants in polyethylene with dissolution by n-heptane in an autoclave

A new procedure for quantitative analysis of phenolic antioxidants in polyolefins was tested. Thereby polyethylene samples were dissolved in pure n-heptane or in n-heptane/isopropanol mixture (1 000/5, v/v) in an autoclave at 160–170°C under elevated pressure. After precipitation of the polymer by cooling to room temperature, the obtained solution was either directly analysed by means of HPLC equipped with an UV-detector, or the supernatant solvent used for the extraction was evaporated in vacuum at room temperature and the residue redissolved in acetonitrile. The concentration of antioxidants in the solution was determined by isocratic HPLC. The advantage of this procedure is the short time of about 2 h needed for analysis and the good reproducibility of results (σ = 3–5%).     

Pulmonary surfactant lipid synthesis from ketone bodies,lactate and glucose in newborn rats

The contribution of acetoacetate (AcAc), β-hydroxybutyrate (βOHB), lactate and glucose to pulmonary surfactant lipid synthesis in three-to five-day-old rats was measured. Minced lung tissue was incubated with³H₂O and [3-¹⁴C]AcAc, [3-¹⁴C]βOHB, [U-¹⁴C]lactate or [U-¹⁴C]glucose, and the radioactivity incorporated into surfactant lipids was measured. When expressed as nmol of substrate incorporated/g lung tissue per four hr, lactate was incorporated more rapidly than other substrates into total surfactant lipids and phosphatidylcholine (PC). There was no difference in the rates of incorporation of lactate, AcAc or glucose into disaturated PC (DSPC). Substrates other than glucose were incorporated almost exclusively into fatty acids, whereas 60–80% of glucose incorporated into surfactant phospholipids was found in fatty acids, with the remaining in glyceride-glycerol. When expressed as nmol acetyl units incorporated/g lung tissue per four hr, the rates of AcAc, lactate and glucose incorporation into total surfactant fatty acids were comparable. Glucose incorporation into DSPC and PC was greater than that of AcAc and lactate. When glucose was the only exogenous substrate added to the incubation medium, it contributed 37% of total surfactant fatty acids synthesized de novo. In the presence of other substrates, the contribution of glucose to de novo fatty acid synthesis dropped to 14–20%. In the presence of unlabeled glucose,¹⁴C-labeled AcAc, lactate and βOHB contributed 52%, 40% and 19%, respectively, of the total fatty acids synthesized de novo. The rate of βOHB incorporation into surfactant lipids was only about 50% that of other substrates and was accompanied by low activity of β-hydroxybutyrate dehydrogenase measured for newborn lung. These results demonstrate that AcAc and lactate are important precursors for surfactant lipids in neonatal rat lung.     

Analysis of triacylglycerols in refined edible oils by isocratic HPLC‐ESI‐MS

A simple, fast and reproducible reversed‐phase high performance liquid chromatography (HPLC) method coupled to electrospray ionization mass spectrometry (ESI‐MS) for the analysis of triacylglycerols (TAGs) species in the commercial edible oils has been developed. The TAGs species were separated using isocratic 18% isopropanol in methanol and a Phenomenex C18 column. The ESI‐MS conditions were optimized using flow injection analysis of standard TAG. Fifteen, fourteen, and sixteen TAGs were separated and identified in corn oil, rapeseed oil, and sunflower oil, respectively. The presence of intense protonated molecular (M + H⁺), ammonium (M + ${\rm NH}_{4}^{ + } $ ), and sodium (M + Na⁺) adducts ions and their respective diacylglycerols ions in the ESI‐MS spectra showed correct identification of TAGs. Some minor potassium adducts (M + K⁺) were also found. In addition, the identity of the fatty acid, position of each fatty acid, and the location of the double bond in the fatty acid moiety were explained. It was found that this isocratic method is useful for fast screening and identification of triacylglycerols in lipids.     

Molecular species of choline and ethanolamine glycerophospholipids in rat brain myelin during development

The composition of the molecular species of various phospholipid subclasses was examined in myelin isolated from brain of 15-, 21- and 90-day-old rats. The molecular species of diacylglycerophosphocholine (PtdCho), diacylglycerophosphoethanolamine (PtdEtn) and plasmenylethanolamine (PlsEtn) were quantified by high-performance liquid chromatography (HPLC) after phospholipase C treatment and dinitrobenzoyl derivatization. In rat brain myelin, each phospholipid subclass showed a specific pattern of molecular species that changed during development. PtdCho contained large amounts of saturated/monounsaturated and disaturated species and low amounts of saturated/polyunsaturated species. During brain development, the levels of saturated/monounsaturated molecular species increased whereas those of the disaturated and saturated/polyunsaturated species decreased. PtdEtn were characterized by their low levels of disaturated species and a high content of saturated/monounsaturated and saturated/polyunsaturated species, of which those containing fatty acids of the n−3 series decreased, whereas those containing fatty acids of the n−6 series did not change during brain development. The levels of saturated/monounsaturated species increased in PtdEtn. No disaturated molecular species could be detected in PlsEtn. This alkenylacyl subclass contained large amounts of saturated/polyunsaturated, saturated/monounsaturated and dimonounsaturated molecular species. During development, the levels of saturated/polyunsaturated molecular species decreased while those of the two others increased. The data indicated that myelin sheaths undergo phospholipid changes during brain development and maturation.     

Activation and solubilization by triton X-100 of membrane-bound phospholipase D of rat brain

In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [³H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[³H]choline (dipalmitoyl [³H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium doexycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1–0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1–7.3. Both Ca²⁺ and Mg²⁺ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5–1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.     

Vasopressin stimulates phospholipase D activity against phosphatidylcholine in vascular smooth muscle cells

It is now clear that various hormones and agonists can stimulate the production of lipid mediators from nonphosphoinositide phospholipids. We have investigated the production of diacylglycerol from nonphosphoinositide sources, and we demonstrated that vasopressin and other vasoactive agents stimulate hydrolysis of phosphatidylcholine in a variety of cultured vascular smooth muscle cells of rat and human origin. We used vasopressin to characterize this response and found that vasopressin stimulates phospholipase D activity against phosphatidylcholine in A-10 vascular smooth muscle cells. The vasopressin-stimulated phosphatidylcholine hydrolysis is both time- and concentration-dependent. The half-maximal dose of vasopressin required for phosphatidylcholine hydrolysis (ED₅₀∼1 nM) correlates well with vasopressin binding to A-10 cells (K_d∼2 nM). The phosphatidylcholine in A-10 cells can be preferentially radiolabeled with [³H]myristic acid; subsequent treatment with vasopressin stimulates a rapid increase in³H-labeled phosphatidate (∼4×control values at 3 min), and after a short lag,³H-labeled diacylglycerol rises and reaches maximal levels at 10 min (∼2×control values). Similar temporal elevations of phosphatidate and diacylglycerol occur in A-10 cells labeled with [³H]glycerol. In A-10 cells radiolabeled with [³H]choline the elevation of cellular phosphatidate and diacylglycerol is concomitant with the release of [³H]choline metabolites (predominately choline) to the culture medium. The temporal production of phosphatidate and diacylglycerol as well as the release of choline to the culture medium are consistent with vasopressin activating phospholipase D. In addition, vasopressin stimulates a transphosphatidylation reaction that is characteristic of phospholipase D. The transphosphatidylation reaction is detected by the production of phosphatidylethanol that occurs when A-10 cells are incubated with ethanol and stimulated with vasopressin. The phospholipase D is active in the absence of extracellular Ca⁺⁺ whereas the vasopressin-stimulated mobilization of arachidonic acid is dependent on extracellular Ca⁺⁺. The data indicate that vasopressin stimulates phospholipase D which hydrolyzes phosphatidylcholine to phosphatidate. The phosphatidate is then metabolized, presumably by a phosphatidate phosphohydrolase, to produce sustained levels of cellular diacylglycerol. These sustained levels of diacylglycerol may activate protein kinase C and theraby function in the “sustained phase” of cellular responses. Portions of this work were presented in poster form (Abstract No. 6229) at the FASEB meeting in Las Vegas, NV May 1988. The abbreviations for DAG, PA, PEt and PC are used to designate “glycerides” and as such do not differentiate between species containing ether- and/or ester-linked moieties.     

HPLC and Phospholipids Part II: Determination of PC and LPC in Defatted Soyabean Lecithins

The possibility of using HPLC for the analysis of the phosphatidylcholine content of high grade defatted soyabean lecithins of pharmaceutical interest was studied. Using isocratic HPLC in an ammonia-modified aqueous ethanol silica system, PC and LPC lipid class peaks were well separated from other lipids incl. other phospholipids. Defatted lecithin samples can simply be dissolved and injected. However, a purified soyabean PC fraction of similar fatty acid composition is necessary for external standard calibration with Rl detection. Because of the requirement that the sample should dissolve in an aqueousethanol mobile phase, the applications of this method is limited to defatted high grade lecithins (ca. 60-100% PC). A “detection limit” can therefore not be given.     

Isolation and Purification of Geniposide,Crocin-1, and Geniposidic Acid from the Fruit of Gardenia jasminoides Ellis by High-Speed Counter-Current Chromatography

Geniposide, crocin-1, and geniposidic acid were simultaneously separated from the fruit of Gardenia jasminoides Ellis by one step of high-speed counter-current chromatography with the solvent system composed of ethyl acetate-n-butanol-water(1:4:5, v/v/v) within 130 min. The purities of the three compounds were 98.7%, 97.1%, and 90.4%, respectively, as determined by HPLC. Their structures were confirmed by MS, UV, ¹H NMR, and¹³C NMR analysis.     

Anionic synthesis of aromatic carboxyl functionalized polymers. Chain-end functionalization of poly(styryl)lithium with 4,5-dihydro-4,4-dimethyl-2-[4-(1-phenylethenyl)phenyl] oxazole

The novel syntheses of 4-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)benzophenone, 1-[4-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)phenyl]-1-phenylethanol and 4,5-dihydro-4,4-dimethyl-2-[4-(1-phenylethenyl)phenyl]oxazole ( 1 ) are described. ω-Oxazolyl polystyrene ( 2 ) was synthesized in quantitative yields by the reaction of poly(styryl)lithium with stoichiometric amounts of 4,5-dihydro-4,4-dimethyl-2-[4-(1-phenylethenyl)phenyl]oxazole ( 1 ) in toluene/tetrahydrofuran (4 : 1 v/v) at −78°C. Deblocking of the oxazoline protecting group by acid hydrolysis followed by saponification quantitatively provides the corresponding aromatic carboxyl chain-end functionalized polystyrene ( 3 ). The functionalization agent and functionalized polymers were characterized by HPLC, thin layer chromatography, size exclusion chromatography, vapor phase osmometry, spectroscopy (¹H NMR, ¹³C NMR and FTIR), potentiometry and elemental analysis.     

Quantitation of vitamin K in human milk

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K₁ was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partrum. Vitamin K₁ was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.     

中药锦灯笼中酸浆苦素类化合物含量测定

以中药锦灯笼中抗炎活性成分4,7-二去氢新酸浆苦素B为对照品,用HPLC法对该药材进行了含量测定研究。Agilent ZORBAX Eclipse XDB C18色谱柱(4.6×250mm,5μm),以乙腈-0.1%磷酸水溶液(40:60)为流动相,流速1.0mL.min-1,检测波长353nm,柱温为室温。该物质在3.27×10-3～2.62×10-2μg呈良好的线性关系,平均回收率为99.1%。该方法简单易行,结果稳定、可靠,为该药材质量标准的建立提供依据。     

Molecular species composition of phosphatidylcholine fromCrypthecodinium cohnii in relation to growth temperature

The molecular species composition was determined for phosphatidylcholine (PC) isolated from the marine dinoflagellateCrypthecodinium cohnii grown at three different temperatures. At all three temperatures the didocosahexaenoyl species comprised about 25% of the PC with 14∶0/22∶6 and 16∶0/22∶6 also being of major importance; these three species comprised 75–82% of the total. Another 20 species were identified, including several short chain disaturated species. Only small differences in the composition of PC were found in response to growth at 16, 23 and 27°C. On dropping the growth temperature from 27°C to 16°C the largest changes were a decrease of 8.9% in saturated/saturated species and an increase of 5.3% in saturated/PUFA species; the 22∶6/22∶6 content only increased slightly (by 1.9% to 25.4%). This unusual molecular species composition is discussed.     

A rapid high-performance liquid chromatographic method for the determination of sinapine and sinapic acid in canola seed and meal

A high-performance liquid chromatographic (HPLC) method has been developed to separate sinapine and sinapic acid from other phenolics in canola seed and meal in a single run. The separation was achieved with a reverse-phase C18 column. Owing to the higher recovery of phenolics and ease of use, refluxing with 100% methanol for 20 min was selected as the extraction method for HPLC analysis and determination of total phenolics using Folin-Ciocalteu reagent. A 10-min isocratic/linear/concave gradient and a 15-min isocratic/linear gradient were selected as the best gradients for the separation of these phenolic compounds. Peak identities for sinapine and sinapic acid were verified with ion exchange separation followed by HPLC analysis. The method was calibrated using sinapine bisulfate and sinapic acid standards; correlation coefficients (R ²) for the calibration curves were 0.997 and 0.999 for sinapine bisulfate and sinapic acid, respectively. The extinction coefficient of sinapine was determined to be 1.16 times that of sinapic acid at the detector wavelength (330 nm). Applying this method to routine canola phenolic analyses can greatly reduce the cost by simplifying the procedures and reducing the time required for each determination.     

Determination of iodine value of palm oil based on triglyceride composition

The triglyceride (TG) composition of palm oil is normally determined by high-performance liquid chromatography (HPLC). The HPLC chromatograms indicated a good separation of most of the TG components in the oil. The TG can be classified based on either the TG groups, i.e., triunsaturated, monosaturated, disaturated, or trisaturated, or the number of double bonds, i.e., zero, one, two, three, or four double bonds. The more unsaturated the fatty acid, the greater the iodine value (IV). Therefore, it is hypothesized that the IV of an oil can be determined based upon the TG composition of the oil. Based on the TG groups, stepwise regression analysis showed that the areas of the disaturated, trisaturated, and triunsaturated TG peaks could predict the IV with a coefficient of determination (R ²) of 0.990. The regression based on the number of double bonds yielded a good regression equation with R ²=0.992. The important variables were the peak area of the fatty acids that contained zero, one, two, and three double bonds. This study concludes that the TG composition can be used to predict the IV of palm oil. The best prediction model is obtained by using the number of double bonds in the TG as the independent variable.