Specific binding of antibodies to platelet-activating factor (PAF) as demonstrated by thin-layer chromatography/Immunostaining

The specificity of rabbit antibodies produced by injection of 1-O-(15'-carboxypentadecyl)-2-N,N-dimethylcar-bamoyl-sn-glycero-3-phosphocholine bovine serum albumin (BSA) conjugates was examined by a thin-layer chromatography (TLC)/immunostaining method. Phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC), lyso platelet-activating factor (lysoPAF), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidic acid (PA) and cardiolipin (CL) were not immunostained. Among several synthetic PAF-related compounds, the antibodies only bound to PAF agonists which have the activity to induce washed rabbit platelet aggregation. The results suggest that the binding sites of the antibodies on the PAF molecule are the acetyl group at thesn-2 position and the choline moiety at thesn-3 position of glycerol, both of which are essential for exerting the biological function of PAF and for binding to the PAF receptors located on cellular membranes. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Molecular analysis of intact preen waxes of Calidris canutus (Aves: Scolopacidae) by gas chromatography/mass spectrometry

The intact preen wax esters of the red knot Calidris canutus were studied with gas chromatography/mass spectrometry (GC/MS) and GC/MS/MS. In this latter technique, transitions from the molecular ion to fragment ions representing the fatty acid moiety of the wax esters were measured, providing additional resolution to the analysis of wax esters. The C₂₁−C₃₂ wax esters are composed of complex mixtures of hundreds of individual isomers. The odd carbon-numbered wax esters are predominantly composed of even carbon-numbered n-alcohols (C₁₄, C₁₆, and C₁₈) esterified predominantly with odd carbon-numbered 2-methyl fatty acids (C₇, C₉, C₁₁, and C₁₃), resulting in relatively simple distributions. The even carbon-numbered wax esters show a far more complex distribution due to a number of factors: (i) Their n-alcohol moieties are not dominated by even carbon-numbered n-alcohol moieties are not dominated by even carbon-numbered n-alcohols esterified with odd carbon-numbered 2-methyl fatty acids, but odd and even carbon-numbered n-alcohols participate in approximately equal amounts; (ii) odd carbon-numbered methyl-branched alcohols participate abundantly in these wax ester clusters; and (iii) with increasing molecular weight, various isomers of the 2,6-, 2,8-, and 2,10-dimethyl branched fatty acids also participate in the even carbon-numbered wax esters. The data demonstrate that there is a clear biosynthetic control on the wax ester composition although the reasons for the complex chemistry of the waxes are not yet understood.     

Vitamin E. Deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129–240, 131–227 and 248–354 pmol/10⁶ cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [³H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [³H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28±0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06±0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26±0.71 and 4.26±0.06 nmol/min/mg protein, respectively), measured as degradation of [³H]PAF to [³H]lysoPAF.In vitro addition of α-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, in-dicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by α-tocopherol. The acetyltransferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 μM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 μM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.     

Double-bond localization in heneicosapentaenoic acid by a gas chromatography/mass spectrometry (GC/MS) method

Double-bond locations in heneicosapentaenoic acid from eel lipids were determined by a method involving gas chromatography/mass spectrometry (GC/MS). The methyl esters of the pentaenoate fraction were first partially reduced with hydrazine. The resultingcis-monoenoates were then methylthiolated, and the resultant adducts were analyzed by GC/MS. The key fragmentation ions generated by the cleavage between the methylthio-substituted carbons were used to ascertain the original double-bond positions in the native fatty acid esters. Based on mass spectral evidence, the acid was identified as all-cis-6,9,12,15,18-heneicosapentaenoic acid.     

Role of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes

The effect of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes (PMN) was studied. Cypridina luciferin analog (CLA) dependent chemiluminescence was used to detect superoxide anion radicals. PAF induced superoxide generation in human PMN in a dose-dependent manner. Preincubation with a small amount of PAF (5 x 10⁻⁹ M) enhanced PMN superoxide release induced by various stimuli, such as phorbol myristate acetate (PMA), opsonized zymosan (OZ), calcium ionophore (A23187) andN-formyl-methionyl-leucyl-phenylalanine (FMLP). The PAF antagonist, CV-6209, inhibited superoxide production induced by PAF, but not that induced by other stimuli. These findings would indicate that PAF may play an important role at inflammatory reaction sites and that CV-6209 may inhibit excessive inflammatory reaction.     

Pooling of blood in postischemic shock is modulated by platelet-activating factor (PAF)

In experiments on dogs,i.v. administration of platelet-activating factor (PAF) (500 ng/kg) was shown to induce hypotension which, apart from decreased myocardial contractility, was characterized by blood pooling in veins (82.6±6.8 mL/kg). This was accompanied by restriction of venous return to the heart and reduction of cardiac output (CO). During postischemic shock the cardio- and hemodynamic disturbances were similar to those induced byi.v. administration of PAF. In the postischemic shock model, preliminary blockage of PAF receptors with the PAF receptor antagonist BN 52021 (6 mg/kg,i.v.) significantly decreased the amount of blood pooled in shock from 38.7±5 to 18.3±2 mL/kg (p<0.01). Simultaneously, the reduction of CO and blood pressure, induced by reperfusion of the continuously ischemized tissues of a rear limb, was less significant in pretreatedvs. the nontreated group. The data suggest that PAF may be involved in postischemic blood pooling and that PAF antagonists could be used to correct postischemic cardio- and hemodynamic disturbances. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Fatty acid composition of oil in snow crab (Chionoecetes opilio) by gas chromatography/mass spectrometry

The hepatopancreatic fatty acid extract of the snow crab contains a high percentage (26%) of odd-carbon-numbered fatty acids and a substantial quantity (29%) of methyl-branched fatty acids, as indicated by gas chromatography/mass spectrometry (GC/MS) and gas liquid chromatography (GLC). A wide distribution in chain length of the fatty acids (C₁₀ to C₂₆) and in positional isomers of the linear monoenes are also indicated by GC/MS.     

Involvement of platelet-activating factor (PAF) in septic shock and priming as indicated by the effect of hetrazepinoic PAF antagonists

Pharmacological data obtained with hetrazepinoic platelet-activating factor (PAF) antagonists, such as apafant (WEB 2086) and bepafant (WEB 2170), indicate a role for PAF in septic shock and in the priming process. The effect of PAF antagonists in different models of shock states favors a role for PAF in endotoxin associated lethality, activation of inflammatory blood cells with release of mediators, cardiovascular failure and increased vascular permeability, and in the development of shock organs and organ failure. The priming process (e.g., by endotoxin or tumor necrosis factor) towards an increased susceptibility towards minute amounts of PAF has to be taken into account when considering the pathophysiological significance of PAF underin vivo conditions and in septic shock. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Primary study of volatiles composition of Rhodiola sachalinensis by using gas chromatography and mass spectrometry (GC/MS)

To determine the volatile compounds in Rhodiola sachalinensis, hydro-distillation (HD) and headspace liquid-phase micro-extraction (HS-LPME) were used to extract 75 and 68 volatiles, respectively. Geraniol (24.73%), n-octanol (15.56%), and linalool (14.51%) were the most abundant essential oils detected in the HD samples, while geraniol (24.17%) and n-octanol (15.81%) were also detected at high levels in the HS-LPME samples. The main chemical classes of the essential oils were monoterpene alcohols in both the HD and HS-LPME samples at 59.02% and 37.64%, respectively. The O-heterocyclic (8.52%) and aromatic (5.92%) compounds were more abundant in the HS-LPME samples than in the HD samples.     

大体积顶空-气相色谱/质谱联用测试人造革中的氯乙烯单体含量

建立了大体积顶空-气相色谱/质谱联用测定人造革中氯乙烯单体的方法。采用正交试验优化了影响氯乙烯检测的大体积顶空仪参数(如吹扫时间、吹扫温度、载气流速、解吸温度和解吸时间),并对影响氯乙烯检测的色谱条件进行了优化。在最佳条件下,氯乙烯在66.1～6610mg/L范围内呈较好的线性关系,检测限低至0.07 mg/kg(以S/N≥13计算);重现性试验(N=6)相对标准偏差为6.17%,平均回收率高于98.13%。指出该方法操作简单、实用性强。     

Characterisation of end groups in poly(2-hydroxyethyl methacrylate) by means of electrospray ionisation-mass spectrometry/mass spectrometry (ESI-MS/MS)

Poly(2-hydroxyethyl methacrylate) (poly(HEMA)) has been characterised by means of electrospray ionisation-mass spectrometry/mass spectrometry (ESI-MS/MS), in order to evaluate this technique for the generation of end group information. Low energy collision-induced dissociation (CID) data from poly(HEMA) enabled information on both end groups of the polymer chain to be gleaned, in a similar fashion to that proposed previously for other methacrylate polymer systems. Exact-mass CID information was employed to aid the understanding of the dissociation mechanism of the polymer. Some additional fragmentation pathways, compared to other methacrylate polymer systems, are proposed. An example of how software can aid the interpretation of the MS/MS data is also shown.     

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag⁺-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11–18∶2 cis/trans and trans, trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were sepatated by GC and Ag⁺-HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis, 13 trans-18∶2; 26.3% 10 trans, 12 cis-18∶2; 20.4% 9 cis, 11 trans-18∶2; and 16.1% 8 trans, 10 cis-18∶2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar patterns to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18∶2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18∶2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18∶2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18∶2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18∶2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18∶2 and 8 trans,10 cis-18∶2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18∶2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18∶2 naturally found in foods have not been established.     

Identification and quantitation of cis/trans C16:1 and C17:1 fatty acid positional isomers in German human milk lipids by thin‐layer chromatography and gas chromatography/mass spectrometry

The intake of trans C18:1 as well as of trans hexadecenoic acids (trans C16:1) is believed to be related with numerous physiological disadvantages, such as the risk of coronary heart disease. Since most of the existing data on trans C16:1 contents in human milk fat have been determined without a pre‐separation by thin‐layer chromatography (TLC), the gas chromatographically determined contents of trans C16:1 frequently are too high due to overlaps with C17 fatty acids. Using a highly polar column with a length of 100 m after AgNO₃‐TLC allowed to establish an average content of total trans C16:1 of 0.15 ±0.04% from 39 samples of human milk fat. Moreover, the C16:1 positional isomers trans Δ4, Δ5, Δ6/7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13 and Δ14 could be quantified from 15 samples exhibiting mean relative contents of 2.6, 3.5, 7.6, 7.2, 24.7, 10.4, 10.1, 14.3, 8.4 and 11.3% related to the total trans C16:1 content, respectively. Also, the C16:1 isomer trans Δ3 could be identified occurring in traces with a mean absolute content of 2 mg/100 g fatty acids. A baseline separation of almost all trans isomers could be achieved for the first time. Further, mass spectrometric analyses of FAME and DMOX derivatives allowed to identify the isomer trans Δ4. Among the C16:1 isomers cis Δ7 to cis Δ14 the isomer cis Δ9 predominated with a relative proportion of 68.3% and an absolute content of 1.88% of all fatty acids. Correspondingly, among the C17:1 isomers cis Δ7 to cis Δ11 the isomer cis Δ9 with 82.6% had the highest relative content.     

Configurational sequence lengths in polyacrylonitrile and poly(acrylonitrile-CO-haloalkyl acrylate/methacrylate)s determined by 13C n.m.r.

The tacticity and number average sequence length of like ($\text{n}\text{?}{}_{\mathrm{<mtext>o</mtext>}}$), meso ($\text{n}\text{?}{}_{\mathrm{<mtext>m</mtext>}}$) and racemic ($\text{n}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$) acrylonitrile (AN) triad units in polyacrylonitrile (PAN) prepared in both water and water—acetone (2:1 v/v) media and AN-3-chloro, 2-hydroxypropyl acrylate/methacrylate, AN-2-bromoethyl methacrylate and AN-2-chloroethyl acrylate copolymers have been calculated using ¹³C n.m.r. spectra of the polymer solutions concerned at a field strength of 24.99 MHz. The spectra reveal that PAN prepared in water medium has a greater percentage (33.4%) of isotactic units than PAN prepared in water-acetone (2:1 v/v) medium (28.3%). The tacticity distribution of AN sequences in PAN and the copolymers is found to be random ($\text{n}\text{?}{}_{\mathrm{<mtext>m</mtext>}}\text{?}\text{n}\text{?}{}_{\mathrm{<mtext>r</mtext>}}\text{?2.0}$) and the number average sequence length of AN sequences in a copolymer containing 14.8 mole% of 3-chloro, 2-hydroxypropyl methacrylate was 15.2.     

Effect of stereoregularity and solvent upon molecular motion and structure of stereoregular poly(methyl methacrylates) in solution. 13C and 1H n.m.r. relaxation study

Spin-lattice relaxation times T₁ of ¹³C and ¹H nuclei, as well as nuclear Overhauser enhancement (NOE) values of stereoregular poly(methyl methacrylates) (PMMA) in CD₃CN and CDCl₃ were measured. Analysis of these data has shown that the mobility of PMMA in solution is affected by stereoregularity of PMMA and by solvent. Comparison of ¹³C and ¹H n.m.r. relaxation data has further shown that the solvent affects also the local conformational structure of stereoregular PMMA in solution; this conclusion is supported by preliminary measurements of infra-red spectra. Based on this finding, the effect of solvent upon formation of the ordered structure of the so-called stereocomplex of PMMA is discussed.     

Reduction of Zr (IV) in (KCl/NaCl) eutectic (50/50 mol%) containing KF/ZrCl4 in molar ratios of 6/1 or 4/1 at 750° C. Characterization of the dissolved species by IR spectroscopy

Infrared analysis of baths which are mixtures of (KCl/NaCl) (50/50) with KF and ZrCl₄ in the molar ratio 4 or 6, indicates that the species present at 750° C are ZrF₄ and K₃ZrF₇. Voltammetric study of these melts containing 1 wt % Zr shows that ZrF₄ reduces in three steps: ZrF₄ ZrF₂, ZrF₄ Zr, ZrF₂ Zr, whilst K₃ZrF₇ is reduced via: K₃ZrF₇ Zr or K₃ZrF₇ ZrF₂, ZrF₂ Zr. It appears that ZrF₄ vanishes completely by increasing the KF/ZrCl₄ ratio until the KF concentration is able to stabilize K₃ZrF₇ in the melt.     

Analysis of low erucic acid turnip rapeseed oil (Brassica campestris) by negative ion chemical ionization tandem mass spectrometry. A method giving information on the fatty acid composition in positionssn-2 andsn-1/3 of triacylglycerols

A tandem mass spectrometric method is described for the rapid analysis of fatty acid combinations in mixtures of triacylglycerols. Triacylglycerols were introduced into a triple quadrupole mass spectrometervia a direct exposure probe and deprotonated using ammonia negative ion chemical ionization. Collisionally activated spectra were obtained and the resulting fragments used to identify the fatty acid constituents, and the fatty acids preferentially located at thesn-2 position of the triacylglycerols. Fourteen major molecular weight species of purified triacylglycerols of a supercritical fluid extract of low erucic acid turnip rapeseed oil (Brassica campestris) were analyzed. The five major combinations of fatty acids comprised two thrids of the total triacylglycerols and contained oleic, linoleic and α-linolenic acids with linoleic acid favoring thesn-2 position.     

Molecular motion of poly(methyl methacrylate), polystyrene and poly(propylene oxide) in solution studied by 13C n.m.r. spin-lattice relaxation measurements: effects due to distributions of correlation times

The ¹³C n.m.r. spin lattice relaxation times and Nuclear Overhauser Enhancements have been measured for solutions of poly(methyl methacrylate) (PMMA) in o-dichlorobenzene, polystyrene (PS) in pentachloroethane and poly(propylene oxide) (PPO) in CDCI₃ as a function of temperature. For PS and PMMA a T₁ minimum is observed close to ambient temperature. The single correlation time theory of relaxation is inadequate to explain the relaxation data, but within experimental error, the data can be interpreted in terms of either the Cole-Cole distribution of correlation times, the log ?χ² distribution or a conformational jump model of chain dynamics.