Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5°C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual subtrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.     

Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis

We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from ¹⁴C-oleate of 1-palmitoyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[¹⁴C]oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.     

Reactions of dinitrogen pentoxide and nitrogen dioxide with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

The reactions of gaseous dinitrogen pentoxide (N₂O₅) and nitrogen dioxide (NO₂) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) coated on the inside surface of a glass reaction cell were studied at 298 K. Unsaturated phosphatidylcholines are significant components of pulmonary surfactant in the alveolar region of the lung and hence serve as a simple model to examine reactions of pulmonary surfactant with these oxidant air pollutants. Using high-performance liquid chromatography (HPLC), Fourier transform infrared and fast atom bombardment mass spectroscopy, the major products of reactions of POPC with N₂O₅ and NO₂ were separated and identified. In the POPC-N₂O₅ reaction using either air or helium as a buffer gas, the nitronitrate, vinyl nitro and allylic nitro derivatives, as well as a small amount of thetrans-isomer of the starting material, were obtained. The nature of the products obtained from the POPC-NO₂ reaction depends on the concentration of NO₂ as well as whether air is present. At low NO₂ concentrations (P_{NO 2}/_{N 2}O₄≤3.8 Torr) in air or in helium, thetrans-isomer of POPC was formed almost exclusively. At higher NO₂ concentrations (P_{NO 2}/N₂O₄≥20 Torr) in helium, the dinitro, vinyl nitro and nitro alcohol derivatives were formed. In the presence of air (or 24%¹⁸O₂ in helium), a nitronitrate and a dinitronitrate were additional products. Mechanisms for the formation of the observed products and implications for the inhalation of oxides of nitrogen are discussed.     

Production of the criegee ozonide during the ozonation of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes

It is likely that Criegee ozonides are formed in small amounts in the lungs of animals breathing ozone-containing air. This makes these compounds potential candidates to act as secondary toxins which relay the toxic effects of ozone deeper into lung tissue than ozone itself could penetrate. Therefore, we have determined the yields of Criegee ozonides from unsaturated lipids in liposomal systems as a model of the types of yields of Criegee ozonides that might be expected both in the lung lining fluid layer and in biological membranes. Ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes produced bothcis- andtrans-Criegee ozonides. These ozonides have been isolated by solid phase extraction and high-performance liquid chromatography of the ozonized lipid, and the products have been identified by two-dimensional¹H nuclear magnetic resonance. The combined yield of thecis- andtrans-Criegee ozonides is 10.7±2.8% (avg. ±SD, n=7) with small unilamellar liposomes and 10.6±2.7% (n=3) with large multilamellar liposomes. We had previously reported (Chem. Res. Toxicol. 5 505–511, 1992) that ozonation of methyl oleate in sodium dodecylsulfate micelles also produces an 11% yield of the Criegee ozonides. Thus, ozonation in a variety of models gives about 11% of the Criegee ozonide, suggesting that these products also would be formed in small but significant amounts in the lungs of animals breathing polluted air. Further research on the pharmacokinetics and possible toxicity of the Criegee ozonides of fatty acids is suggested.     

Biochemical characterization of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase in rat spleen microsomes

Acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) ultrasonic disruption in the presence of 25% glycerol from rat spleen microsomes. About 26% of the enzymatic activity was recovered in the 225,000×g supernatant by this treatment, although the specific activity was slightly decreased compared with the original microsomes. The solubilized enzyme was remarkably susceptible to various kinds of metal ions. Sulfhydryl reagents such as p-chloromercuribenzoate and N-ethyl-maleimide significantly inhibited the enzyme reaction, suggesting that the enzyme is an SH enzyme. Based on the sedimentation pattern in sucrose density centrifugation, the isoelectric point, the kinetic characteristics and the sensitivity to tryptic digestion of microsomes, it appears that acetyl-CoA:lyso-PAF acetyltransferase does not differ from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to 1-acyl-2-lyso-sn-glycero-3-phosphocholine.     

Growth inhibitory effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

The growth inhibitory effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃) and various liposome compositions of ET-18-OCH₃ were compared in a standardized growth inhibition assay utilizing a diverse tumor cell line panel including cell lines expressing multidrug resistance. ET-18-OCH₃ and ELL-12 (4∶3∶1∶2, dioleoylphosphatidylcholine/cholesterol/dioleoylphosphatidylethanolamine-glutaric acid/ET-18-OCH₃), an optimal liposomal ET-18-OCH₃ formulation, inhibited growth in the micromolar range in drug-sensitive and-resistant cells. In general, ET-18-OCH₃-liposomes were about twofold less growth inhibitory than ET-18-OCH₃. However, the known hemolytic effects of ET-18-OCH₃ were greatly reduced, up to 20 or more times, by liposome association. The effects of ET-18-OCH₃ and ELL-12 were compared in intracellular [Ca²⁺] modulation and DNA fragmentation assays. ET-18-OCH₃ elicited both concentration- and serum-dependent transient and permanent increases in intracellular [Ca²⁺]. In contrast, ELL-12 did not modulate intracellular [Ca²⁺]. ET-18-OCH₃ and ELL-12 similarly affected DNA fragmentation, which may be indicative of apoptosis. The results suggest that, although the specific growth inhibitory effects of ET-18-OCH₃ and ELL-12 are similar, associating ET-18-OCH₃ with stable well-characterized liposomes eliminates nonspecific cell membrane-associated lytic effects.     

H3PW12O40/Al2O3催化合成丙烯醛

采用等体积浸渍法制备了具有Keggin结构的杂多酸H3PW12O40/Al2O3催化剂,用IR、XRD、BET等方法对催化剂进行了表征。同时利用常压连续流动的固定床反应器考察该催化剂对甘油制备丙烯醛的工艺条件,重点考察了催化剂的种类、杂多酸的负载量、反应温度等条件对反应的影响。结果表明,由甘油制备丙烯醛的较优条件为：当催化剂为H3PW12O40/Al2O3,负载量为40%,反应温度为330℃时,丙烯醛的选择性可达83.7％,甘油的转化率为100％。     

Characterization of an HL-60 cell variant resistant to the antineoplastic ether lipid 1-O-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine

A resistant cell line (HL-60R) was selected by incubating HL-60 cells with increasing concentrations of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and used to examine the mechanism of resistance to the antineoplastic ether-linked lipid. The HL-60R cells exhibited a>10-fold increase in resistance when measured by [³H]-thymidine incorporation in comparison to the HL-60 cell line. ET-18-OCH₃ binding occurred at 4°C and was not saturable at the concentrations tested (1–100 μM), indicating that the binding was receptor-independent. At 4°C, association of ET-18-OCH₃ was low for each cell line. At 37°C, uptake in the HL-60 cells was approximately 5-fold greater in comparison to HL-60R cells at each concentration tested. However, when the cellular content of ET-18-OCH₃ was equal, both cell lines experienced similar declines in cell growth. Cellular incorporation of ether lipid was determined using serum-free media and in the presence of serum albumin or lipoproteins. Reduced uptake by the resistant cell line was observed only in the presence of albumin. A greater proportion of ether lipid could be removed from prelabeled HL-60R cells than from HL-60 cells, by an albumin wash procedure, indicating an increased rate of internalization and retention by the sensitive cell line. ET-18-OCH₃ uptake in the HL-60 cell line was also more sensitive to treatment with endocytic (chloroquine, monensin) or metabolic (NaF, KCN) inhibitors. These results suggest that uptake is the principal determinant influencing sensitivity of the resistant cell line and consists of receptor-independent binding followed by internalization. Differential uptake requires the presence of serum albumin and is dependent on the energy-dependent endocytosis of the ether lipid.     

H3PW12O40/SiO2催化合成糠醛乙二醇缩醛的研究

采用溶胶-凝胶法制备出具有Keggin结构的杂多酸H3PW12O40/SiO2复合催化剂,用FT-IR,XRD等手段表征了其结构形态。考察了复合催化剂催化合成糠醛乙二醇缩醛的工艺条件,其中重点考察了反应物配比、催化剂用量、反应时间及催化剂的重复使用性能等对产品收率的影响。结果表明:合成糠醛乙二醇缩醛的较优反应条件为:n(醛)/n(醇)=1.0/1.2,催化剂的用量为糠醛质量的6.0%,环己烷为带水剂,反应时间4.0 h。上述条件下,产物的收率可达85.0%以上。     

Analysis of sn-1(3)- and sn-2-short-chain acyl isomers of triacylglycerols in butteroil by gas-liquid chromatography

The aim of the study was to determine major triacylglycerols (TG), and sn-1(3) and sn-2 isomers of butyryl and caproyl TG in butteroil (BO) and interesterified butteroil (IBO) by gas-liquid chromatography (GLC) and silver ion column chromatography. Altogether, 112 molecular species of TG were synthesized by interesterification and their retention indices were determined. Molar empirical correction factors for TG were determined using linear calibration. Retention indices showed that sn-1(3) and sn-2 isomers of the TG containing one short-chain acyl (butyrate, caproate) and two long-chain acyls (lauroate, myristate, palmitate, stearate, and oleate) were separated on a phenyl (65%) methylsilicone column. The difference between retention indices of 1(3)- and 2-short-chain acyl isomers ranged from 14 to 19, and from 9 to 16 for butyrates and caproates, respectively. The proportion of sn-2 isomers of butyrates averaged 1.4%, but only traces of sn-2 isomers of caproates were detected in butteroil. The ratio of sn-1(3)- to sn-2-butyrates and caproates in interesterified butteroil averaged 2.0:1. The most abundant molecular species of mono-short-chain TG in butteroil were BPP + BMS (5.6 mol%), BPO + BSPo (4.8 mol%), BMP + BLaS (3.4 mol%), BMO + BPPo (2.7 mol%), BPS (2.5 mol%), and CoPP + CoMS (2.3 mol%).     

十二烷基三乙氧基硅烷改性H3PW12O40/MCM-41在蓖麻油脱水反应中催化性能研究

采用十二烷基三乙氧基硅烷(DTES)改性H_3PW_(12)O_(40)/MCM-41催化剂,研究了硅氧烷偶联剂改性对催化剂上蓖麻油脱水制备脱水蓖麻油反应性能的影响。结果表明,硅氧烷偶联剂改性有利于显著提高催化剂上H_3PW_(12)O_(40)物相的分散度,主要原因是由于硅氧烷偶联剂增加了相邻H_3PW_(12)O_(40)分子的空间位阻;分散度的提高导致了催化剂上总酸量以及Brnsted酸占总酸量的比值显著增加,且酸强度减弱,从而有利于蓖麻油转化率、脱水蓖麻油选择性和催化剂稳定性的提高。在220℃,H_3PW_(12)O_(40)和DTES最佳负载量分别为15%和9%的条件下反应1. 5 h,羟值、碘值分别达到8. 3 mg KOH/g和149. 3 g I_2/100 g。     

Crystallization kinetics of the pure triacylglycerols glycerol-1,3-dipalmitate-2-oleate,glycerol-1-palmitate-2-oleate-3-stearate,and glycerol-1,3-distearate-2-oleate

Crystallization kinetics of the three main components of cocoa butter, the triacylglycerols POP, POS, and SOS (where P, O, and S stand for palmitic, oleic, and stearic acids, respectively) were studied by combined differential scanning calorimetry and polarized light microscopy. The morphologies, nucleation kinetics, growth kinetics, and phases of the grains formed were identified with this system. The experimental data, as well as two different models to simulate crystallization and to predict behavior of the pure triacylglycerols, are presented. The first model is based on a macroscopical approach to solidification by using time-temperature-transformation (TTT) diagrams and the additivity principle. It allows prediction of the proportion of the different phases formed for any given thermal path imposed on the sample once the TTT diagram is known for the product. It is illustrated for SOS at constant cooling rates and is compared with experimental results. The second model directly simulates growth of the spherulites in the sample by using nucleation and growth rates that are determined experimentally. It provides a view of the structure as it would be observed with a microscope and shows evolution of the heat released in the sample. Isothermal solidification of POP at 15°C is displayed. The experiment and the model are in good agreement.     

Mass spectrometric evidence for the presence of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human amniotic fluid during labor

Evidence is presented for the existence of platelet-activating factor (PAF) in human amniotic fluid during labor by gas-liquid chromatographic (GLC) and mass-spectrometric (MS) analysis. The unique ether-linked phospholipid was identified only in an amniotic fluid obtained from women during labor, and its alkyl side chain was composed exclusively of octadecyl residue.     

Capillary gas chromatography and tandem mass spectrometry of paf-acether and analogs: Absence of 1-O-alkyl-2-propionyl-sn-glycero-3-phosphocholine in human polymorphonuclear neutrophils

Fast atom bombardment-tandem mass spectrometry was used to identify molecular species of paf-acether (paf) produced by human polymorphonuclear neutrophils. Using this biological material, normal phase high performance liquid chromatography was necessary prior to the fast atom bombardment-tandem mass spectrometry step. Gas liquid chromatography/electron capture detection after hydrolysis with phospholipase C and conversion to heptafluorobutyrate derivatives was used to confirm the results. The results indicated the presence of mainly 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine, acyl analogs of paf and only trace amounts of other alkyl analogs of paf. We did not detect the 2-propionyl analog of paf. Moreover, supplementation of human polymorphonuclear neutrophils with sodium propionate did not result in formation of the 2-propionyl analog of paf.     

Phosphatidylcholine as substrate for human pancreatic phospholipase A2. Importance of the physical state of the substrate

The long-chain phosphatidylcholine/sodium cholate aqueous system as substrate for human pancreatic phospholipase A₂ (PLA₂) was investigated. At a constant phosphatidylcholine (PC) concentration of 8 mM, the enzyme activity increased with a decrease in cholate (C) concentration up to a PC/C ratio of approximately 0.8 and then rather abruptly decreased to lower values at a ratio above 1.5. At ratios between 0.8 and 1.5, an increasing lag phase in the PLA₂ activity was seen, indicating a progressive decrease in substrate availability to the enzyme. Reaction mixtures with a PC/C ratio of up to 0.67 were optically clear solutions composed of mixed bile salt/PC micelles of increasing mixed micellar aggregate size. Ratios between 0.67 and 1.5 were characterized by an increase in turbidity (at 330 and 450 nm) due to increasing formation of vesicles or liposomes. Above a PC/C ratio of 1.5, a sharp increase in turbidity was seen due to increasing formation of bilayer structures other than vesicles. Pure vesicles obtained by dialysis of mixed micellar solutions were not hydrolyzed by the enzyme. Addition of bile salts reversed the inhibition which was accompanied by a decrease in turbidity. Phosphatidylcholine was preferred as substrate for human PLA₂ when present in large mixed disc-like bile salt micelles. Vesicular or other types of lamellar liquid-crystalline phases of long-chain phosphatidylcholine did not serve as substrate for PLA₂.     

Simultaneous modeling of VLE,LLE and VLLE of CO2 and 1, 2, 3 and 4 alkanol containing mixtures using GC-PPC-SAFT EOS

A polar version of the group contribution PC-SAFT equation of state (GC-PPC-SAFT; Tamouza et al., 2004; NguyenHuynh et al., 2008) combined with a method for correlation/prediction of binary interaction parameters k_ij (NguyenHuynh et al., 2008) is here applied to model vapor–liquid, liquid–liquid and vapor–liquid–liquid phase equilibria of CO₂ + alkanol mixtures simultaneously.A cross-association interaction between CO₂ and alkanol had to be taken into account to model/predict the mixtures equilibria accurately. The cross-association parameters were evaluated using the so-called CR1 mixing rules supported by ab initio computations.Extensive prediction tests on CO₂ + alkanol mixtures involving linear and branched alkanols are carried out. The results obtained showed that in most cases, the correlation and prediction calculations are qualitatively and quantitatively satisfactory: the overall deviations on liquid phase and vapor phase are respectively ΔX = 3–4% and ΔY = 1–2%.     

Conversion of 18∶3 Δ9cis, 12cis, 15trans in rat liver microsomes

Several years ago, it was established that the Δ15 trans isomer of α-linolenic acid is converted in vivo into fatty acids containing 20 and 22 carbons (geometrical isomers of eicosapentaenoic and docosahexaenoic acids). The present study focused on the in vitro Δ6 desaturation, the first step of the biosynthesis of the n-3 long-chain polyunsaturated fatty acids from 18:3n-3. For that purpose, rat liver microsomes were prepared and incubated with radiolabeled 18∶3 Δ9cis, 12cis, 15cis (18∶3 c,c,c) or 18∶3 Δ9cis, 12cis, 15trans (18∶3c,c,t) under desaturation conditions. The data show that 18∶3c,c,t is converted at a lower rate compared with α-linolenic acid. The product of conversion of 18∶3 c,c,t may be 18∶4 Δ6cis, 9cis, 12cis, 15trans resulting from a Δ6 desaturation of the trans substrate. Moreover, the conversion of radiolabeled 18∶3c,c,t was strongly decreased by the presence of 18∶3c,c,c (up to 48%) while the 18∶3c,c,t only slightly decreased the conversion of radiolabeled 18∶3c,c,c. Thus, the desaturation enzyme presented a higher affinity for the native all-cis n-3 substrate.