Structural analysis of hydroperoxides formed by oxidation of phosphatidylcholine with singlet oxygen

Soybean phosphatidylcholine (PC) and dilinoleoyl PC (di-18∶2 PC) were oxidized with singlet molecular oxygen using methylene blue as the photosensitizer. The oxidation products, PC monohydroperoxides (PC-MHP) and PC dihydroperoxides (PC-DHP), were isolated by reverse phase liquid chromatography, and their structures were analyzed by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). Signals for the hydroperoxy proton appeared downfield in NMR spectra of PC-MHP and PC-DHP. Soybean PC-MHP and di-18∶2 PC-MHP were converted to trimethylsilyl (TMS) derivatives of hydrogenated diglycerides when treated with phospholipase C and hydrogenated. Thetert-butyldimethylsilyl (TBDMS) derivatives of hydrogenated diglycerides were also prepared from di-18∶2 PC-MHP. Fragmentation of the TMS and TBDMS derivatives was obtained in electron impact mass spectra. The isomeric composition of hydroperoxylinoleate component in di-18∶2 PC-MHP was determined by methanolysis of the hydrogenated diglyceride and mass chromatographic analysis of the resulting isomeric hydroxy octadecanoates.     

Simultaneous determination of the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their corresponding hydroperoxides obtained by lipoxygenase treatment

A method for the simultaneous determination of the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their corresponding hydroperoxides is described. Hydroperoxides were formed by incubation of phospholipids with lipoxygenase at pH 9.2. Silicic acid column chromatography (silica Sep-Pak column) was used to separate the phospholipids into phosphatidylcholine and phosphatidylethanolamine. A single C−18 reverse-phase column was employed to separate the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their hydroperoxides by high-performance liquid chromatography. The mobile phase consisted of 5% 10 mM ammonium acetate at pH 5 and 95% methanol. The molecular species of phosphatidylcholine and phosphatidylethanolamine were detected at 205 nm; the eluate was mixed with a chemiluminescence reagent (isoluminol and microperoxidase) and monitored by fluorometry. Under the experimental conditions used, three individual molecular species of both soybean phosphatidylethanolamine and phosphatidylcholine (18∶3/18∶2, 18∶2/18∶2 and 16∶0/18∶2), together with their corresponding hydroperoxides, were identified and quantitated.     

Mechanisms of grinding modification by chemical additives: organic reagents

The effects of dodecylammonium chloride on properties of quartz slurries that are relevant to grinding, such as pulp fluidity, flocculation and dispersion, and primary breakage, have been investigated in this study. These tests were conducted under chemical conditions (pH, ionic strength, additive concentration, etc.) similar to those used in wet ball milling tests. The data obtained were correlated with the grinding results. Amine was found to improve grinding, pulp fluidity, and primary breakage of quartz suspensions, especially in the alkaline pH range. These beneficial effects are attributed to the formation of highly surface-active amineaminium complexes in this pH range. In contrast to the above, amine decreased the grinding rate in the acidic pH range owing to increased flocculation of quartz fines in the presence of amine in this pH range. These results show that grinding of quartz can be improved by amine if other conditions such as pH are controlled. Also, the results indicate that the effect of chemical additives on grinding cannot be attributed to a single mechanism, but that the cumulative effect on different pulp properties has to be taken into consideration. Examination of the effect of all properties has led to a better understanding of the mechanisms responsible for the observed grinding aid effects in the present study.     

Enzymatic acyl modification of phosphatidylcholine using immobilized lipase and phospholipase A2

Ethanol‐soluble (ES) lecithin mainly contains phosphatidylcholine (PC). The incorporation of caprylic acid into PC using immobilized phospholipase A₂ (PLA₂) and lipase was investigated. The Rhizomucor meihei lipase and the porcine pancreatic PLA₂ were immobilized on the hydrophobic resin Diaion HP‐20 and the modification was carried out in hexane as solvent. HPTLC with densitometer technique was successfully used for monitoring the production of structured phospholipids (PL) (ML‐type PC, MM‐type PC, and lysophosphatidylcholine; L: long‐chain fatty acid, M: medium‐chain fatty acid). The various parameters such as the effects of reaction temperature, enzyme loading, and the effect of molar proportion of substrate were studied in order to determine the optimum reaction conditions for the acidolysis reaction. The optimal operating conditions for the PLA₂‐catalyzed reaction were obtained as 50°C temperature, 50% (wt/wt of substrate) enzyme loading, and a 1:12 molar proportion of PC/caprylic acid. For the lipase‐catalyzed reaction, the optimized temperature was the same as for PLA₂, but the enzyme loading and molar proportion were slightly lower, i.e., 40 % w/w of substrate and 1:9 PC/caprylic acid, respectively. The effects of these parameters on the production of structured PL were compared. Under these optimal conditions, the ML‐type PC content was higher in the PLA₂‐catalyzed reaction, i.e., 45.29 mol%, and in the lipase‐catalyzed reaction it was 38.74 mol%.     

Synthesis ofE,E-diene hydroperoxides via photoisomerization of protected hydroperoxides

A general approach toE,E-diene hydroperoxides is described based upon photoisomerization of readily availableZ,E-diene monoperoxyketals. Protection of aZ,E-diene hydroperoxide as the 2-methoxypropyl peroxyketal is followed by iodine-mediated photoisomerization to produce a mixture enriched in theE,E isomer. After chromatographic purification, deprotection of the peroxyketal with mild acid furnishes theE,E-diene hydroperoxide.     

Quantification of carbonyls produced by the decomposition of hydroperoxides

Carbonyls produced by the decomposition of cyclohexene hydroperoxide and various hydroperoxides of linoleic and linolenic acids and their methyl esters were determined by gas chromatography of the 2,4,6-trichlorophenylhydrazones. The effect of temperature, iron and copper ions, ethanol and several antioxidants on the rate of decomposition, the nature of the products and their yield was observed. The hydroperoxides of methyl esters decomposed more slowly than those of free fatty acids. Ethanol slowed, and metal ions accelerated the rates of decomposition. Metal ions, especially copper, increased the yield and complexity of the carbonyls formed, but ethanol decreased carbonyl yields. Antioxidants and decomposition temperatures changed the relative yields of carbonyls produced. The 9- and 13-hydroperoxides of linoleic acid gave similar carbonyls, but those of linolenic acid did not. The carbonyl mixtures produced from autoxidized fatty acid methyl esters were more complex than those produced from lipoxygenase-treated fatty acids.     

Reduction of fatty acid hydroperoxides by human parotid saliva

Arachidonic acid hydroperoxide (15-hydroperoxyeicosatetraenoic acid; 15-HPETE) was introduced into human parotid saliva and incubated at 37°C. Straight phase high-performance liquid chromatography analysis of the reaction mixture showed that 15-HPETE was detoxified to its reduced form, 15-hydroxyeicosatetraenoic acid, in the presence of glutathione. Therefore, it is concluded that human parotid saliva possesses fatty acid hydroperoxide-reducing ability. However, its effectiveness was found to be lower than that of blood plasma.     

Detection and measurement of hydroperoxides by near infrared spectrophotometry

Summary Near-infrared spectra have been measured on a group of hydroperoxides of fatty acid esters and related substances. Only those substances having an −OOH group were found to absorb at 1.46 and 2.07 μ. Dialkyl peroxides and ozonized unsaturated substances had no such maxima in their near infrared spectra although they had high iodometric peroxide values. In a study of the thermal decomposition of methyl oleate hydroperoxide and a study of the autoxidation of methyl linoleate, the intensity of absorption at 1.46 and 2.07 μ paralleled the iodometric peroxide value. This work was aided by grants-in-aid from the Hormel Foundation, the Atomic Energy Commission (Contract AT-11-1-108), the Office of Naval Research (Contract N8 onr66218), the National Live Stock and Meat Board, and the National Diary Council. Hormel Institute Publication No. 170.     

Quantitative determination of methyl octadecadienoate hydroperoxides by infrared spectroscopy

The determination of peroxide values has been used for many years to estimate the concentration of the -OOH group in hydroperoxides formed during autoxidation of unsaturated fatty acid esters. Generally a considerable amount of a sample is required for this determination. The thiobarbituric acid test for estimating oxidative deterioration in various fats has also been used but mainly to determine malonaldehyde in an oxidized sample. From the IR spectra of fatty acid methyl esters, -OOH groups can be detected, but not estimated in general, because of association of the -OOH groups by hydrogen bonds. The present study shows that the -OOH group, even in small amounts of intact methyl octadecadienoate hydroperoxides, can be quantitatively determined by IR spectroscopy using a 1 cm cell (100 times the depth of usual cells) and a solution usually of concentration 1:100. Thus, a new quantative determination method for the -OOH group by IR spectroscopy has been developed. Presented at Ninth ISF Congress, Rotterdam, September 1968.     

Transformation of fatty acid hydroperoxides by alkali and characterization of products

It has previously been determined that (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid was mainly converted into (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid by 5 N KHO with preservation of the stereochemistry of the reactant [Simpson, T.D., and Gardner, H.W. (1993)Lipids 28, 325–330]. In addition, about 20–25% of the reactant was converted into several unknown by-products. In the present work it was confirmed that the stereochemistry was conserved during the hydroperoxy-diene to hydroxydiene transformation, but also, novel by-products were identified. It was found that after only 40 min reaction (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid accumulated to as much as 7% of the total. Later, (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid began to disappear, and several other compounds continued to increase in yield. Two of these compounds, 2-butyl-3,5-tetradecadienedioic acid and 2-butyl-4-hydroxy-5-tetradecenedioic acid, were shown to originate from (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid, and they accumulated up to 2–3% each after 4 to 6 h. Some other lesser products included 11-hydroxy-9,12-heptadecadienoic acid, 3-hydroxy-4-tridecenedioic acid, 13-oxo-9,11-octadecadienoic acid and 12,13-epoxy-11-hydroxy-9-octadecenoic acid. Except for the latter two, most or all of the compounds could have originated from Favorskii rearrangement of the early product, (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid, through a cyclopropanone intermediate.     

Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits <0.5 pmol for cholesterol hydroperoxides and <50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm² (which reduced clonally assessed survival by 30%) showed 12–15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (<12 min) were assigned to the following species by virtue of comigration with authentic standards: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH), and 3β-hydroxycholest-5-ene-7α/7β-hydroperoxide (7α/7β-OOH). Formation of 5α-OOH and 6β-OOH (singlet oxygen adducts) was confirmed by subjecting [¹⁴C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18–22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18–22 min peak during Ca²⁺/phospholipase A₂ treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts ofPCOOH and ChOOH at a light fluence that clonally inactivated <10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing. This paper is based on a dissertation submitted by G.J. Bachowski in partial fulfillment of the requirements for a Ph.D. degree in Biochemistry at the Medical College of Wisconsin (Milwaukee, WI).     

Thermal degradation of single methyl oleate hydroperoxides obtained by Photosensitized oxidation

A peroxidation mixture containing methyl 9- and 10-hydroperoxy-trans-octadecenoates (MOHP) was obtained by singlet oxygen oxidation of methyl oleate. The two hydroperoxides were collected by solid phase extraction and purified separately by high-performance liquid chromatography. Identification and single-isomer purity evaluations were carried out by comparing the chromatographic and gas chromatography-mass spectrometry parameters of the corresponding reduced hydroxy derivatives. Each purified MOHP was thermally degraded and new reaction mechanisms were proposed from the identification of the degradation products. Thermal rearrangement of each hydroperoxide isomer involved an allylic 3-carbon intermediate before further degradation steps. The two MOHP isomers obtained from singlet oxygen oxidation produced all eight hydroperoxide isomers by thermal degradation in the condensed phase at high temperature (200°C). This result supports the assumption of singlet oxygen as a promoter of the first steps of oxidation of food lipids and also reconsiders the Khan mechanism.     

Inhibition of lipoxygenase 1 by phosphatidylcholine micelles-bound curcumin

Curcumin (diferuloyl methane) from rhizomes of Curcuma longa L. binds to phosphatidylcholine (PC) micelles. The binding of curcumin with PC micelles was followed by fluorescence measurements. Curcumin emits at 490 nm with an excitation wavelength of 451 nm after binding to PC-mixed micelles stabilized with deoxycholate. Curcumin in aqueous solution does not inhibit dioxygenation of fatty acids by Lipoxygenase 1 (LOX1). But, when bound to PC micelles, it inhibits the oxidation of fatty acids. The present study has shown that 8.6 μM of curcumin bound to the PC micelles is required for 50% inhibition of linoleic acid peroxidation. Lineweaver-Burk plot analysis has indicated that curcumin is a competitive inhibitor of LOX1 with K _l of 1.7 μM for linoleic and 4.3 μM for arachidonic acids, respectively. Based on spectroscopic measurements, we conclude that the inhibition of LOX1 activity by curcumin can be due to binding to active center iron and curcumin after binding to the PC micelles acts as an inhibitor of LOX1.     

Analysis of phosphatidylcholine in soy lecithins by HPLC

A simple and rapid high pressure liquid chromatographic method with RI detector was developed to determine the content of phos-phatidylcholine in soy lecithins. To whom all correspondence should be addressed.     

Thermal degradation of single methyl oleate hydroperoxides obtained by photosensitized oxidation

A peroxidation mixture containing methyl 9- and 10-hydroperoxy-trans-octadecenoates (MOHP) was obtained by singlet oxygen oxidation of methyl oleate. The two hydroperoxides were collected by solid phase extraction and purified separately by high-performance liquid chromatography. Identification and single-isomer purity evaluations were carried out by comparing the chromatographic and gas chromatography-mass spectrometry parameters of the corresponding reduced hydroxy derivatives. Each purified MOHP was thermally degraded and new reaction mechanisms were proposed from the identification of the degradation products. Thermal rearrangement of each hydroperoxide isomer involved an allylic 3-carbon intermediate before further degradation steps. The two MOHP isomers obtained from singlet oxygen oxidation produced all eight hydroperoxide isomers by thermal degradation in the condensed phase at high temperature (200°C). This result supports the assumption of singlet oxygen as a promoter of the first steps of oxidation of food lipids and also reconsiders the Khan mechanism.     

Reduction of polyunsaturated fatty acid hydroperoxides by human brain glutathione peroxidase

Glutathione peroxidase (GSHPx) activity in the normal human brain was investigated using lipid hydroperoxides as substrates. Samples were obtained from autopsied frontal gray matter of 5 normal human males with no known central nervous system (CNS) disease. Aliquots were homogenized in 0.9% NaCl-0.5% Triton X-100, and the supernatant solution, obtained after centrifugation at 105,000 × g, was used for GSHPx assay. Glutathione peroxidase was measured by following the oxidation of NADPH at 340 nm. Hydroperoxides of linoleic, linolenic, gamma linolenic, 11,14 eicosodienoic, homo gamma linolenic, arachidonic, docosotetraenoic and docosohexaenoic acids were prepared and used as substrates. All these hydroperoxides were reduced by the brain GSHPx system, but at different rates. Gamma linolenic and docosotetraenoic hydroperoxides were reduced rapidly, whereas the peroxides of docosohexaenoic and 11,14 eicosodienoic were reduced at the lowest rate. Arachidonic hydroperoxide had the highest affinity for the enzyme and linolenic the lowest. Our results suggest that the brain GSHPx system is capable of reducing hydroperoxides of polyunsaturated fatty acids.