Identification of adduct radiolysis products from ethyl palmitate and ethyl oleate

Radiolysis induced adduct products have been separated and identi-fied from irradiated ethyl palmitate, ethyl α-d₂-palmitate and ethyl oleate. In the saturated compounds, adduct formation was observed mainly at the position α to the carbonyl group. The three major adduct products identified in irradiated ethyl palmitate were ethyl α-tetradecylpalmitate, ethyl α-pentadecylpalmitate and the α,α′- dimer of ethyl palmitate. Corresponding compounds were identified from the irradiated ethyl α-deuteropalmitate. Adduct radiolysis products formed in ethyl oleate were identified as the monoene and diene dimers.     

Identification of adduct radiolysis products from pork fat

Mass spectrometric evidence is given to show the formation of adduct radiolysis products in pork fat. A variety of adduct radiolysis products were identified. Only the major recombination products are considered and discussed herein. These compounds consist of triglyceride dimers, propanedioldiester-triglyceride adducts, propanedioldiester dimers and branched alkyl substituted triglycerides.     

Addition compounds of oxidizing tocopherol and soybean lecithin

Evidence is presented for the formation of addition compounds from tocopherol and soybean lecithin, when adsorbed in monolayer on silica from a mixed solution in chloroform and oxidized at 80 C for 72 hr. Tocopherol was present at 7 mol % of the lecithin in the monolayer. The compounds produced are analogous to linoleic acid-tocopherol adducts previously reported by us. UV spectra of the mixed lecithin and tocopherol monolayers while in silica gel slurry in a solvent were obtained by the method previously reported by us for oxidized linoleic acid and tocopherol monolayers. The monolayer spectra show no evidence for tocopherol dimer. A minor amount of quinone is indicated, but the spectrum has a maximum at 287 nm with no other major peaks. The proposed addition compounds have been characterized by transesterification and hydrogenation, UV, IR, thin layer chromatography and gas chromatography retention behavior, mass spectrometry and elemental analysis. IR spectra of twice-chromatographed addition compounds show specific lecithin absorption bands (5.75 μ; 10.3 μ) together with much enhanced 3.4–3.5, 6.8, 7.25 and 9.14 μ peaks, the latter two being characteristic of α-tocopherol. The UV spectrum of the adducts showed λ _max ^CHCl 3 287 nm, with shoulders at 276 and 300 nm. Hydrogenation removed the peaks at 287 and 276 nm, leaving a peak at 300 nm similar to that of the linoleic acid-tocopherol adduct. Esters of the adducts prepared by transesterification and hydrogenation were similar by all our chemical, chromatographic and spectral tests to the previously characterized esters of the linoleic acid-tocopherol adduct. Paper No. TP-1225 in the U.S. Army Natick Labs. Series.     

Mass spectrometry of triglycerides: II. Specifically deuterated triglycerides and elucidation of fragmentation mechanisms

Deuterium labeled monoacid triglycerides were synthesized and their mass spectra were measured. The spectra provided further support for proposed (1) structures of principal ions, knowledge about the formation of [M-18]⁺, the interexchange of hydrogen atoms between 2 and the 5,6 or 7 positions and the explusion of part of the alkyl chain. Presented in part at the AOCS Meeting, New Orleans, April 1970. Part VI of a series on Mass Spectrometry of Lipids. For V see Lipids 5:861–868 (1970).     

Mass spectrometric analysis of mono- and dialkyl ethers of diols

Mass spectra of homologous series of long chain mono- and dialkyl ethers of ethanediol and propanediols were measured and general patterns of fragmentation were established. Both classes of diol lipids produce ions which are characteristic for the alkoxy moieties as well as ions which are typical of the constituent short chain diols. Prominent ions are formed by cleavages α and β to the ether oxygen and by rearrangement of one or two hydrogens and concurrent fission. High resolution mass spectrometry and deuterium labeling techniques were used to verify the composition of ions and to substantiate fragmentation mechanisms. Part VIII in the series “Naturally Occurring Diol Lipids” (part VII is Reference 1) and part IX in the series “Mass Spectrometry of Lipids” (part VIII is Reference 1).     

Direct evidence of oxygen evolution from Li1+ x (Ni1/3Mn1/3Co1/3)1? x O2 at high potentials

Li_1+x (Ni_1/3Mn_1/3Co_1/3)_1−x O₂ (NMC) oxides are among the most promising positive electrode materials for future lithium–ion batteries. A voltage “plateau” was observed on the first galvanostatic charging curve of NMC in the extended voltage region positive to 4.5 V vs. Li/Li⁺ for compounds with x > 0 (overlithiated compounds). Differences were observed in the cycling stability of the overlithiated and stoichiometric (x = 0) NMC oxides in this potential region. A differential plot of the charge vs. potential profile in the first cycle revealed that, for the overlithiated compounds, a large irreversible oxidative peak arises positive to 4.5 V vs. Li/Li⁺, while in the same potential region only a small peak due to the electrolyte oxidation is detected for the stoichiometric material. Differential Electrochemical Mass Spectrometry (DEMS) was used to investigate the high voltage region for both compounds and experimental evidence for oxygen evolution was provided for the overlithiated compounds at potentials positive to 4.5 V vs. Li/Li⁺. No oxygen evolution was detected for the stoichiometric compound.     

Double-bond location in long-chain polyunsaturated fatty acids by chemical lonization-mass spectrometry

For determination of the double-bond position in polyunsaturated C_24–30 fatty acids from marine organisms, methoxy derivatives were prepared. Diagnostic mass spectral fragment as well as molecular ion intensities were obtained by adjusting the ion source optics in the presence of ammonia at a lower source pressure than used conventionally. A lower detection limit was observed compared to conventional methane chemical ionization, which is a more favorable condition for capillary gas chromatography. Analysis of fatty acids from the spongeCalyx niceaensis showed the double-bond position of 8 unsaturated fatty acids, including two new ones. In addition, structural proof is provided for the presence of a new cyclopropane-containing fatty acid: 19,20-methylene-hexacosanoic acid. “Mass Spectrometry in Structural and Stereochemical Problems 262.” For preceding paper in this series, see Patterson, D.G., Haley, M.J., Midgley, J., and Djerassi, C., Org. Mass. Spectrom., submitted for publication. Recipient of a travel grant from the Queen Wilhelmina Fund, The Netherlands Cancer Foundation     

Degradation of MMEA at absorber and stripper conditions

This study examines the degradation of N-methylethanolamine (MMEA) under different experimental conditions. Thermal degradation with and without CO₂, and oxidative degradation are investigated. Samples of the degraded solution were taken at regular intervals and analyzed. The percentage of amine loss was determined by Liquid Chromatography–Mass Spectrometry (LC–MS) while the degradation compounds were identified and quantified by Gas Chromatography–Mass Spectrometry (GC–MS). MMEA degradation at absorber and stripper conditions is compared with previous work on 2-ethanolamine (MEA). Degradation mechanisms are proposed and discussed in order to understand the differences compared to MEA.     

Characterization of toxic compound in thermally oxidized oil

In preliminary experiments, soybean oil was heated at 275 C for 12 hr in the presence of N₂ or air. Feeding studies with rats showed that the oil heated in the presence of air, oxidatively polymerized oil, retarded weight gain more than that heated with N₂, thermally polymerized oil. Oxidatively polymerized oil was fractionated with silicic acid column chromatography, and the fraction eluted with ether (fraction III) proved to be most toxic to mice. For chemical studies combined with bioassays, additional oxidatively polymerized oil was prepared by aeration at 185 C for 90 hr, and the product was fractionated by silicic acid column chromatography. The material originally eluted with ether (fraction III) was eluted in stages, and fraction IIIc proved to be most toxic in a mouse bioassay. IR, UV and NMR analyses did not indicate the presence of C−O−O−C or C−O−C linkages or aldehyde groups or aromatic compounds. Fraction IIIc was converted to its methyl esters and molecularly distilled to yield two fractions: one containing unpolymerized fatty acid esters and one giving evidence of more functional groups per molecule and of dimeric material. This “dimeric” fraction was more toxic to mice. IR analysis of this fraction revealed carbonyl groups, and NMR showed several functional groups on alkyl chains. Treatment with sodium borohydride and with hydroiodic acid revealed no C−O−O−C and no C−O−C linkages. Mass spectrography showed peaks at mass 586 and 293; in the reduced fraction a peak also occurred at mass 143, suggestive of cleavage of some of the chains. It is tentatively concluded that a highly toxic material formed in oil heated in air is a dimer of triglyceride molecules. One of seven papers presented in the symposium “Biological Significance of Autoxidized and Polymerized Oils,” JOCS-AOCS Joint Meeting, Los Angeles, April 1972.     

Pyrolysis chromatography of lipids. I. Mass spectrometric identification of pyrolysis products of hydrocarbons

The products of pyrolysis at 600C of normal paraffins C₁₀−C₁₈, 2-methyl octadecane, 4-methyl octadecane, 6-methyl octadecane, cyclohexyl decane, cyclopentyl decane, 2,2,4,4,6,8,8-heptamethyl nonane, pristane and phytane were studied by means of a pyrolysis gas chromatograph directly coupled to a mass spectrometer. n-Paraffins yield a homologous series of n-olefins. Branched paraffins yield two homologous series, one of n-olefins and one of branched olefins. The n-olefin corresponding to the position of the branch is not formed. Interpretation of pyrolograms is similar in principle to the interpretation of mass spectra. Presented at the Symposium on “Mass Spectrometry of Lipids”, AOCS Cincinnati, October 1965.     

Mass spectrometry of triglycerides: I. Structural effects

Mass spectra of several triglycerides of specific structure or with specific deuterium labeling have been measured with a low resolution mass spectrometer. With saturated triglycerides the abundances of ions characteristic of the component acids, [M-RCO₂]⁺, increase with increasing chain length, and [M-RCO₂CH₂]⁺ decrease with increasing chain length. Unsaturation in the acyl moiety causes the abundant formation of [RCO-1]⁺. Structures have been suggested for a number of the main peaks obtained from saturated triglycerides, and high resolution spectra of one triglyceride agree with the postulated structures. The peaks, [RCO+74]⁺, [RCO+115]⁺ and [RCO+128+14n]⁺, represent structures which contain the glyceryl portion of the triglyceride, since in case of the replacement of its hydrogens with deuteriums, these peaks are shifted accordingly. Evidence which indicates the possibility of determining the location of unsaturation by the interruption of homology of the [RCO+128+14n]⁺ series, brought about by the addition of deuterium to the unsaturated linkages, is introduced. Further evidence is also presented, which indicates that the [M-RCO₂CH₂]⁺ ions arise from the positions 1 and 3 and, in agreement with earlier studies from other laboratories, it is thus possible to identify the acyl groups attached to the 1 and 2 positions of the glyceryl moiety. Presented in part at the AOCS Meeting, New Orleans, April 1970. Part V of a series on Mass Spectrometry of Lipids. For IV see Lipids 4:421–427 (1969).     

Separation of α-tocopherol and its oxidation products by high performance liquid chromatography

A very sensitive high performance liquid chromatographic (HPLC) method was developed for the separation of α-tocopherol (α-T) and its five oxidation products: α-tocopheryl quinone (TQ), dimer (D), dihydroxy dimer (DHD), trimer (T) and 9-methoxy-α-tocopherone commonly called α-tocopheroxide (TO). The separation was achieved on a normal-phase silica-based column (Ultrasphere-Si), using a mobile phase of hexane/chloroform/isopropanol (95∶4.5∶0.5, v/v/v) at a flow rate of 0.4 ml/min, and the eluants were monitored simultaneously at their maximum absorptions using a variable-wavelength UV detector. The minmum detection limit is 0.01 μg for α-T, TQ and TO, 0.05 μg for DHD and D, and 0.1 μg for T/injection. This normal-phase method has the combined advantages of being very sensitive, fast and capable of separating all six compounds at the same time.     

Polymerization of drying oils. V. Further observations on the reaction of unsaturated dibasic anhydrides with methyl linoleate

Summary Adducts of methyl linoleate with itaconic and citraconic anhydrides have been prepared. Each adduct was separated into monomeric and polymeric components. With ethylene diamine, the itaconic and citraconic polymeric adducts gave gelled products at 170° in one hour and 3.5 hours, respectively. The adduct of citraconic anhydride and monomeric distillate gave a tough, tacky resin which did not gel during a reaction time of 20 hours at 170°–180°. The effect of dimethylaniline on the condensation of methyl linoleate and maleic anhydride was studied. Increasing amounts of dimethylaniline were found to promote the formation of a heteropolymer and to decrease the yield of liquid adduct. Presented before the American Oil Chemists’ Society Meeting, May 10–12, 1949, New Orleans, Louisiana. Trainee sponsored by the Government of India, 1947–1948. One of the Laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Oxidation products of α-tocopherol formed in autoxidizing methyl linoleate

The oxidation products of¹⁴C-α-tocopherol formed by heating with methyl linoleate in an air atmosphere at 60 C or 100 C were investigated. The products included a dimer, trimer and dihydroxy dimer, α-tocopherol quinone and unidentified degradation compounds. The dimer and trimer constituted the major products present after heating for 70 hr at 60 C. After 70 hr at 100 C most of the¹⁴C-α-tocopherol had been converted to degradation products; part of the¹⁴C originallt present in the 5-methyl group was recovered as¹⁴CO₂ and¹⁴CH₃OH.     

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

We previously demonstrated by X-ray crystallography and electrospraymass spectrometry that D52E mutant hen lysozyme formed a covalentenzyme–substrate adduct on reaction with N-acetylglucosamineoligomer. This observation indicates that D52E lysozyme mayacquire a catalytic pathway via a covalent adduct. To explainthis pathway, the formation and hydrolysis reactions of thecovalent adduct were investigated. Kinetic analysis indicatedthat the hydrolysis step was the rate-limiting step, 60-foldslower than the formation reaction. In the formation reaction,the pH dependence was bell-shaped, which was plausibly explainedby the functions of the two catalytic pK_as of Glu35 and Glu52.On the other hand, the pH dependence in the hydrolysis was sigmoidalwith a transition at pH 4.5, which was identical with the experimentallydetermined pK_a of Glu35 in the covalent adduct, indicating thatGlu35 functions as a general base to hydrolyze the adduct. Toimprove the turnover rate of D52E lysozyme, the mutation ofN46D was designed and introduced to D52E lysozyme. This mutationreduced the activation energy in the hydrolysis reaction ofthe covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C butdid not affect the formation reaction. Our data may providea useful approach to understanding the precise mechanism ofthe function of natural glycosidases, which catalyze via a covalentadduct.     

Heated fat,vitamin E and vascular eicosanoids

A semisynthetic diet containing adequate amounts of vitamin E and 10% (w/w) of a mixture of polyunsaturated oils subjected to heating and characterized by elevated indexes of thermal alteration (polar component, dimer triglyceride, altered triglyceride contents and reduced α-tocopherol levels) was fed to growing male rats for a period of eight weeks. It resulted in a selective alteration of the production of vascular eicosanoids (elevation of platelet thromboxane formation and decrease of vascular prostacyclin release) compared to the values found in rats fed a diet containing a fresh mixture of polyunsaturated oils. Major nutritional parameters, plasma lipids and the fatty acid profiles of plasma, liver and heart lipids were not different in the two groups of animals. Supplementation of an excess vitamin E (300 mg/kg) to the diet containing heated fat neutralized the adverse effects of heated fat on vascular eicosanoid production.     

Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking

In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a ‘back–to–back’dimer that occurs in different crystal forms of wild–typeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wild–type, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof cross–linked dimers had thermal factors somewhat lowerthan wild–type, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent cross–Unking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the ‘back’of the molecule and formation of the cross–linked dimerappears to leave the active sites completely unobstructed. Nevertheless,the cross–linked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate     

Mass spectrometric analysis of long chain alk-1-enyl ether esters and alkyl ether esters of diols

The mass spectra of homologous series of long chain alk-1-enyl ether esters and alkyl ether esters of short chain diols were determined, and general patterns of fragmentation were established. Both types of diol lipids yielded ions characteristic of the alkoxy or alk-1-enyloxy moiety and the acyl moiety, as well as ions indicative of the constituent short chain diol. Prominent ions were formed from both types of ether esters due to the loss of the alkoxy or alk-1-enyloxy moieties giving rise to ions for which cyclic structures are proposed. High resolution mass spectrometry and deuterium labeling techniques were used to verify the composition of ions and to substantiate fragmentation mechanisms. This is part VII in the series “Naturally Occurring Diol Lipids” (part VI is Reference 1) and part VIII in the series “Mass Spectrometry of Lipids” (part VII is Reference 23).     

The urinary excretion of thiobarbituric acid reactive substances and malondialdehyde by normal adult males after consuming a diet containing salmon

In this study we investigated the output of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA), as thiobarbituric acid (TBA)-MDA adduct, in the urine from subjects eating a diet in which the only source of n−3 long-chain, polyunsaturated fatty acids was fresh salmon. Nine healthy men, ages 30–65, were confined in the United States Department of Agriculture Western Human Nutrition Research Center, San Francisco, CA, for 100 d; food intake and exercise levels were controlled. All subjects were placed on a stabilization diet (StD) for 20 d, then six were fed the salmon diet for 40 d. The others remained on the StD. The groups switched diets for the last 40 d. Both diets were isocaloric (16% protein, 54% CHO and 30% fat by energy %). The salmon diet contained 7.5% of calories from n−6 fatty acids (FAs) and 2% from n−3 FAs, primarily eicosapentaenoic acid and docosahexaenoic acid in a 50∶60 ratio, while the StD contained 7.5% from n−6 FAs and <0.3% n−3 FAs (with presumably no significant amounts of C₂₀ or C₂₂ n−3 FAs). Twenty-four hour urinary output was collected, and 2% 3−d pool samples prepared for analysis of urinary TBARS and the TBA-MDA adduct. The total urinary output of each individual varied considerably, and on a daily basis the concentration of autoxidation products in an individual's urine varied also. However, the mean daily output (in μmoles TBA-MDA equivalents/day) at the end of the salmon diet feeding period was significantly greater (7.05±1.33 TBARS,P<0.05; and 7.07±1.73 TBA-MDA adduct,P<0.01) compared to when the subjects were eating the StD (5.65±1.09 TBARS and 4.65±0.76 TBA-MDA adduct). When the TBARS and TBA-MDA adduct values were normalized relative to creatinine output (in nmoles TBA-MDA equivalents/μmole creatinine), the data achieved even greater statistical significance. The mean output of the group eating the salmon diet was 0.478±0.076 for TBARS (P<0.01) and 0.476±0.082 for the TBA-MDA adduct (P<0.001)vs. 0.345±0.059 for TBARS and 0.283±0.041 for the TBA-MDA adduct when the subjects were consuming the StD. Thus, the consumption of cooked fish may increase one's exposure to MDA and other autoxidation products, compounds that may be carcinogenic or mutagenic.     

N-2-mercaptoethyl amides of fatty acids—A new class of derivatives

Twelve N-2-mercaptoethyl amides have been prepared by reacting 2-aminoethyl mercaptan with a carboxylic acid in refluxing xylene or toluene. All products were well-defined crystalline compounds except for the dimer acid derivatives. Addition of dithiol amides, prepared from dimer acids and 2-aminoethyl mercaptan, to diolefins gave a new class of potential protective coatings, a polyamide of a dimerized fatty acid with a β-thio linkage. Oxidation of the dithiols gave the corresponding disulfides, another new class of compounds, a polyamide with a β-disulfide structure. Film properties have been obtained with both classes of polymers. Air-dried films were soft and tacky, but baking the films improved hardness. Dry-to-touch times of less than 2 hr at 150C and good alkali resistance were obtained. Presented at the AOCS Meeting in Chicago, October, 1964. No. Util. Res. and Dev. Div., ARS, USDA.