Membrane-stabilizing effect of vitamin E: effect of alpha-tocopherol and its model compounds on fluidity of lecithin liposomes

The effects of vitamin E (alpha-tocopherol) and its model compounds on the fluidity of liposomes composed of dipalmitoylphosphatidylcholin (DPPC) and fatty acids were investigated by the measurement of the fluorescent polarization (P) using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plobe. Although all tocopherols decreased the fluidity of liposomes which was perturbed by the inclusion of an unsaturated fatty acid having more than one double bond, alpha-tocopherol was more effective than the others. The fluidity in arachidonic acid-containing liposomes was decreased most in the presence of alpha-tocopherol and was decreased considerably by the inclusion of model compounds having a side chain at least one isoprene unit or a long straight chain instead of isoprenoid side chain. However, the chromanol with methyl group instead of the above side chain, and phytol, having no chromanol moiety, had no effect. These results show that a structural requirement for a membrane stabilization is to be either the chromanol moiety with methyl groups born on its aromatic ring or a side chain of appropriate length; an isoprenoid side chain of full length or one containing 4'a- and 8'a-methyl groups is not necessarily needed.     

Functional diversity of tocochromanols in plants

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. They accumulate in photooxidative organisms, e.g. in some algae and in plants, where they localize to thylakoid membranes and plastoglobules of chloroplasts. Tocochromanols contain a polar chromanol head group with a long isoprenoid side chain. Depending on the nature of the isoprenoid chain, tocopherols (containing a phytyl chain) or tocotrienols (geranylgeranyl chain) can be distinguished in plants. The tocochromanol biosynthetic pathway has been studied in Arabidopsis and Synechocystis in recent years, and the respective mutants and genes were isolated. Mutant characterization revealed that tocopherol protects lipids in photosynthetic membranes and in seeds against oxidative stress. In addition to its antioxidant characteristics, tocopherol was shown be involved in non-antioxidant functions such as primary carbohydrate metabolism. A considerable proportion of tocopherol is synthesized from free phytol suggesting that excess amounts of phytol released from chlorophyll breakdown during stress or senescence might be deposited in the form of tocopherol in chloroplasts.     

Separation and characteristics of the components of the antibiotic virenomycin

The composition of virenomycin, a new antitumor antibiotic was studied. Two components V and M were detected with high resolution liquid chromatography and thin layer chromatography on siluphol (Czechoslovakia) and silica gel (Merk, BRD). A preparative method for separation of the antibiotic components with the use of chromatography on columns with silica gel was developed. Biological and physicochemical properties of separate components were studied to show that they significantly differed by their antibacterial action in vitro: virenomycin V was 2 to 4 times more active than virenomycin M against a number of microbes. The physicochemical properties of the components are similar. It was shown with mass spectrometry that the molecular weight of virenomycin is 12 units higher than that of virenomycin M. The PMR spectra showed that this difference is due to the presence of a vinyl group in the chromophore moiety of the virenomycin V molecule and a methyl group at the similar site of the virenomycin M molecule.     

Antioxidant Action of 2,2,4,6-Tetra-Substituted 2,3-Dihydro-5-hydroxybenzofuran Against Lipid Peroxidation: Effects of Substituents and Side Chain

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di- tert -butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di- tert -butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), &#102 -tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of &#102 -tocopherol on the efficacy of radical scavenging within the membranes. Di- tert -butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert -butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.     

Antioxidant action of 2,2,4,6-tetra-substituted 2,3-dihydro-5-hydroxybenzofuran against lipid peroxidation: effects of substituents and side chain

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di-tert-butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of alpha-tocopherol on the efficacy of radical scavenging within the membranes. Di-tert-butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert-butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.     

High performance liquid chromatography of phospholipids on modified silica gel

A sensitive method for the separation of phospholipids by a HPLC procedure is described. The separation was carried out on a 30 cm prepacked column with aminopropyl bonded silica under isocratic conditions. The effective separation of major phospholipid classes was carried out for less than 5 min. These chromatographic conditions were used for analysis of natural phospholipid mixtures.     

Preparative liquid chromatography of carbohydrates: mono- and di-saccharides, uronic acids, and related derivatives

The general principles and practical aspects of preparative high-performance liquid chromatography (l.c.) of mono- and di-saccharides, sugars acids, lactones, and N-acetylated amino sugar derivatives are described. Milligram to gram quantities of these carbohydrates were isolated on semi-preparative (0.78 X 30 cm) or preparative (approximately 2.0 X 30 cm) columns packed with aminopropyl silica gel provided better resolution of individual mono- and di-saccharides, but columns of cation-exchange resin had higher capacity and were more durable and economical to use. Preparative, cation-exchange columns were operated at flow rates of less than 5 mL/min and pressures of approximately 1-2 MPa, allowing them to be used on unmodified analytical l.c. systems. Details are given for the efficient packing, use, and care of these columns, and on the effects of column selectivity, packing technique, and sample size on chromatographic resolution. Isolation of naturally occurring sugars from biological sources on a laboratory-packed column is described.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Vitamin E and its function in membranes

Vitamin E is a fat-soluble vitamin. It is comprised of a family of hydrocarbon compounds characterised by a chromanol ring with a phytol side chain referred to as tocopherols and tocotrienols. Tocopherols possess a saturated phytol side chain whereas the side chain of tocotrienols have three unsaturated residues. Isomers of these compounds are distinguished by the number and arrangement of methyl substituents attached to the chromanol ring. The predominant isomer found in the body is alpha-tocopherol, which has three methyl groups in addition to the hydroxyl group attached to the benzene ring. The diet of animals is comprised of different proportions of tocopherol isomers and specific alpha-tocopherol-binding proteins are responsible for retention of this isomer in the cells and tissues of the body. Because of the lipophilic properties of the vitamin it partitions into lipid storage organelles and cell membranes. It is, therefore, widely distributed in throughout the body. Subcellular distribution of alpha-tocopherol is not uniform with lysosomes being particularly enriched in the vitamin compared to other subcellular membranes. Vitamin E is believed to be involved in a variety of physiological and biochemical functions. The molecular mechanism of these functions is believed to be mediated by either the antioxidant action of the vitamin or by its action as a membrane stabiliser. alpha-Tocopherol is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions. The unpaired electron of the tocopheroxyl radical thus formed tends to be delocalised rendering the radical more stable. The radical form may be converted back to alpha-tocopherol in redox cycle reactions involving coenzyme Q. The regeneration of alpha-tocopherol from its tocopheroxyloxyl radical greatly enhances the turnover efficiency of alpha-tocopherol in its role as a lipid antioxidant. Vitamin E forms complexes with the lysophospholipids and free fatty acids liberated by the action of membrane lipid hydrolysis. Both these products form 1:1 stoichiometric complexes with vitamin E and as a consequence the overall balance of hydrophobic:hydrophillic affinity within the membrane is restored. In this way, vitamin E is thought to negate the detergent-like properties of the hydrolytic products that would otherwise disrupt membrane stability. The location and arrangement of vitamin E in biological membranes is presently unknown. There is, however, a considerable body of information available from studies of model membrane systems consisting of phospholipids dispersed in aqueous systems. From such studies using a variety of biophysical methods, it has been shown that alpha-tocopherol intercalates into phospholipid bilayers with the long axis of the molecule oriented parallel to the lipid hydrocarbon chains. The molecule is able to rotate about its long axis and diffuse laterally within fluid lipid bilayers. The vitamin does not distribute randomly throughout phospholipid bilayers but forms complexes of defined stoichiometry which coexist with bilayers of pure phospholipid. alpha-Tocopherol preferentially forms complexes with phosphatidylethanolamines rather than phosphatidylcholines, and such complexes more readily form nonlamellar structures. The fact that alpha-tocopherol does not distribute randomly throughout bilayers of phospholipid and tends to form nonbilayer complexes with phosphatidylethanolamines would be expected to reduce the efficiency of the vitamin in its action as a lipid antioxidant and to destabilise rather than stabilise membranes. The apparent disparity between putative functions of vitamin E in biological membranes and the behaviour in model membranes will need to be reconciled.     

Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase

Human cytochrome P450 4F2 (CYP4F2) catalyzes the initial omega-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is, to date, the only enzyme shown to metabolize vitamin E. The objective of this study was to characterize this activity, particularly the influence of key features of tocochromanol substrate structure. The influence of the number and positions of methyl groups on the chromanol ring, and of stereochemistry and saturation of the side chain, were explored using HepG2 cultures and microsomal reaction systems. Human liver microsomes and microsomes selectively expressing recombinant human CYP4F2 exhibited substrate activity patterns similar to those of HepG2 cells. Although activity was strongly associated with substrate accumulation by cells or microsomes, substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration. Methylation at C5 of the chromanol ring was associated with markedly low activity. Tocotrienols exhibited much higher Vmax values than their tocopherol counterparts. Side chain stereochemistry had no effect on omega-hydroxylation of alpha-tocopherol (alpha-TOH) by any system. Kinetic analysis of microsomal CYP4F2 activity revealed Michaelis-Menten kinetics for alpha-TOH but allosteric cooperativity for other vitamers, especially tocotrienols. Additionally, alpha-TOH was a positive effector of omega-hydroxylation of other vitamers. These results indicate that CYP4F2-mediated tocopherol-omega-hydroxylation is a central feature underlying the different biological half-lives, and therefore biopotencies, of the tocopherols and tocotrienols.     

An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates

An improved, HPLC with electrochemical detection method for the isocratic separation and determination of amino acids from post-mortem brain tissue and from microdialysates of awake-behaving animals is described. Optimal conditions that maximize stability, resolution, and sensitivity were determined for the pre-column derivatization of amino acids using o-phthalaldehyde and B-mercaptoethanol. Ten different amino acids including aspartate, glutamate, taurine, tyrosine and GABA were effectively resolved within 18 min. Tissue measurements from caudate, globus pallidus and substantia nigra showed regional variations in amino acid content. Microdialysis of the striatum yielded significant amounts of all amino acids examined, including GABA, from only 25 microliter of perfusate.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

Enantioseparation of dipeptides and tripeptides by micro-HPLC comparing teicoplanin and teicoplanin aglycone as chiral selectors

Chiral separation of glycyl- and diastereomeric dipeptides and tripeptides was performed by micro-HPLC using macrocyclic antibiotics as chiral selectors. Teicoplanin was compared with teicoplanin aglycone (TAG) regarding selectivity, efficiency and separation time. The stationary phases are based on teicoplanin and TAG chemically bonded to 3.5 mum silica gel. The material was packed into 10 cm x 1 mm stainless steel microcolumns. Different mobile phases were checked using the reversed phase mode. Both teicoplanin and TAG were found to show good chiral separation ability for dipeptides. Glycyl-dipeptides were baseline resolved and most of the diastereomeric dipeptides and tripeptides were separated into their four isomers. In this study, teicoplanin was found to be advantageous compared to TAG regarding separation time, although TAG showed the higher resolution power. Baseline resolution for some glycyl-dipeptides was obtained within 3 min, diastereomeric dipeptides were resolved in 7 min. This method was also shown to be applicable for enantiomer purity control.     

Normal- and reverse-phase HPLC separations of fluorescent (NBD) lipids

We have developed two high-performance liquid chromatography methods for separating a number of fluorescent 4-nitrobenzo-2-oxa-1,3-diazole (NBD) analogs of glycerolipids and sphingolipids. Samples of fluorescent lipid analogs containing NBD-aminocaproyl (C6-NBD) or NBD-aminododecanoyl (C12-NBD) acyl chains were synthesized and analyzed by the following HPLC methods. An isocratic normal-phase method permitted resolution of a mixture of the 1,2-(palmitoyl, C6-NBD)-analogs of triacylglycerol, diacylglycerol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in less than 10 min, while a mixture of the (C6-NBD)-labeled analogs of ceramide, glucocerebroside, and sphingomyelin was separated in approximately 15 min. This method also detected various (C6-NBD)-phosphatidylcholine and -phosphatidylethanolamine molecules which differed only in their nonfluorescent acyl (oleoyl or palmitoyl) chains, and readily separated nonfluorescent dipalmitoylphosphatidylcholine from both (C6-NBD)- and (C12-NBD)-phosphatidylcholine derivatives. An isocratic reverse-phase system permitted separation of isomers of fluorescent phosphatidylcholine, -ethanolamine, -glycerol, -inositol, -serine, and phosphatidic acid in which the NBD-fatty acid was present in either the sn-1 or sn-2 position of the glycerol backbone.     

Membrane stabilization of vitamin E; interactions of alpha-tocopherol with phospholipids in bilayer liposomes

13C Spin-lattice relaxation times (T1) of 13C-labeled alpha-tocopherol in three kinds of liposomes varying in their contents of arachidoyl residues have been measured by 13C-NMR spectroscopy. On the basis of T1 values, it is proved that the segmental motion of isoprenoid side chain of alpha-tocopherol tends to increase with an increase in the distance from the chromanol moiety, and that three methyl groups attached on the aromatic ring, have some affinity to unsaturated fatty acid residues rather than those of the isoprenoid side chain. These results are incompatible with the hypothesis of Diplock et al. (1) which 4'a- and 8'a-methyl groups of isoprenoid side chain are fitted in the Z-pockets of arachidoyl chain of polyunsaturated lipids in membrane.     

Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors

Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. The four forms of tocopherols (??-, ??-, ??- and ??-tocopherols) consist of a polar chromanol ring and lipophilic prenyl chain with differences in the position and number of methyl groups. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. The regulation of tocopherol biosynthesis in photosynthetic organisms occurs, at least partially, at the level of key enzymes as such including p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), homogentisate phytyltransferase (HPT, EC 2.5.1.-), tocopherol cyclase (TC, EC 5.4.99.-), and two methyltransferases. Tocopherol biosynthesis changes during plant development and in response toward different stresses induced by high-intensity light, drought, high salinity, heavy metals, and chilling. It is supposed that scavenging of lipid peroxy radicals and quenching of singlet oxygen are the main functions of tocopherols in photosynthetic organisms. The antioxidant action of tocopherols is related to the formation of tocopherol quinone and its following recycling or degradation. However, until now, the mechanisms of tocopherol degradation in plants have not been established in detail. This review focuses on mechanisms of tocopherols biosynthesis and its regulation in photosynthetic organisms. In addition, available information on tocopherol degradation is summarized.     

Preparation of enantiopure Wieland-Miescher ketone and derivatives by the MalphaNP acid method: substituent effect on the HPLC separation

Enantiopure Wieland-Miescher ketone (4, W-M ketone) and derivatives were prepared by the enantioresolution with 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1). Various racemic derivatives of 4 were esterified with acid (S)-(+)-1 yielding diastereomeric MalphaNP esters, which were separated by HPLC on silica gel. It was clarified that the HPLC separation of diastereomers depended on the substituent of the derivatives, leading to the working hypothesis that MalphaNP acid esters of alcohols with less polar and more bulky aliphatic substituents are more effectively separated. The best separation was obtained in the case of tert-butyldimethylsilyl (TBDMS) ether derivative (12a/12b): separation factor alpha=1.80, and resolution factor, Rs=1.30. The (1)H NMR spectra of separated MalphaNP esters showed anomalously large magnetic anisotropy effects, from which their absolute configurations were determined. Solvolysis or reduction of the separated MalphaNP esters yielded alcohols, which were converted to enantiopure W-M ketones 4. The results thus provided another route for preparation of enantiopure ketones (8aR)-(-)-4 and (8aS)-(+)-4.     

A rapid, isocratic method for phospholipid separation by high-performance liquid chromatography

A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.     

Separation and structural analysis of vinyl- and isovinyl-azobilirubin derivatives

The coupling reaction of bilirubin with the diazonium salts of ethyl anthranilate or of aniline yields two isomeric azopigments. These can be separated by t.l.c. as their methyl esters. The mass spectra of each pair of azopigments are very similar, showing that they are isomers. Proton-magnetic-resonance spectrometric studies show that they differ in the positions of the substituents on the pyrrolenone end ring; in one compound the methyl and vinyl groups are interposed compared with the other compound. These azo compounds were used as reference standards for determination of the site of conjugation in bilirubin monoglucuronide prepared enzymically. Analysis showed that conjugation occurs at the carboxyethyl side chain of both sides of the bilirubin molecule. During the preparation of the ethyl anthranilate reference compounds a series of minor azopigments were isolated by t.l.c. Analysis of the mass spectra of many of these showed that three side reactions can occur: (1) methylation of the imide carbonyl group; (2) addition of methanol or water to the vinyl substituent; (3) transmethylation of the ethoxycarbonyl group.     

The effect of vitamin E analogues and long hydrocarbon chain compounds on calcium-induced muscle damage. A novel role for α-tocopherol?

Previous studies have demonstrated that supplemental α-tocopherol inhibited calcium-induced cytosolic enzyme efflux from normal rat skeletal muscles incubated in vitro and suggested that the protective action was mediated by the phytyl chain of α-tocopherol [1]. In order to investigate this further a number of hydrocarbon chain analogues of tocopherol (7.8-dimethyl tocol, 5,7-dimethyl tocol, tocol, α-tocotrienol, α-tocopherol [10], vitamin K1, vitamin K1 [10], vitamin K1 diacetate, vitamin K2 [20], phytyl ubiquinone and retinol) were tested for any ability to inhibit calcium ionophore, A23187, induced creatine kinase (CK) enzyme efflux. Some compounds were found to be very effective inhibitors and comparison of their structures and ability and to inhibit TBARS production in muscle homogenates revealed that the effects did not appear related to antioxidant capacity or chromanol methyl groups, but rather the length and structure of the hydrocarbon chain was the important mediator of the effects seen.