Autoxidation of conjugated linoleic acid methyl ester in the presence of α-tocopherol: the hydroperoxide pathway

The autoxidation of conjugated linoleic acid (CLA) is poorly understood in spite of increasing interest in the beneficial biological properties of CLA and growing consumption of CLA-rich foods. In this thesis, the autoxidation reactions of the two major CLA isomers, 9-cis,11-trans-octadecadienoic acid and 10-trans,12-cis-octadecadienoic acid, are investigated. The results contribute to an understanding of the early stages of the autoxidation of CLA methyl ester, and provide for the first time a means of producing and separating intact CLA methyl ester hydroperoxides as well as basic knowledge on lipid hydroperoxides and their hydroxy derivatives. Conjugated diene allylic monohydroperoxides were discovered as primary autoxidation products formed during autoxidation of CLA methyl esters in the presence and absence of α-tocopherol. This established that one of the autoxidation pathways of CLA methyl ester is the hydroperoxide pathway. Hydroperoxides were produced from the two major CLA methyl esters by taking advantage of the effect of α-tocopherol to promote hydroperoxide formation. The hydroperoxides were analysed and separated first as methyl hydroxyoctadecadienoates and then as intact hydroperoxides by HPLC. The isolated products were characterized by UV, GC-MS, and NMR techniques. In the presence of a high amount of α-tocopherol, the autoxidation of CLA methyl ester yields six kinetically-controlled conjugated diene monohydroperoxides and is diastereoselective in favour of one particular geometric isomer as a pair of enantiomers. The primary autoxidation products produced from the two major CLA isomers include new positional isomers of conjugated diene monohydroperoxides, the 8-, 10-, 12-, and 14-hydroperoxyoctadecadienoates. Furthermore, two of these new positional isomers have an unusual structure for a cis,trans lipid hydroperoxide where the allylic methine carbon is adjacent to the cis instead of the usual trans double bond. The 1H and 13C NMR spectra of nine isomeric methyl hydroxyoctadecadienoates and of ten isomeric methyl hydroperoxyoctadecadienoates including the unusual cis,trans hydroperoxides, i.e. Me 8-OOH-9c,11t and Me 14-OOH-10t,12c, were fully assigned with the aid of 2D NMR spectroscopy. The assigned NMR data enabled determination of the effects of the hydroxyl and hydroperoxyl groups on the carbon chemical shifts of CLA isomers, identification of diagnostic signals, and determination of chemical shift differences of the olefinic resonances that may help with the assignment of structure to as yet unknown lipid hydroperoxides either as hydroxy derivatives or as intact hydroperoxides. A mechanism for the hydroperoxide pathway of CLA autoxidation in the presence of a high amount of α-tocopherol was proposed based on the characterized primary products, their relative distribution, and theoretical calculations. This is an important step forward in CLA research, where exact mechanisms for the autoxidation of CLA have not been presented before. Knowledge of these hydroperoxide formation steps is of crucial importance for understanding the subsequent steps and the different pathways of the autoxidation of CLA. Moreover, a deeper understanding of the autoxidation mechanisms is required for ensuring the safety of CLA-rich foods. Knowledge of CLA oxidation and how it differs from the oxidation of nonconjugated polyunsaturated fatty acids may also be the key to understanding the biological mechanisms of CLA activity.

Effect of phenolic-rich plant materials on protein and lipid oxidation reactions

The antioxidant activity of natural plant materials rich in phenolic compounds is being widely investigated for protection of food products sensitive to oxidative reactions. In this thesis plant materials rich in phenolic compounds were studied as possible antioxidants to prevent protein and lipid oxidation reactions in different food matrixes such as pork meat patties and corn oil-in water emulsions. Loss of anthocyanins was also measured during oxidation in corn oil-in-water emulsions. In addition, the impact of plant phenolics on amino acid level was studied using tryptophan as a model compound to elucidate their role in preventing the formation of tryptophan oxidation products. A high-performance liquid chromatography (HPLC) method with ultraviolet and fluorescence detection (UV-FL) was developed that enabled fast investigation of formation of tryptophan derived oxidation products. Byproducts of oilseed processes such as rapeseed (Brassica rapa L.), camelina (Camelina sativa) and soy meal (Glycine max L.) as well as Scots pine bark (Pinus sylvestris) and several reference compounds were shown to act as antioxidants toward both protein and lipid oxidation in cooked pork meat patties. In meat, the antioxidant activity of camelina, rapeseed and soy meal were more pronounced when used in combination with a commercial rosemary extract (Rosmarinus officinalis). Berry phenolics such as black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins showed potent antioxidant activity in corn oil-in-water emulsions toward lipid oxidation with and without β-lactoglobulin. The antioxidant effect was more pronounced in the presence of β-lactoglobulin. The berry phenolics also inhibited the oxidation of tryptophan and cysteine side chains of β-lactoglobulin. The results show that the amino acid side chains were oxidized prior the propagation of lipid oxidation, thereby inhibiting fatty acid scission. In addition, the concentration and color of black currant anthocyanins decreased during the oxidation. Oxidation of tryptophan was investigated in two different oxidation models with hydrogen peroxide (H2O2) and hexanal/FeCl2. Oxidation of tryptophan in both models resulted in oxidation products such as 3a-hydroxypyrroloindole-2-carboxylic acid, dioxindolylalanine, 5-hydroxy-tryptophan, kynurenine, N-formylkynurenine and β-oxindolylalanine. However, formation of tryptamine was only observed in tryptophan oxidized in the presence of H2O2. Pine bark phenolics, black currant anthocyanins, camelina meal phenolics as well as cranberry proanthocyanidins (Vaccinium oxycoccus) provided the best antioxidant effect toward tryptophan and its oxidation products when oxidized with H2O2. The tryptophan modifications formed upon hexanal/FeCl2 treatment were efficiently inhibited by camelina meal followed by rapeseed and soy meal. In contrast, phenolics from raspberry, black currant, and rowanberry (Sorbus aucuparia) acted as weak prooxidants. This thesis contributes to elucidating the effects of natural phenolic compounds as potential antioxidants in order to control and prevent protein and lipid oxidation reactions. Understanding the relationship between phenolic compounds and proteins as well as lipids could lead to the development of new, effective, and multifunctional antioxidant strategies that could be used in food, cosmetic and pharmaceutical applications.

Occurrence and properties of steryl ferulates and glycosides in wheat and rye

Cereal kernels are known to contain a number of minor components that possess beneficial health attributes. In this thesis rye and wheat were studied as sources of steryl ferulates and steryl glycosides and their behaviour in processing were evaluated. Further, enzymatic hydrolysis of these conjugates was studied, as well as the capacity of steryl ferulates to inhibit lipid oxidation at different temperatures. Steryl ferulates were shown to have a strong positive correlation with dietary fibre contents in milling fractions from the outer parts of the kernels obtained from a commercial scale mill. Highest contents of steryl ferulates were found in the bran in both cereals, with the content decreasing once moving towards the inner parts of the kernel. Variation in the contents of steryl ferulates was higher in wheat fractions than rye fractions. Steryl glycosides, on the other hand, had either negative or no correlation with dietary fibre, and the range of the steryl glycoside contents was much narrower than that of steryl ferulates in both cereals. There were significant differences in the sterol compositions of these steryl conjugates when compared with each other or with the total plant sterols in the corresponding fractions. Properties of steryl ferulates and steryl glycosides were evaluated after common processing methods and in enzymatic hydrolysis. Thermal and mechanical processing had only minor or no effects on the contents of steryl conjugates from rye and wheat bran. Enzymatic treatments on the other hand caused some changes, especially in the contents of glycosylated sterols. When steryl ferulates extracted from rye or wheat bran were subjected to enzymatic treatments by steryl esterase, significant differences in the rates of hydrolysis were observed between steryl ferulates from different sources with differing sterol compositions. Further, differences were also observed between enzymes from different sources. Steryl glycosides were shown to be hydrolysed by β-glucosidase (cellobiase) from A. niger, but less with β-glucosidases from other sources. Steryl ferulates showed good antioxidant activity at both moderate and high temperatures. In bulk and emulsion systems of methyl linoleate at 40°C steryl ferulates extracted from rye and wheat bran inhibited hydroperoxide formation much more effectively than synthetic steryl ferulates or those extracted from rice (γ-oryzanol), demonstrating that the sterol composition has an effect on the activity. At cooking (100°C) and frying temperatures (180°C) sitostanyl ferulate was shown to inhibit polymer formation significantly and, especially at 100°C, comparably to α-tocopherol. The rate of antioxidant degradation was slower for sitostanyl ferulate, showing higher heat stability than α-tocopherol. When evaluated as a mixture, no synergistic effect was observed between these two antioxidants. The data presented in this thesis provides information that may henceforth be applied when evaluating the intakes of steryl conjugates from cereal sources, as well as their possible influences as minor bioactive components. Wheat and rye both are good sources of steryl ferulates and steryl glycosides and, especially with steryl ferulates, what may be lost out to some other cereals on quantity is compensated with quality of the sterol composition.

Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

ORP2 is a sterol receptor that regulates cellular lipid metabolism

ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.

Lipid-Containing Icosahedral dsDNA Bacteriophages: Entry, Exit and Structure

In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.