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.

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.

Antioxidants, weight change and risk of type 2 diabetes

The incidence of type 2 diabetes has increased rapidly worldwide. Obesity is one of the most important modifiable risk factors of type 2 diabetes: weight gain increases and weight loss decreases the risk. However, the effects of weight fluctuation are unclear. Reactive oxygen species are presumably part of the complicated mechanism for the development of insulin resistance and beta-cell destruction in the pancreas. The association of antioxidants with the risk of incident type 2 diabetes has been studied in longitudinal prospective human studies, but so far there is no clear conclusion about protective effect of dietary or of supplementary antioxidants on diabetes risk. The present study examined 1) weight change and fluctuation as risk factors for incident type 2 diabetes; 2) the association of baseline serum alpha-tocopherol or beta-carotene concentration and dietary intake of antioxidants with the risk of type 2 diabetes; 3) the effect of supplementation with alpha-tocopherol or beta-carotene on the risk of incident type 2 diabetes; and on macrovascular complications and mortality among type 2 diabetics. This investigation was part of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a randomized, double-blind, placebo-controlled prevention trial, which has undertaken to examine the effect of alpha-tocopherol and beta-carotene supplementation on the development of lung cancer, other cancers, and cardiovascular diseases in male smokers aged 50-69 years at baseline. Participants were assigned to receive either 50 mg alpha-tocopherol, 20mg beta-carotene, both, or placebo daily in a 2 x 2 factorial design experiment during 1985-1993. Cases of incident diabetes were identified through a nationwide register of drug reimbursements of the Social Insurance Institution. At baseline 1700 men had a history of diabetes. Among those (n = 27 379) with no diabetes at baseline 305 new cases of type 2 diabetes were recognized during the intervention period and 705 during the whole follow-up to 12.5 years. Weight gain and weight fluctuation measured over a three year period were independent risk factors for subsequent incident type 2 diabetes. Relative risk (RR) was 1.77 (95% confidence interval [CI] 1.44-2.17) for weight gain of at least 4 kg compared to those with a weight change of less than 4 kg. The RR in the highest weight fluctuation quintile compared to the lowest was 1.64 (95% CI 1.24-2.17). Dietary tocopherols and tocotrienols as well as dietary carotenoids, flavonols, flavones and vitamin C were not associated with the risk of type 2 diabetes. Baseline serum alpha-tocopherol and beta-carotene concentrations were not associated with the risk of incident diabetes. Neither alpha-tocopherol nor beta-carotene supplementation affected the risk of diabetes. The relative risks for participants who received alpha-tocopherol compared with nonrecipients and for participants who received beta-carotene compared with nonrecipients were 0.92 (95% CI 0.79-1.07) and 0.99 (95% CI 0.85-1.15), respectively. Furthermore, alpha-tocopherol or beta-carotene supplementation did not affect the risk of macrovascular complications or mortality of diabetic subjects during the 19 years follow-up time. In conclusion, in this study of older middle-aged male smokers, weight gain and weight fluctuation were independent risk factors for type 2 diabetes. Intake of antioxidants or serum alpha-tocopherol or beta-carotene concentrations were not associated with the risk of type 2 diabetes. Supplementation with of alpha-tocopherol or beta-carotene did not prevent type 2 diabetes. Neither did they prevent macrovascular complications, or mortality among diabetic subjects.