A study of unsaturated fat oxidase systems and their inhibition

Shearer, George (1964) A study of unsaturated fat oxidase systems and their inhibition. PhD thesis, University of Glasgow.

Abstract

Unsaturated fats readily oxidise, giving rise to hydroperoxides, which, in turn, oxidise with formation of a complex mixture of monomeric and polymeric oxy-compound fatty acids such as linolenic, linolenic and arachidonic, and their derivatives, contain methylene-interrupted unsaturated centres which on oxidation form initially conjugated diene hydroperoxide products. The oxidation of these acids may occur autoxidatively or may be catalysed by trace metals, Haematin compounds or by the enzyme lipoxidase. The two former catalysts are non-specific in that, they catalyse the oxidation of unsaturated fats in general, but the action of lipoxidase is specific for compounds containing methylene-interrupted, unsaturated centres. Concurrent with unsaturated fat oxidation, many other substances undergo degradative oxidation. Among such compounds are carotene and vitamin A, The loss of such compounds from stored foods is undesirable, as is unsaturated fat oxidation which produces the unacceptable odours and tastes associated with rancidity. The work described in this thesis is largely concerned with lipoxidase catalysed oxidation of systems consisting of linoleate, linoleate-carotene, or linoleate-vitamin A. Some observations on the autoxidation and haematin catalysis of some of these systems are also included. The systems used were biphasic emulsions of fat in aqueous media. Initial studies, in which linoleic acid was enzymically oxidised by lipoxidase normally present in crude aqueous extracts of defatted soya, confirmed the presence of a lipoperoxidase or hydroperoxide breakdown factor. This factor caused the loss of hydroperoxide content without corresponding loss of diene content from the initial conjugated linoleic acid hydroperoxide product. This factor was shorn to act similarly with methyl linoleate hydroperoxide provided that a sufficiently high initial reaction rate could be induced to cause exhaustion of dissolved oxygen in reaction mixtures. Under anaerobic conditions, the lipoperoxidase factor was shown to break down both linoleic acid hydroperoxide and methyl linoleate hydroperoxide. Which had been produced by lipoxidase oxidation of the respective substrates, but, it could not cause the breakdown of pure trans-trans methyl linoleate hydroperoxide. Haemoglobin caused loss of peroxide and diene contents with both the lipoxidase produced linoleate hydroperoxide and the pure trans-trans linoleate hydroperoxide. Studies of the inhibition of lipoxidase activity were carried out. A series of unsaturated substances were examined and several shorn to inhibit the enzymic activity. The structure of these inhibitors closely resembled that of the lipoxidase substrates in that they were long chain methylene-interrupted unsaturated compounds. The unsaturation was acetylenic rather than the ethylenic found in the lipoxldase substrates. These inhibitors are considered to be the most efficient competitive inhibitors of lipoxidase that have been so far demonstrated by anyone. Further inhibitory studies included polyphenolic antioxidants which are considered to be the best inhibitors of lipoxidase. These compounds were shown to be largely ineffective under the reaction conditions employed. These conditions were low antioxidant levels in the presence of low substrate concentrations and the oxidation of the substrate was relatively rapid. A notable exception to this was the antioxidant, nordihydroguaiaretic acid (NDGA) which completely inhibited the enzymic reaction until it was itself oxidised. An assay was developed to study the effect of antioxidants on the rate of bleaching of carotene in the absence and presence of fats. The carotene, antioxidants and fats were suspended in agar gels to produce systems which would remain stable over a period of days. The assay was used to study the effect of fats, both saturated and unsaturated, on the rate of autoxidative bleaching of carotene and also to study the efficacy of antioxidants in preventing such bleaching in the absence and presence of the various fatty substrates. This assay was also applied to studies incorporating the unsaturated fat oxidases, lipoxidase and haemoglobin. Inhibitory studies were carried out in the presence of these catalysts. Finally, the effect of antioxidants on the coupled linoleate-vitamin. A reaction catalysed by lipoxidase was studied. It was found possible with several antioxidants to inhibit vitamin A degradation without inhibiting primary linoleate oxidation, and, with NDGA both primary and secondary oxidation of linoleate and vitamin k were inhibited.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J A Blain
• Keywords: Biochemistry
• Date of Award: 1964
• Depositing User: Enlighten Team
• Unique ID: glathesis:1964-73229
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73229