MECHANISMS OF OXIDATION INHIBITION

V. MECHANISMS OF OXIDATION INHIBITION

complex biochemical pathways that have evolved to prevent, delimit, or respond to oxida- tion: (1) Within tissues, oxidation is isolated and compartmentallized within subcellular organelles such as mitochondria and peroxisomes. (2) Activated oxidants are scavenged by redox-active phenolics, including the lipid-soluble species α-tocopherol and ubiqui- none, and water-soluble hydrogen donors such as ascorbate, uric acid, polyphenolics, vari- ous flavonoids and their polymers, amino acids, and protein thiols. (3) Various enzymes reduce reactive oxygen and reactive oxygen–containing molecules such as peroxides. Cat- alase, superoxide dismutases, and peroxidases all detoxify active-oxygen species in vivo and in vitro after harvest. (4) Enzymatic repair systems are ubiquitous in cells. For exam- ple, proteases, lipases, ribonucleases, etc., constantly turn over cellular constituents, and degradative enzymes often have higher affinities for modified molecules. Substrate affinit- ies of synthetic enzymes discriminate against oxidized forms of lipids, proteins, and nucle- otides. This discriminatory process removes damaged molecules from the living cell; how- ever, these enzymes typically remain active to various extents and their hydrolytic activities can pose significant problems to the integrity of tissues post-harvest. The com- plexity and interdependence of these antioxidative protection systems is highly dependent on the structural integrity of tissues, and the disruption associated with food processing abolishes many of the oxidant defenses that are present in natural tissues. As a result, the major antioxidant protection remaining in biomaterials destined to be foods are the redox- active free radical–scavenging phenolics. These phenolic molecules are also most effective when added back to food materials as either natural or synthetic antioxidants. The mecha- nisms by which this class of antioxidants acts to slow oxidation will therefore be discussed in greater detail. Importantly, antioxidant is a broad classification for molecules that may act prior to or during a free radical chain reaction, at initiation, propagation, termination, decomposition, or the subsequent reaction of oxidation products with sensitive targets. Antioxygenic compounds are broadly defined as those molecules that can participate in any of the protective strategies. However, differences between antioxidant molecules, es- pecially in the stage or site of activity at which they act, are not trivial and influence various aspects of the efficacy of a given compound to act as a net antioxidant or protectant. How and where different molecules act can affect the rate of oxidation, its reaction course, the products formed, and the final targets damaged.

A. Metal Inactivation

The oxidation reactions of polyunsaturated lipids are most successfully prevented by avoiding initiation. Because metals are the single most important contributors to the initia- tion reactions in most foods, the most effective inhibitors of oxidation initiation are the metal chelators. Metal chelators are structurally diverse, from simple polyacids such as ethylenediaminetetraacetic acid (EDTA) to slightly more elaborate peptides such as carno- sine and even a variety of proteins such as lactoferrin and ceruloplasmin. The fundamental property of these molecules that renders them effective inhibitors of the initiating activities of metals is their ability to bind metals and either completely occupy their liganding orbit- als or alter their redox potential so as to prevent them from participating in the redox chemistry necessary to form initiating radicals. These structural considerations are not all met by just any compound that binds a metal. Ironically, some proteins actually activate metals to promote their oxidative properties. The most stunning example is lactoferrin, which binds and inactivates two moles of iron per mole of protein, but subsequent moles The oxidation reactions of polyunsaturated lipids are most successfully prevented by avoiding initiation. Because metals are the single most important contributors to the initia- tion reactions in most foods, the most effective inhibitors of oxidation initiation are the metal chelators. Metal chelators are structurally diverse, from simple polyacids such as ethylenediaminetetraacetic acid (EDTA) to slightly more elaborate peptides such as carno- sine and even a variety of proteins such as lactoferrin and ceruloplasmin. The fundamental property of these molecules that renders them effective inhibitors of the initiating activities of metals is their ability to bind metals and either completely occupy their liganding orbit- als or alter their redox potential so as to prevent them from participating in the redox chemistry necessary to form initiating radicals. These structural considerations are not all met by just any compound that binds a metal. Ironically, some proteins actually activate metals to promote their oxidative properties. The most stunning example is lactoferrin, which binds and inactivates two moles of iron per mole of protein, but subsequent moles

B. Inhibition of Hydroperoxide Formation

The alkyl radical R • is too reactive in an oxygen-rich environment for any competing species to successfully re-reduce R • to RH before oxygen adds to form the peroxy radical ROO • . At this point, however, ROO • is a relatively stable free radical that reacts relatively slowly with targets such as PUFA. This is the most widely accepted point of action for free radical-scavenging antioxidants such as the phenolic tocopherol. Tocopherol can re- duce ROO • to ROOH with such ease that tocopherol is competitive with biologically sensitive targets such as unsaturated lipids, RH, even at 10,000-fold lower concentration. The tocopheroxyl radical A • is in general a poor oxidant and reacts significantly more slowly than ROO • . Conversion of ROO • to ROOH and formation of A • effectively impart

a kinetic hindrance on the propagating chain reaction. The tocopheroxyl radical can be either re-reduced by reductants such as ascorbate, dimerized with another radical, or be oxidized further to a quinone. These free radical–scavenging functions of tocopherol are well documented (Buettner, 1993).