Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

N epsilon-(carboxymethyl)lysine, N epsilon-(carboxymethyl)hydroxylysine, and the fluorescent cross-link pentosidine are formed by sequential glycation and oxidation reactions between reducing sugars and proteins. These compounds, termed glycoxidation products, accumulate in tissue collagen with age and at an accelerated rate in diabetes. Although glycoxidation products are present in only trace concentrations, even in diabetic collagen, studies on glycation and oxidation of model proteins in vitro suggest that these products are biomarkers of more extensive underlying glycative and oxidative damage to the protein. Possible sources of oxidative stress and damage to proteins in diabetes include free radicals generated by autoxidation reactions of sugars and sugar adducts to protein and by autoxidation of unsaturated lipids in plasma and membrane proteins. The oxidative stress may be amplified by a continuing cycle of metabolic stress, tissue damage, and cell death, leading to increased free radical production and compromised free radical inhibitory and scavenger systems, which further exacerbate the oxidative stress. Structural characterization of the cross-links and other products accumulating in collagen in diabetes is needed to gain a better understanding of the relationship between oxidative stress and the development of complications in diabetes. Such studies may lead to therapeutic approaches for limiting the damage from glycation and oxidation reactions and for complementing existing therapy for treatment of the complications of diabetes.

Role of Oxidative Stress in Development of Complications in Diabetes

Radicals are species containing one or more unpaired electrons. The oxygen radical superoxide (O 2 - ) and the non-radical oxidants hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are produced during normal metabolism and perform several useful functions. Excessive production of O 2 - and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxy 1 radical (· OH) and other oxidants in the presence of “catalytic” iron or copper ions. A major form of antioxidant defence is the storage and transport of iron or copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e. g., by ischaemia or trauma, can cause increased iron availability and accelerate free radical reactions. This may be especially important in the brain, since areas of this organ are rich in iron and cerebrospinal fluid cannot bind released iron ions. Oxidative stress upon nervous tissue can produce damage by several interacting mechanisms, including rises in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminium and in damage to the substantia nigra in Parkinson’s disease are reviewed. Finally, the nature of antioxidants is discussed, with a suggestion that antioxidant enzymes and chelators of iron ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be taken in the design of antioxidants for therapeutic use.

Reactive Oxygen Species and the Central Nervous System

1. A new method has been developed for measuring the total antioxidant capacity of body fluids and drug solutions, based on the absorbance of the ABTS.+ radical cation. 2. An automated method for use on a centrifugal analyser, as well as a manual method, is described. 3. The procedure has been applied to physiological antioxidant compounds and radical-scavenging drugs, and an antioxidant ranking was established based on their reactivity relative to a 1.0 mmol/l Trolox standard. 4. The Trolox equivalent antioxidant capacity of plasma from an adult reference population has been measured, and the method optimized and validated. 5. The method has been applied to investigate the total plasma antioxidant capacity of neonates and how this may be compromised in prematurity.

A Novel Method for Measuring Antioxidant Capacity and its Application to Monitoring the Antioxidant Status in Premature Neonates

Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury.

Is hyperviscosity a treatable component of diabetic microcirculatory disease

Impaired red cell deformability in peripheral vascular disease

SUMMARY 1. Fresh ox-brain homogenate prepared under standard conditions at 4°C and stored at - 20°C autoxidizes spontaneously and reproducibly when re-heated to 37°C. 2. The preparation can be used for assaying the antioxidant activity of serum and of other biological fluids. The preparatory and assay procedures are described. 3. The antioxidant activity of normal serum, cerebrospinal fluid, synovial fluid and of various known antioxidants have been compared.

T.L. Dormandy

Papers

Is hyperviscosity a treatable component of diabetic microcirculatory disease

Impaired red cell deformability in peripheral vascular disease

Assay using brain homogenate for measuring the antioxidant activity of biological fluids

Free-radical oxidation and antioxidants

An approach to free radicals