Richard C. Rose

Biology of free radical scavengers: an evaluation of ascorbate.

Reactive free radical species (R∗) are associated with several forms of tissue damage and disease, and also with the process of aging. Protection is thought to be available in the form of endogenous compounds that react with and thereby “scavenge” the R∗. Because many R∗ are reactive forms of oxygen, an effective scavenger is often referred to as an antioxidant. To be an effective antioxidant physiologically, a substance must have certain chemical and biological properties: it must be present in adequate amounts in the body; it must react with a variety of R∗; it must be suitable for compartmentation; it must be readily available; it might be suitable for regeneration; it must be conserved by the kidneys; and it must have tolerable toxicity. Several water‐soluble candidates are mentioned, with most having no more than one or two of the attributes listed. Ascorbic acid is discussed in detail, and an analysis is made of whether it has the properties mentioned.—Rose, R. C., Bode, A. M. Biology of free radical scavengers: an evaluation of ascorbate. FASEB J. 7: 1135‐1142; 1993.

OCULAR OXIDANTS AND ANTIOXIDANT PROTECTION

Abstract Oxidative damage and antioxidant protection in ocular tissues has not been reviewed recently. Metabolism in the eye is of increasing interest because the organ is highly susceptible to damage by sunlight, oxygen, various chemicals, and pollutants. Interest is expected to increase because of an aging Western world population and a continued depletion of stratospheric ozone. Hydrogen peroxide is discussed because it is both a byproduct and a source of free radical reactions and is normally present in the aqueous humor. The metabolism of reactive oxygen species by enzymes, nutrients, pigments, and low molecular weight scavengers is evaluated. Ascorbic acid, because of its high concentration in the eye, is thought to be a primary substrate in ocular protection; progress in determining the mechanisms by which it is recycled and maintained in the useful, reduced state is discussed. Recent information is included about antioxidants not previously known to be present in the eye, and some importance is placed on the properties of the vitreous humor and tear fluid because of the previous lack of emphasis on these.

Ocular ascorbate transport and metabolism

1. The concept is reviewed that the eye is subject to photo-oxidative damage through chemical free radical species that interact with sensitive tissue components. 2. The role of ascorbic acid may be to protect the eye by scavenging free radicals. 3. Ascorbic acid is present at a high concentration in various ocular compartments of diurnal animals, regardless of whether the animal synthesizes the compound or extracts it from the diet. 4. Ascorbic acid accumulates in the eye by active transport through the iris-ciliary body into aqueous humor, and subsequent transport into the lens and cornea. 5. Conservation of ascorbic acid occurs by reduction of dehydro-L-ascorbic acid and the ascorbate free radical through processes that appear to be enzymatic.

Water soluble antioxidants in mammalian aqueous humor: interaction with UV B and hydrogen peroxide

HPLC/electrochemical detection was used to identify five major low MW water soluble electrochemically active molecules from the aqueous humor of three species of mammals: New Zealand White rabbits and humans (diurnal) and Sprague-Dawley rats (nocturnal). These molecules are L-cysteine (CYS), L-ascorbic acid (AA), glutathione (GSH), uric acid (UA) and L-tyrosine (TYR); all of these molecules have known antioxidant properties. Nocturnal rat aqueous humor is concentrated in two thiols: GSH (125 microM; n = 24 pooled eyes) and CYS (63 microM), in contradistinction to diurnal species which have high concentrations of AA. No deterioration of any of these antioxidants occurs in a synthetic aqueous humor mixture irradiated with a physiologically relevant spectral UV B dose of 30 mJ/cm2/h (5.5 UV equivalent sunlight hours). The same result occurred with addition of the endogenous aqueous humor UV B photosensitizer L-tryptophan. In a second set of experiments, human synthetic aqueous humor was subjected to hydrogen peroxide induced oxidant stress. The decay of antioxidants was CYS > GSH > AA > UA > TYR. The second highest concentrated antioxidant in human aqueous humor is TYR. Yet TYR failed to protect AA against H2O2-induced free radical damage in a synthetic aqueous humor model system (P = 0.10; ANOVA). The existence of multiple electrochemically active constituents and their thermodynamic interactions must be recognized when choosing animal models to evaluate human aqueous humor antioxidant defense.

Cerebral metabolism of oxidized ascorbate

The brain has a high level of ascorbic acid which is thought to act as a reducing agent, e.g. in protecting tissues against oxidative stress. The mechanism by which ascorbate is maintained in the useful, reduced state in the CNS is evaluated herein. Cerebrum from rat or calf was minced and homogenized in buffer. The endogenous levels of ascorbic acid, dehydro-L-ascorbic acid (DHAA) and reduced glutathione (GSH) were determined by HPLC with coulometric electrochemical detection. We also quantitated tissue capacity to regenerate ascorbic acid from DHAA, which is a product of electron transfer reactions of ascorbic acid. The homogenate was fractionated by centrifugation in steps up to 110,000 x g and dialyzed free of low molecular weight components. The activity for reducing DHAA was approximately equal in the various supernatants; resuspended pellets had little activity. The active component has several properties of a protein, including being precipitated by solid ammonium sulfate addition to the tissue extract; most activity appeared in the 40-80% saturated fraction. The activity was stable up to a temperature of 80 degrees C, but was lost at 95 degrees C. The protein was digested by trypsin. The results suggest that a cytosolic component of cerebrum regenerates ascorbic acid in a step that preferentially uses GSH and NADPH as reducing cofactors. At least one form of DHAA reductase exists in brain.

Intestinal Absorption of Water-Soluble Vitamins

Abstract Vitamin utility is a scientific/medical topic that appears to be pursued as ardently by the lay public as by scientists and medical practitioners. A group of epidemiologists evaluates the effects on health of vitamin intake in the natural diet and by supplementation. The role in ocular disease, cancer of the breast or colon, and cardiovascular disease are a few of the concerns. The results and recommendations concerning dietary vitamin intake will likely continue to change. However, the processes by which dietary vitamins are delivered from intestinal chyme to the blood are more certain. The concept of homeostasis might apply to various of the vitamins as it does to minerals, water, etc. This review will discuss some common methods used to study vitamin absorption and the proposed mechanisms of absorption, and will conclude with a section about dietary regulation.

Tissue-mediated regeneration of ascorbic acid : is the process enzymatic ?

Ascorbic acids function in animals is attributed in part to the ease with which it reduces potentially damaging components, such as reactive free radicals. After more than six decades of speculation and laboratory efforts, the mechanisms by which ascorbic acid is maintained in the useful, reduced state remain uncertain. Previous attempts to isolate the enzymes that reduce the partially and the fully oxidized metabolites of vitamin C are reviewed. Some speculation on why dehydroascorbate reductase (EC 1.8.5.1) has not been purified from animal tissues is presented.

Short term effects of oxidized ascorbic acid on bovine corneal endothelium and human placenta

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.

Glutathione-dependent ascorbate recycling activity of rat serum albumin.

An efficient regeneration of vitamin C (ascorbate) from its oxidized byproduct, dehydroascorbate (DHAA), is necessary to maintain sufficient tissue levels of the reduced form of the vitamin. Additionally, the recycling may be more significant in mammals, such as guinea pigs and humans, who have lost the ability to synthesize ascorbate de novo, than it is in most other mammals who have retained the ability to synthesize the vitamin from glucose. Both a chemical and an enzymatic reduction of DHAA to ascorbate have been proposed. Several reports have appeared in which proteins, including thioltransferase, protein disulfide isomerase, and 3-alpha-hydroxysteroid dehydrogenase, characterized for other activities have been identified as having DHAA reductase activity in vitro. Whether these previously characterized proteins catalyze the reduction of DHAA in vivo is unclear. In the present study, a 66 kD protein was purified strictly on the basis of its DHAA-reductase activity and was identified as rat serum albumin. The protein was further characterized and results support the suggestion that serum albumin acts as an antioxidant and exerts a significant glutathione-dependent DHAA-reductase activity that may be important in the physiologic recycling of ascorbic acid.

ANALYSIS OF WATER-SOLUBLE ANTIOXIDANTS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

Publisher Summary This chapter discusses the analysis of water-soluble antioxidants by high-performance liquid chromatography (HPLC) with electrochemical detection (HPLC/EC). Water-soluble antioxidants—such as ascorbic acid and glutathione—receive substantial attention because of their potential role in maintaining optimal health. The endogenous level of one or more antioxidants may reflect the health of a particular tissue. Compared to colorimetric or spectrophotometric methods, HPLC/EC provides a relatively fast, quantitative method that is reliable, highly sensitive, specific, and allows no interference from nonrelated substances within the sample and is currently the method of choice. The sensitivity and selectivity of HPLC/EC are appropriate for evaluating small tissue samples. Vitreous or aqueous humor samples are obtained by a needle connected to a syringe. The size of the needle depends on the animal species and whether vitreous humor or aqueous humor is being obtained. The chapter concludes with a discussion of the hydrodynamic voltammetry plot.