K. U. Ingold

The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma.

The Total (Peroxyl) Radical-trapping Antioxidant Parameter (TRAP) of six freshly prepared human plasma samples and 45 frozen plasma samples has been determined. It is shown that contributions from urate (35-65%), plasma proteins (10-50%), ascorbate (0-24%) and vitamin E (5-10%) to TRAP account for all of the peroxyl radical-trapping antioxidant activity in the majority of the samples. The changes in concentrations of the plasma antioxidants during peroxyl radical attack show that the first line of defense is provided by the plasma sulfhydryl groups, even urate being spared during the initial stages of the reaction. The modes of action of all of these plasma antioxidants and possible interactions between them are discussed, with particular emphasis on the abilities of the water-soluble antioxidants to regenerate or spare the only lipid-soluble antioxidant, vitamin E.

Is vitamin E the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes?

The concentration of lipid-soluble, chain-breaking antioxidants in human plasma and in erythrocyte ghosts have been determined for the first time by an inhibited-autoxidation method. The results are very similar to the concentrations of vitamin E measured for the same blood components by the HPLC method. It is concluded that vitamin E, which is largely present as alpha-tocopherol, is the only significant lipid-soluble, chain-breaking type of antioxidant present in human blood. The concentration of vitamin E in the plasma lipids divided by the concentration of vitamin E in the ghost membrane lipids is approximately a constant despite the large differences in vitamin E-intake and in plasma lipid concentrations in different individuals. Vitamin E/lipid ratios for plasma and ghosts were larger for subjects taking a supplement of alpha-tocopherol acetate of 100 IU per week, compared to nonsupplemented subjects (based on data from a limited number of subjects). A larger supplement of 2800 IU per week did not significantly increase the vitamin E/lipid ratios.

Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation: The important contribution made by plasma proteins

Plasma obtained from fasted humans has been analysed for total radical‐trapping antioxidant content by subjecting it to controlled peroxidation using the thermal decomposition of water‐soluble azobis (2‐amidinopropane hydrochloride) at 37°C to produce peroxyl radicals at a known, steady rate. It is found that the total radical‐trapping antioxidant content is rather similar for the 7 subjects that have been tested and, furthermore, it is 10–20‐times larger than the effect attributable to vitamin E alone. Although it is shown that urate and ascorbate augment the contribution from vitamin E, their contributions (21–34 and 0–2%, respectively) still leave 57–73% of the total antioxidant content unaccounted for. Evidence is presented to show that this previously unrecognized large reserve of antioxidant capacity is attributable to the plasma proteins.

Vitamin E as an in Vitro and in Vivo Antioxidanta

alpha-Tocopherol has near optimal activity as a chain-breaking antioxidant. Inherent antioxidant activity plays an important part in determining overall biological activity but the phytyl tail also exerts a very important influence. The new deuterated alpha-tocopherol/GC-MS technique is providing unprecedented insight into the importance of the stereochemistry of the phytyl tail in determining bioavailability, as well as helping to discover how rapidly and effectively absorption, transport, uptake, and loss occur. Measurements of rate of turnover in tissues indicate that differences exist between different types of animals. It is possible that these tissue differences may explain the diverse range of vitamin E deficiency symptoms observed across a wide variety of animals. It is not known what is responsible for the differences in biokinetic behavior.

A mild, rapid, and efficient method of lipid extraction for use in determining vitamin E/lipid ratios

A new, general method for lipid extraction and measurement of vitamin E/total lipid ratios in tissue and cell samples has been developed. The new extraction procedure use a combination of sodium dodecylsulfate, ethanol andn-heptane, and is mild, clean, convenient, efficient and rapid (≤5 min). The efficiency of the new method has been confirmed for human plasma, red blood cells and rat liver homogenate by the comparison of the yields of vitamin E, O-acyl lipid and cholesterol with the yields obtained following conventional extraction procedures. Extraction efficiency also has been confirmed for multilamenllar vesicles composed of known quantities of vitamin E, egg lecithin and cholesterol.

Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes.

Thermally labile azo-initiators, dissolved in either the aqueous or lipid phase, have been used to generate peroxyl radicals at a known, steady rate in an aqueous dispersion of dilinoleoylphosphatidylcholine multilamellar liposomes at 37 degrees C in order to study the antioxidant behaviour of ascorbate itself and ascorbate in combination with either alpha-tocopherol or a water-soluble alpha-tocopherol analogue (TROLOX(-]. It is found that ascorbate is an effective inhibitor of peroxidations initiated in the aqueous phase, with each ascorbate terminating 0.6 radical chains (i.e., n = 0.6), but it is a very poor inhibitor of peroxidations initiated in the lipid phase. Peroxidations initiated in the lipid-phase in the presence of either alpha-tocopherol or TROLOX(-) indicate that ascorbate is an excellent synergist with both phenolic antioxidants (n = 0.4). In peroxidations initiated in the aqueous phase ascorbate acts as a co-antioxidant with TROLOX(-) (n = 0.7), but the interpretation of the approximately additive effect obtained in the presence of alpha-tocopherol is complicated by the fact that under the experimental conditions employed alpha-tocopherol alone does not give a distinct, measurable inhibition period. The latter problem is shown to be due to a non-uniform distribution of the water-soluble initiator within the liposome. Other examples of the complicating effects of non-uniform distributions of reactants in kinetic studies of the autoxidation of organic substrates dispersed in water are described.

Biokinetics of and discrimination between dietaryRRR- andSRR-α-tocopherols in the male rat

The net rates of uptake of the natural (2R,4′R,8′R) diastereoisomer of α-tocopherol (α-T) and the biodiscrimination relative to its 2S-epimer (2S,4′R,8′R) have been measured, in two experiments, for the blood and 21 tissues of male Sprague-Dawley rats fed over a period of several months diets containing deuterium-substituted forms of the α-T acetates. Gas chromatography-mass spectrometry was used to measure the amount of deuterated tocopherols taken up relative to the amount of nondeuterated tocopherol remaining. The measurements were performed at different times after the rats, placed for one month on a basal diet containing nondeuterated, natural α-T acetate, were switched to a diet containing the same total quantity of deuterated forms of either natural α-T acetate or a mixture of the acetates of the 2R- and 2S-epimers (i.e.,ambo-α-T acetate). In experiment 1 the source of vitamin E in the replacement diet was trideuterio-2R,4′R,8′R-α-T acetate. The data obtained provide the first direct measure of the rate at which natural vitamin E is replaced and augmented in the tissues of growing animals under normal laboratory dietary conditions. There are dramatic differences in the tissue kinetics; for example, the apparent half-life of vitamin E, i.e., the time at which the total amount of ingested trideuterio-α-T taken up is the same as the amount of nondeuterated α-T remaining, varies from ca. 1 wk for the lung to ca. 11 wk for the spinal cord. In experiment 2 the vitamin E in the replacement diet was an equimolar mixture of trideuterio-2S,4′R,8′R- and hexadeuterio-2R,4′R,8′R-α-T acetates. The results show that there is a preferential uptake of the natural diastereoisomer of α-T by all tissues (except the liver during the first month). Examination of fecal material reveals that the biodiscrimination begins in the gut; the incomplete hydrolysis of the acetates shows clearly that this reaction proceeds to a greater extent with the natural diastereoisomer. The greatest discrimination of all the tissues examined was found to occur in the brain. After five months, the level of the deuterated natural diastereoisomer was more than five times that of the deuterated 2S-epimer. These results have potential implications for human nutrition.

Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency☆

The chain-breaking (peroxyl radical-trapping) antioxidant activity of plasma obtained from several patients with a very severe vitamin E deficiency has been measured. The total chain-breaking antioxidant activity in lipid extracts has been shown to be approximately equal to the concentration of vitamin E. For whole plasma there is no significant difference in the concentrations of water-soluble, chain-breaking antioxidants between the E-deficient patients and healthy adults. It is concluded that even in cases of very severe vitamin E deficiency the requirement for this vitamin is not met by some other exogenous or endogenous antioxidant.

Biokinetics of dietaryRRR-α-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C does not “spare” vitamin Ein vivo

The net rates of uptake of “new” and loss of “old”2R,4′ R,8′ R-α-tocopherol (RRR-α-TOH, which is natural vitamin E) have been measured in the blood and in nine tissues of male guinea pigs over an eight week period by feeding diets containing deuterium-labelled α-tocopheryl acetate (d6-RRR-α-TOAc). There was an initial two week “lead-in” period during which 24 animals [the “high” vitamin E (HE) group] received diets containing 36 mg of unlabelled (d0)RRR-α-TOAc and 250 mg of ascorbic acid per kg diet, while another 24 animals [the “low” vitamin E (LE) group] received diets containing 5 mgd0-RRR-α-TOAc and 250 mg ascorbic acid per kg diet. The HE group was then tivided into three equal subgroups, which were fed diets containing 36 mgd6-RRR-α-TOAc and 5000 mg [the “high” vitamin C (HEHC) subgroup], 250 mg [the “normal” vitamin C (HENC) subgroup] and 50 mg [the “low” vitamin C (HELC) subgroup] ascorbic acid per kg diet. One animal from each group was sacrificed each week and the blood and tissues were analyzed ford0- andd6-RRR-α-TOH by gas chromatography-mass spectrometry. The LE group was similarly divided into three equal subgroups with animals receiving diets containing 5 mgd6-RRR-α-TOAc and 5,000 mg (LEHC), 250 mg (LENC) and 50 mg (LELC) ascorbic acid per kg diet with a similar protocol being followed for sacrifice and analyses. In the HE group the totald0-+d6-)RRR-α-TOH concentrations in blood and tissues remained essentially constant over the eight week experiment, whereas in the LE group the totalRRR-α-TOH concentrations declined noticeably (except in the brain, an organ with a particularly slow turnover of vitamin E). There were no significant differences in the concentrations of “old”d0-RRR-α-TOH nor in the concentrations of “new”d6-RRR-α-TOH found in any tissue at a particular time between the HEHC, HENC and HELC subgroups, nor between the LEHC, LENC and LELC subgroups. We conclude that the long-postulated “spring” action of vitamin C on vitamin E, which is well documentedin vitro, is of negligible importancein vivo in guinea pigs that are not oxidatively stressed in comparison with the normal metabolic processes which consume vitamin E (e.g., by oxidizing it irreversibly) or elminate it from the body. This is true both for guinea pigs with an adequate, well-maintained vitamin E status and for guinea pigs which are receiving insufficient vitamin E to maintain their body stores.The biokinetics of vitamin E uptake and loss in the HE guinea pigs are compared with analogous data for rats reported previously (Lipids 22, 163–172, 1987). For most guinea pig tissues the uptake of vitamin E under “steadystate” conditions was faster than for the comparable rat tissues. However, the brain was an exception with the turnover of vitamin E occurring at only one-third of the rate for the rat.

The antioxidant efficiency of vitamin C is concentration-dependent

The inhibition of autoxidation of plasma lipids by vitamin C (ascorbic acid) has been studied. The ascorbate stoichiometric factor, n, i.e., the number of peroxyl radicals trapped by each ascorbate molecule, decreases as the concentration of ascorbate increases. This is attributed to the fact that ascorbate not only acts as a radical-trapping antioxidant, but can also undergo autoxidation. The data indicate that n----2.0 as [ascorbate]----0 and that n----0 as [ascorbate]----infinity. This concentration-dependent behaviour accounts for the wide variation of n values reported in the literature. It is suggested that this autoxidative destruction of ascorbate may play a role regulating its concentration in blood plasma.