The folates are made up of a pterdine ring attached to a p-aminobenzoate and a polyglutamyl chain. The active form is tetrahydrofolate which can have C1 units enzymically attached. These C1 units (as a formyl group) are passed on to enzymes in the purine pathway that insert the C−2 and C−8 into the purine ring. A methylene group (−CH2−) attached to tetrahydrofolate is used to convert the uracil-type pyrimidine base found in RNA into the thymine base found in DNA. A further folate cofactor, i.e. 5-methyltetrahydrofolate, is involved in the remethylation of the homocysteine produced in the methylation cycle back to methionine. After activation to S-adenosylmethionine this acts as a methyl donor for the dozens of different methyltransferases present in all cells. Folate deficiency results in reduction of purine and pyrimidine biosynthesis and consequently DNA biosynthesis and cell division. This process is most easily seen in a reduction of erythrocytes causing anaemia. Reduction in the methylation cycle has multiple effects less easy to identify. One such effect is certainly on the nerve cells, because interruption of the methylation cycle causing neuropathy can also happen in vitamin B12 deficiency due to reduced activity of the vitamin B12-dependent enzyme methionine synthase (EC 2.1.1.13). In vitamin B12 deficiency, blocking of the methylation cycle causes the folate cofactors in the cell to become trapped as 5-methyltetrahydrofolate. This process in turn produces a pseudo folate deficiency in such cells, preventing cell division and giving rise to an anaemia identical to that seen in folate deficiency.

Epidemiological investigations repeatedly show decreased morbidity from regular exercise compared with sedentary life. A large number of investigations have demonstrated increased oxidation of important cellular macromolecules, whereas other investigators have found no effects or even signs of lowering of oxidation of macromolecules. In particular, extreme and long-duration strenuous exercise appears to lead to deleterious oxidation of cellular macromolecules. The oxidation of DNA is important because the oxidative modifications of DNA bases, particularly the 8-hydroxylation of guanine, are mutagenic and have been implicated in a variety of diseases such as ageing and cancer. The methodologies for further investigation of the relationship between DNA oxidation and exercise are available. The preferred methods rely on HPLC or GC-mass spectrometry; whereas the theoretically-attractive liquid chromatography- tandem mass spectrometry is being developed. Caution should be taken to avoid artifacts because of the six orders of magnitude of difference between oxidized and non-oxidized DNA bases in tissues. The methods can be used to estimate tissue levels, i.e. a local concentration of oxidized DNA, or to estimate the rate of body DNA oxidation by the urinary output of repair products, the latter being a method that is independent of repair. During exercise there appears to be a shifting of dietary-dependent antioxidant, e.g. vitamin C and vitamin E, from muscle to plasma, and an increased oxidation in plasma of these antioxidants. Supplementation trials with antioxidants have not been able to increase exercise performance; however, optimum nutrition with antioxidants and possibly supplementation, could be important in the prevention of diseases in the long term. The pattern from these observations appears to be quite consistent; immediately after exercise, regardless of how intense, there do not appear to be any signs of oxidative damage to DNA. Acute or prolonged moderate exercise does not produce signs of oxidative DNA damage and might even be associated with lowering of the levels of oxidation of tissue DNA; however, after long-duration and intense exercise an increase in oxidative DNA modifications is apparent. We suggest as a hypothesis that the relationship between exercise and health is U-shaped. This hypothesis needs to be tested in detail in order to establish the maximum beneficial exercise level with regard to oxidative DNA modification, and also the level that could be deleterious and might even increase the risk for cancer and other diseases.

There are two major dietary sources of vitamin A: easily absorbed retinyl palmitate in foods of animal origin, and poorly bioavailable carotenoids from plant foods. Plasma retinol is tightly controlled, probably by regulation of retinol-binding protein (RBP) formation in the liver, and only hormonal factors (e.g. oral contraceptives) and infection will alter the homeostasis. Delivery of retinol to the tissues is facilitated by the RBP-retinol complex; however, there is evidence that this mechanism can be bypassed when very high doses of vitamin A are given. Some retinyl ester may be released to tissues from chylomicrons when the latter bind to tissue lipoprotein receptors during their passage from the gut to the liver following a meal. High-dose vitamin A therapy is a means of rapidly improving vitamin A status in persons with sub-optimal vitamin A nutrition but there are dangers of toxic symptoms (e.g. teratogenicity) from excess vitamin A usage. Evidence is presented to suggest that the plasma retinol : RBP may be a guide to optimal vitamin A status, since values less than one frequently occur in less-developed countries and during infection. In contrast to plasma retinol, plasma carotenoids reflect the dietary intake of plant foods. However, absorption is limited by poor bioavailability and a saturable uptake mechanism in competition with other phytochemicals. Recent work on bioavailability suggests that the calculation of plant food vitamin A activity should be re-examined. Illness has little influence on plasma levels except by suppressing appetite. Carotenoids are generally regarded as non-toxic yet intervention studies with β-carotene in smokers have been associated with increased lung cancer and heart disease. Some carotenoids are important as vitamin A precursors, but the physiological importance of their antioxidant properties is not known and consequently the amount needed for optimal nutrition is uncertain.

Evidence from biochemical and animal models suggests that nutritional antioxidants should inhibit the development of diseases such as CHD and certain cancers. This evidence is not clearly corroborated by intervention studies in human subjects, due, in part, to inadequacies in current analytical methodologies. Although in vitro assays can give useful information on the attributes required by a compound to act as an antioxidant, results may have little nutritional relevance due to limited bioavailability. The determination of antioxidants in blood is often used as a measure of antioxidant status in vivo, but may not necessarily reflect concentrations in target tissues where oxidative stress is greatest. In addition, the accumulation of antioxidants in selective tissues may not be apparent from plasma measurements. Participation in quality-control schemes for antioxidant determination by HPLC allows inter-laboratory comparison of results. Moderation of indices of oxidative damage to lipids, proteins and DNA can provide information on the effectiveness of compounds as nutritional antioxidants. However, most current methods of assessing oxidative stress are subject to confounding factors of non-oxidative origin. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. They give different results, should never be used in isolation, and results should be interpreted with caution. Until more is known about the activity and metabolic fate of antioxidants, caution should be exercised in the consumption of large amounts of commercially-available antioxidant preparations.

Interest in the role of vitamin E in disease prevention has encouraged the search for reliable indices of vitamin E status. Most studies in human subjects make use of static markers, usually a-tocopherol concentrations in plasma or serum. Plasma or serum α-tocopherol concentrations of < 11.6, 11.6–16.2, and > 16.2 mmol/l are normally regarded as indicating deficient, low and acceptable vitamin E status respectively, although more recently it has been suggested that the optimal plasma α-tocopherol concentration for protection against cardiovascular disease and cancer is > 30 μmol/l at common plasma lipid concentrations in combination with plasma vitamin C concentrations of > 50 μmol/l and > 0.4 mmol β-carotene/l. Assessment of vitamin E status has also been based on α-tocopherol concentrations in erythrocytes, lymphocytes, platelets, lipoproteins, adipose tissue, buccal mucosal cells and LDL, and on α- tocopherol: γ-tocopherol in serum or plasma. Erythrocyte susceptibility to haemolysis or lipid oxidation, breath hydrocarbon exhalation, oxidative resistance of LDL, and α-tocopheryl quinone concentrations in cerebrospinal fluid have been used as functional markers of vitamin E status. However, many of these tests tend to be non-specific and poorly standardized. The recognition that vitamin E has important roles in platelet, vascular and immune function in addition to its antioxidant properties may lead to the identification of more specific biomarkers of vitamin E status.

Muscular exercise results in an increased production of radicals and other forms of reactive oxygen species (ROS). Recent evidence suggests that radicals and other ROS are an underlying aetiology in exercise-induced disturbances in muscle redox status. These exercise-induced redox disturbances in skeletal muscle are postulated to contribute to both muscle fatigue and/or exercise-induced muscle injury. To defend against ROS, muscle cells contain complex cellular defence mechanisms to reduce the risk of oxidative injury. Two major classes (enzymic and non-enzymic) of endogenous protective mechanisms work together to reduce the harmful effects of oxidants in the cell. Primary antioxidant enzymes include superoxide dismutase (EC 1.15.1.1; SOD), GSH peroxidase (EC 1.11.1.9; GPX), and catalase (EC 1.11.1.6); these enzymes are responsible for removing superoxide radicals, H2O2 and organic hydroperoxides, and H2O2 respectively. Important non-enzymic antioxidants include vitamins E and C, β-carotene, GSH and ubiquinones. Vitamin E, β-carotene and ubiquinone are located in lipid regions of the cell, whereas GSH and vitamin C are in aqueous compartments of the cell. Regular endurance training promotes an increase in both total SOD and GPX activity in actively-recruited skeletal muscles. High-intensity exercise training has been shown to be generally superior to low-intensity exercise in the upregulation of muscle SOD and GPX activities. Also, training-induced upregulation of antioxidant enzymes is limited to highly-oxidative skeletal muscles. The effects of endurance training on non-enzymic antioxidants remain a relatively uninvestigated area.

Most species of plants and animals synthesize ascorbic acid, but human subjects cannot, making vitamin C an essential component of our diet. Relationships between vitamin C intake and status, and between status and health are not yet clear. There is evidence, however, that higher intake of vitamin C is associated with lower risk of disease, supporting the concept that optimal intake is needed for optimal vitamin C status, and that both factors are required for optimal health. Vitamin C has low toxicity in healthy subjects, but a clear definition of optimal status and the dietary intake required to meet and maintain this status is needed before a change in the current recommended intake can be considered. Available evidence suggests that intake of 200 mg vitamin C/d saturates tissues and maintains fasting plasma levels above the proposed threshold (50 μmol/l) for minimum risk of CHD. However, the issue of whether or not these levels produce ‘optimal vitamin C status’ awaits the clear and accepted definition of the term. This definition in turn awaits the development of reliable functional markers capable of assessing the effects of varying levels of vitamin C nutriture. In the present paper the relationship between intake and body stores of vitamin C and the role of vitamin C in human health are reviewed briefly. The requirements of a reliable functional marker of human vitamin C status are defined, three classes of functional markers (molecular, biochemical and physiological) are described, and possible candidate markers are examined.

In the past, a major challenge for nutrition research was in defining indicators of nutritional adequacy. More recently, the research base related to the role of nutrition in chronic disease has expanded sufficiently to permit moving beyond deficiency indicators to other indicators with broader functional significance. Thus, nutrition research is faced with the new challenge of defining ‘optimal nutrition’. One definition of optimal nutrition with respect to any particular nutrient could be when a functional marker reaches an ‘optimal value’ or plateau beyond which it is no longer affected by intake or stores of the nutrient. A functional marker of nutrient status could be defined as a physiological or biochemical factor which (1) is related to function or effect of the nutrient in target tissue(s) and (2) is affected by dietary intake or stores of the nutrient (which may include markers of disease risk). Examples of such indicators or markers are those related to risk of chronic diseases such as osteoporosis, CHD, or hypertension. The present review focuses on the concept of optimal nutrition with respect to three nutrients, Ca, Mg and P. However, for P and Mg there are as yet no functional indicators which respond to dietary intake, and in such cases nutrient requirements are established using more traditional approaches, e.g. balance data. For Ca, there has been interest in using maximal Ca retention, which is based on balance data, bone mass measurements and biomarkers of bone turnover as useful functional indicators of the adequacy of Ca intake.

The major liabilities of Fe lack include defects in psychomotor development in infants, impaired educational performance in schoolchildren, increased perinatal morbidity, and impaired work capacity. Few if any of the relevant investigations have demonstrated these abnormalities in the absence of anaemia. Consequently, adequate Fe nutrition can be defined as a normal haemoglobin concentration. On the other hand, optimal Fe nutrition should be regarded as sufficient body Fe to avoid any limitation in tissue Fe supply, termed Fe-deficient erythropoiesis. A variety of laboratory measurements have been used to identify this milder form of Fe deficiency, including serum ferritin, transferrin saturation, erythrocyte protoporphyrin, mean corpuscular volume, and more recently the concentration of the soluble fragment of transferrin receptor in serum. Recent studies indicate that the serum transferrin receptor is the preferred measurement, because enhanced synthesis of the transferrin receptor represent the initial cellular response to a declining Fe supply. Moreover, unlike other methods, it is not affected by chronic inflammation or infection which are often confused with Fe deficiency. In an otherwise normal healthy population the transferrin receptor: ferritin value provides a useful quantitative index of body Fe over a wide spectrum of Fe status, ranging from Fe repletion to Fe-deficiency anaemia. It is concluded that optimal Fe nutrition is best defined as a normal haemoglobin, serum ferritin and transferrin receptor concentration.

Defining optimal dietary intakes of Cu and Zn throughout the life cycle continues to present a considerable challenge for nutrition scientists. Although the daily intake of these micronutrients is below that currently recommended for many groups, traditional biochemical indicators of nutritional status for these trace metals largely remain within the normal range. Thus, it is unclear whether the recommended daily intakes may be unnecessarily high, or if the commonly-used markers simply lack the necessary sensitivity and specificity that are required for accurately assessing Cu and Zn status. The increasing number of reports that daily supplements with these trace metals enhance the activities of selective metalloenzymes and specific cellular and organ processes further points out the need to differentiate between meeting the requirement and providing optimal nutriture. Results from recent studies suggesting that alternative molecular and functional markers possess sufficient sensitivity to better assess Cu and Zn status are discussed. Likewise, recent studies evaluating the impact of very low and excessive levels of dietary Mn and Mo on selective biochemical and metabolic indicators are reviewed.

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