The aim of the present article is to evaluate critically the various methods employed in studies designed to quantify precisely skeletal muscle substrate utilization during exercise. In general, the pattern of substrate utilization during exercise can be described well from O2 uptake measurements and the respiratory exchange ratio. However, if the aim is to quantify limb or muscle metabolism, invasive measurements have to be carried out, such as the determination of blood flow, arterio-venous (a-v) difference measurements for O2 and relevant substrates, and biopsies of the active muscle. As many substrates and metabolites may be both taken up and released by muscle at rest and during exercise, isotopes can be used to determine uptake and/or release and also fractional uptake can be accounted for. Furthermore, the use of isotopes opens up further possibilities for the estimation of oxidation rates of various substrates. There are several methodological concerns to be aware of when studying the metabolic response to exercise in human subjects. These concerns include: (1) the muscle mass involved in the exercise is largely unknown (bicycle or treadmill). Moreover, whether the muscle sample obtained from a limb muscle and the substrate and metabolite concentrations are representative can be a problem; (2) the placement of the venous catheter can be critical, and it should be secured so that the blood sample represents blood from the active muscle with a minimum of contamination from other muscles and tissues; (3) the use of net limb glycerol release to estimate lipolysis is probably not valid (triacylglycerol utilization by muscle), since glycerol can be metabolized in skeletal muscle; (4) the precision of blood-borne substrate concentrations during exercise measured by a-v difference is hampered since they become very small due to the high blood flow. Recommendations are given in order to obtain more quantitative and conclusive data in studies investigating the regulatory mechanisms for substrate choice by muscle.

Microdialysis has been used for 25 years to study brain function in vivo. Recently, it has been developed for investigations on peripheral tissues. A microdialysis catheter is an artificial blood vessel system which can be placed in the extracellular space of various tissues such as adipose tissue and skeletal muscle in order to examine these tissues in situ. Molecules are collected from the tissue by the device and their true interstitial concentration can be estimated. Metabolically-active molecules can be delivered to the interstitial space through the microdialysis probe and their action on the tissue can be investigated locally without producing generalized effects. It is also possible to study local tissue blood flow with microdialysis by adding a flow marker (usually ethanol) to the microdialysis solvent. The microdialysis technique is particularly useful for studies of small and water-soluble molecules. A number of important observations on the in vivo regulation of lipolysis, carbohydrate metabolism and blood flow in human skeletal muscle and adipose tissue have been made recently using microdialysis.

Techniques in human skeletal muscle research are by necessity predominantly 'descriptive'.Microdialysis has raised high expectations that it could meet the demand for a method that allows 'mechanistic' investigations to be performed in human skeletal muscle. In the present review, some views are given on how well the initial expectations on the use of the microdialysis technique in skeletal muscle have been fulfilled, and the areas in which additional work is needed in order to validate microdialysis as an important metabolic technique in this tissue. The microdialysis catheter has been equated to an artificial blood vessel, which is introduced into the tissue. By means of this 'vessel' the concentrations of compounds in the interstitial space can be monitored. The concentration of substances in the collected samples is dependent on the rate of perfusate flow. When perfusate flow is slow enough to allow complete equilibration between interstitial and perfusate fluids, the concentration in the perfusate is maximal and identical to the interstitial concentration. Microdialysis data may be influenced by changes in blood flow, especially in instances where the tissue diffusivity limits the recovery in vivo, i.e. when recovery in vitro is 100 %, whereas the recovery in vivo is less than 100 %. Microdialysis data indicate that a significant arterial-interstitial glucose concentration gradient exists in skeletal muscle but not in adipose tissue at rest. While the concentrations of glucose and lactate in the dialysate from skeletal muscle are close to the expected values, the glycerol values obtained for muscle are still puzzling. Ethanol added to the perfusate will be cleared by the tissue at a rate that is determined by the nutritive blood flow (the microdialysis ethanol technique). It is concluded that microdialysis of skeletal muscle has become an important technique for mechanistic studies in human metabolism and nutrition.

In the study of the regulation of skeletal muscle blood flow during exercise it is useful to obtain information regarding the concentrations of vasoactive substances in the muscle interstitium, a site where the compounds act on the vascular and skeletal muscle cells. The microdialysis technique is a useful tool for measuring interstitial substances in the muscle at rest and during exercise in human subjects, and the technique can also be used to study the effect of both systemic and local interventions in a specific area of an exercising muscle. Probe recovery, which represents the relative amount of a substance that is diffusing to the dialysis membrane, changes from rest to exercise and can be determined by the internal-standard technique which allows for a relatively high time resolution (min). Furthermore, the use of electrodes at the microdialysis outlet makes it possible to perform continuous measurements of interstitial substances. The present review gives examples of how the microdialysis technique has been applied to study potentially important vasodilators such as adenosine, NO and K+ in human skeletal muscles and highlights areas for future research to establish the functional importance of these compounds.

The present article is a review written at a level suitable for students and new workers to the field of techniques in common current use for the measurement of static and dynamic features of metabolism, especially nutritional metabolism. It covers the nature of radioactive and stable-isotope tracers, the means of measuring them, and the advantages and disadvantages of their use. The greater part of the review deals with methods for the measurement of pool sizes and metabolic processes, with the emphasis being on protein metabolism, a field the author knows best. The examples given are from a variety of sources, including the work of the author, but the principles underlying the techniques are universally applicable to all metabolic investigations using tracers.

Our understanding of human energy metabolism has benefited greatly from the application of water labelled with 2H and 18O for the measurement of total body water, water turnover and total daily energy expenditure. Applications include validation of techniques for the assessment of dietary intake and physical activity, assessment of water and energy requirement and the assessment of the effect of dietary and physical activity interventions, including its use with endurance athletes competing at the highest level. Critical aspects of the application are isotope dose preparation, sample collection, sample analysis and the calculation procedure. The labelled-water method can easily be applied in normal living conditions, including exercise, and in the clinical setting. However, sample analysis requires a sophisticated laboratory with an isotope-ratio mass spectrometer and a sample preparation system. Examples of insights based on labelled-water studies are: (1) self-reported dietary intakes often underestimate energy requirements; (2) subjects have problems maintaining energy balance when daily energy expenditure exceeds 2.5 times resting energy expenditure. Devices for the assessment of physical activity validated using labelled water allow the study of activity patterns and strategies to influence the activity level of a sedentary society.

The present review discusses the advantages and limitations of using stable-isotope tracers to assess carbohydrate and fat metabolism at the whole-body level. One advantage of stable-(v. radioactive-) isotope tracers is the relative ease with which the location of a label within a molecule can be determined using selected-ion-monitoring GC-mass spectrometry (SIM-GC- MS). This technique minimizes potential problems due to label recycling, allows the use of multiple-labelled compounds simultaneously (e.g. to quantify glucose cycling), and perhaps most importantly, has led to the development of unique stable-isotope methods for, for example, quantifying gluconeogenesis. However, the limited sensitivity of SIM-GC-MS sometimes requires that relatively large amounts of a stable-isotope tracer be used, thus increasing cost and potentially altering metabolism. At least theoretically, stable- (or radioactive-) isotope tracers can also be used in conjunction with indirect calorimetry to estimate utilization of muscle glycogen or triacylglycerol stores, thus potentially circumventing the need to obtain muscle biopsies. These calculations, however, require certain critical assumptions, which if incorrect could lead to major errors in the values obtained. Despite such limitations, stable-isotope tracers provide a powerful and sometimes unique tool for investigating carbohydrate and fat metabolism at the whole-body level. With continuing advances in availability, instrumentation and methods, it is likely that stable-isotope tracers will become increasingly important in the immediate future.

The contribution of gluconeogenesis to glucose production is estimated from the enrichment of the H bound to C-5 of glucose relative to either that bound to C-2 of glucose or the enrichment in body water on ingesting 2H2O in the fasted state. Contributions of all gluconeogenic substrates are included in the estimate and the limitation of an uncertain precursor enrichment removed. The half-life of 2H2O in body water precludes a repeat study for many weeks. Glycogen cycling could result in underestimation, but there is evidence that glycogen cycling does not occur in liver in the fasted state. Gluconeogenesis has been estimated by mass-isotopomer-distribution analyses, usually by administering 13C-labelled glycerol. Underestimates emphasize the major limitation of the method, i.e. the need to assume a single enrichment of the precursor pool. Estimates of gluconeogenesis from isotopomer distribution in arterial-blood glucose and lactate on infusing [U-13C6] glucose are unreliable, as a proportion of the glucose is formed from glycerol and from amino acids not converted to glucose via pyruvate. Loss of label in the Krebs cycle and relying on enrichment of arterial-blood lactate as a measure of hepatic pyruvate further add to the uncertainty. Estimates of the rate of gluconeogenesis by NMR are obtained by subtraction of the rate of glycogenolysis determined by NMR from the rate of glucose production. Estimates are then the mean rate for the period over which glycogen contents are measured. Technical considerations can limit the accuracy of analyses and result in overestimates.

The rate of appearance of glycerol in the systemic circulation is determined from the enrichment of arterial blood glycerol when labelled glycerol is infused intravenously. This value provides a good measure of whole-body lipolysis during fasting, except that arterial infusion and venous sampling, if feasible, would probably give a higher more-accurate value. Lipolysis occurs primarily in adipose tissue, although other tissues contribute, notably muscle. Measurement is based on the difference in the enrichment of the glycerol entering and leaving the tissue. Lipolysis is underestimated by the extent to which glycerol released by lipolysis does not enter the systemic circulation, as occurs when lipolysis takes place in the non-hepatic tissue of the splanchnic bed. Glycerol released into the systemic circulation is utilized mainly by liver, although kidney and muscle are also major users of glycerol. Measurement of glycerol utilization is based on the amount of labelled glycerol taken up by the tissues. Other tissues probably utilize glycerol to a smaller extent, but in total this represents a significant amount. Most glycerol taken up by liver is converted to glucose. Glucose is probably the major source of glycerol-3-phosphate used in the esterification of fatty acids by adipose tissue.

The oxidation of fatty acids, carbohydrates and amino acids can be measured by quantifying the rate of excretion of labelled CO2 following administration of 14C- or 13C-labelled substrates at whole-body and tissue level. However, there is a theoretical need to correct the oxidation rates for the proportion of labelled CO2 that is produced via oxidation but not excreted. Furthermore, depending on the substrate and position of the C label(s), there may also be a need to correct for labelled C from the metabolized substrate that does not appear as CO2, but rather becomes temporarily fixed in other metabolites. The bicarbonate correction factor is used to correct for the labelled CO2 not excreted. Recently, an acetate correction factor has been proposed for the simultaneous correction of CO2 not excreted and label fixed in other metabolites via isotopic exchange reactions, mainly in the tricarboxylic acid cycle. Changes in metabolic rate induced, for example, by feeding, hormonal changes and physical activity, as well as infusion time, have been shown to affect both correction factors. The present paper explains the theoretical and physiological basis of these correction factors and makes recommendations as to how these correction factors should be used in various physiological conditions.

Three tracer methods have been used to measure protein synthesis, protein breakdown and protein oxidation at whole-body level. The method using L-[1-13C]leucine is considered the method of reference. These methods have contributed greatly to the existing knowledge on whole-body protein turnover and its regulation by feeding, fasting, hormones and disease. How exercise and ingestion of mixed protein-containing meals affect whole-body protein metabolism is still open to debate, as there are discrepancies in results obtained with different tracers. The contribution of whole-body methods to the future gain of knowledge is expected to be limited due to the fact that most physiological disturbances have been investigated extensively, and due to the lack of information on the relative contribution of various tissues and proteins to whole-body changes. Tracer amino acid-incorporation methods are most suited to investigate these latter aspects of protein metabolism. These methods have shown that some tissues (liver and gut) have much higher turnover rates and deposit much more protein than others (muscle). Massive differences also exist between the fractional synthesis rates of individual proteins. The incorporation methods have been properly validated, although minor disagreements remain on the identity of the true precursor pool (the enrichment of which should be used in the calculations). Arterio-venous organ balance studies have shown that little protein is deposited in skeletal muscle following a protein-containing meal, while much more protein is deposited in liver and gut. The amount deposited in the feeding period in each of these tissues is released again during overnight fasting. The addition of tracers to organ balance studies allows the simultaneous estimation of protein synthesis and protein breakdown, and provides information on whether changes in net protein balance are caused primarily by a change in protein synthesis or in protein breakdown. In the case of a small arterio-venous difference in a tissue with a high blood flow, estimates of protein synthesis and breakdown become very uncertain, limiting the value of using the tracer. An additional measurement of the intracellular free amino acid pool enrichment allows a correction for amino acid recycling and quantification of the inward and outward transmembrane transport. However, in order to obtain reliable estimates of the intramuscular amino acid enrichment and, therefore, of muscle protein synthesis and breakdown in this so-called three-pool model, the muscle should be freeze-dried and the resulting fibres should be freed from connective tissue and small blood clots under a dissection microscope. Even when optimal precautions are taken, the calculations in these tracer balance methods use multiple variables and, therefore, are bound to lead to more variability in estimates of protein synthesis than the tracer amino acid incorporation methods. In the future, most studies should focus on the measurement of protein synthesis and breakdown in specific proteins in order to understand the mechanisms behind tissue adaptation in response to various stimuli (feeding, fasting, exercise, trauma, sepsis, disuse and disease). The tracer laboratories, therefore, should improve the methodology to allow the measurement of low tracer amino acid enrichments in small amounts of protein.

Assays which purport to assess free radical activity in biological systems are multiple. However, despite numerous published descriptions of new methods and modifications of methods to assess free radical activity in biological materials, there is still a lack of reliable techniques for quantification of activity in vivo. Analysis of a number of related indicators and use of a variety of approaches appears the only reliable way to evaluate these processes in vivo. In studies of free radical generation by contracting skeletal muscle we have attempted to use a variety of indicators, including measurement of endogenous antioxidant levels, measurement of indirect indicators of free radical activity (e.g. products of lipid peroxidation, DNA oxidation or protein oxidation) and, where possible, measurement of direct indicators of free radical activity by electron spin resonance techniques. In view of the relative lack of specificity of many available techniques, caution should be exerted in evaluating the numerous examples of isolated single measures of free radical activity which are present in the scientific literature.

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.

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.

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.