Biochemical Society Transactions

Biochemical Society Transactions 362 ma1 conditions for the control of key biosynthetic processes during proliferation [ 18, 191. In addition, the link between muscle and brain, and muscle and immune cells, may provide a nutritional mechanism to explain how psychological stress can influence the immune system (i.e. psychoimmunology [ZO]). Of further clinical interest is the report in the Lancet in 1988 of the trial of the administration of branched-chain amino acids to patients suffering from amylotrophic lateral sclerosis (motor neuron disease) [Zl]. The article states Those treated with amino acids showed significant benefit in terms of maintenance of extremity muscle strength and continued ability to walk. The use of the branchedchain amino acids was based on the possibility that glutamate metabolism in the central nervous system may be impaired in this condition and that these amino acids may stimulate glutamate dehydrogenase activity. An alternative explanation might be the maintenance of the normal plasma tryptophan/ branched-chain amino acid concentration ratio, if indeed this is disturbed in this disease. Further studies in this area may be valuable. Newsholme, E. A. & Crabtree, B. (1979) J. Mol. Cell. Cardiol. 11,839-856 Newsholme, E. A. & Leech, A. R. (1988) New Sci. 22 September, 39-43 Parry-Billings, M.. Blomstrand, E., McAndrew, N. & Newsholme, E. A. (1990) Int. J. Sports Med. 11, S 122-S 128 Newsholme, E. A. & Leech, A. R. (1983) The Runner, Oxford Books, 21, Pitts Road, Headington, Oxford 5. Newsholme, E. A. & Leech, A. (1983) Biochemistry for the Medical Sciences, John Wiley, Chichester 5a. Hultman, E., Greenhaff, P. L., Ren, J. M. & Soderlund, K. (1991) Biochem. SOC. Trans. 19,OO-00 6. Young, V. R. (1987) Am. J. Clin. Nutr. 46.709-725 7. Fernstrom, J. D. (1990) J. Nutr. Biochem. 1,508-517 8. Wagenmakers, A. J. M., Brookes, J. H., Coakley, J. H., Reilly, T. & Edwards, R. H. T. (1989) Eur. J. Appl. Physiol. 59, 159-167 9. Sarna, G. S., Kantamaneni, B. D. & Curzon, G. N. (1985) Neurochemistry 44,1575-1580 10. Curzon, G., Friedel, J. & Knott, P. J. (1973) Nature (London) 242,198-200 11. Chaouloff, F., Kennett, G. A., Semrier, B., Merina, D. & Curzon, G. (1986) J. Neurochem. 46,1647-1650 12. Blomstrand, E., Celsing, F. & Newsholme, E. A. (1988) Acta Physiol. Scand. 133, 115-121 13. Blomstrand, E., Parrett, D., Parry-Billings, M. & Newsholme, E. A. (1989) Acta Physiol. Scand. 136, 473-481 14. Blomstrand, E., Hassmen, P., Ekblom, B. & Newsholme, E. A. (1991) Eur. J. Appl. Physiol. in the press 15. Golden, C. J. (1978) The Stroop Colour and Word Test. A Manual for Clinical and Experimental Uses, Staoelting, Chicago 16. Jeejeebhoy, K. M. (1986) Gut 27 (Suppl. l), 25-39 17. Editorial (1990) Lancet ii, 619 18. Newsholme, E. A., Newsholme, P., Curi, R., Challoner, M. A. & Ardawi, M. S. M. (1988) Nutrition 4,261-268 19. Parry-Billings, M., Evans, J., Calder, P. C. & Newsholme, E. A. (1990) Lancet ii, 523-525 20. Newsholme, E. A. (1990) Biol. Psychiatry 27, 1-3 21. Ploitkis, A., Smith, J., Mandeli, J. & Yahr, M. D. (1988) Lancet i, 1015-1018 Received 2 1 November 1990 Effect of diet manipulation on substrate availability and metabolism in trained cyclists Fred Brouns Nutrition Research Center, Department of Human Biology, University of Limburg, Maastricht, The Netherlands Nutrition as one of the factors influencing performance has received attention from athletes and their coaches since the classical athletic competitions in Olympia. However, it is only in the last 50 years that scientific studies have been focused more specifically on the role of nutrients in human performance and physical fitness. In 1939 Christensen & Hansen [l] observed that a change in the proportions of carbohydrate Abbreviations used: CHO, carbohydrate; FFA, free fatty acids. (CHO) and fat in the daily diet influenced the respiratory quotient. They concluded that food intake was one of the determinants of substrate selection for oxidation. It was also observed that physical activity raised the value of the respiratory quotient (R) and the conclusion was that CHO metabolism is enhanced as a result of physical activity. More recently, it has been suggested that fat is the dominant substrate for energy exchange in resting conditions and that CHO plays a role as immediate energy source, whenever energy exchange from fat is too slow to meet the require- Volume 19

Biochemistry of Exercise ments [2]. Based on this suggestion it can be explained that CHO utilization, which leads to a larger energy flow per second than fat degradation, is enhanced at the onset of exercise when aerobic metabolism still has to be increased. It may also explain why increased fatty acid availability in the course of exercise (caused by neural and hormonal adaptations) reduces CHO oxidation so that endogenous CHO stores can be spared [2, 41. Since the studies of Bergstrom & Hultman [S, 61, it is known that local CHO stores may play a limiting role in the ability to perform exercise of a high intensity. This may explain the adaptation of the body to increase fat metabolism and to spare CHO for emergency actions. The same authors also observed that increasing the amount of CHO in the daily diet results in increased glycogen stores and that this increase in glycogen availability is related to longer exercise times before exhaustion is reached. The general advice thereafter was that athletes should consume more CHO. With respect to earlier observations [7, 81, this may sound conflicting. Increasing the amount of CHO in the diet stimulates CHO oxidation and inhibits fat metabolism, whereas the natural adaptations of the body lead to the opposite. Meanwhile a large number of studies with respect to nutritional factors and exercise have been performed, and extensively reviewed. However, only very few studies have been done with highly trained athletes, and practically no information is available on the effect of highly intensive long-lasting exercise performed on sequential days. Therefore, we decided to perform a controlled experiment in which highly trained cyclists performed until exhaustion on sequential days, and in which the effect of a conventional CHO-rich diet on nutritional and metabolic parameters was compared with the effect of the same diet supplemented with a CHO beverage. The idea behind this experiment was to examine the efficacy of diet intervention with respect to meeting the nutritional needs during days of exhausting long-lasting physical exercise. The second aim was to study the rationale for nutritional supplementation under these circumstances. Such a rationale could be obtained from the analysis of the changes in nutritional and biochemical parameters as well as performance capacity. Thirteen highly trained cyclists participated in a 7 day study, in which we determined actual performance capacity [W,. (W)] and maximal oxygen-uptake on day 2 of the experiment. Day 3 was a standardized active rest day, followed by 2 days of intensive endurance cycling comparable to energy expenditure and intensity levels as observed in the Tour de France [9] (days 4 and S), followed by a repetition of the standardized rest day (day 6). The study was performed in a cross-over design with a randomized order of treatment and was conducted over 7 sequential days using a semiautomated respiration chamber system, allowing metabolic-balance studies (Fig. 1). The subjects were asked not to participate in vigorous training or competition during the 2 days preceding the arrival in the laboratory and to ingest a normal but CHO-rich diet (dietary advice was given by a registered dietician). After amval at the laboratory, the subjects received either a normal diet (N) or a normal diet supplemented with CHOconcentrated liquids (). In a case of N, CHO-rich food and ordinary drinks of known quantity and composition were selected by each subject from a variety of available foods for breakfast, lunch and dinner. This food was continuously provided until the end of the experiment, 6 days later. There were no quantitative limitations and extras could be obtained throughout the day upon request. The cyclists were informed about the importance of adequate food and fluid intake and encouraged to eat and drink whenever they liked during exercise. To study the effects of diet manipulation by supplementing the normal CHO-rich diet with CHO-concentrated solutions, we divided the total group of cyclists into two sub-groups. One group of 6 cyclists was supplemented with a solution,, containing 17% (w/v) maltodextrin, 3% (w/v) fructose (Perform, Wander, Bern). The other group was supplemented with a solution containing 10% (w/v) Fig. I Semi-automated respiration chamber for metabolicbalance studies Analysis ofi I I I I Room air L Energy Expenditure I I CHO oxidation Food consumption Energy intake I Protein intake I CHO inuke Urine Faeces f Energy iosses I Sweat Room air Protein losses I I I I Energy balance I 'Protein balance' I THO balance' Respiration Chamber I I Fresh air in 363 1991

Biochemical Society Transactions 364 maltodextrin, 10% (w/v) fructose, which will not be discussed here. During exercise CHO-rich food and fluids (tea, coffee, milk, water and lemonade which consisted of artificially sweetened, coloured and additionally mineralized water) were available ad libitum. In the case of supplementation, the cyclists received equal amounts of food and fluids; however, the lemonade was exchanged for the [20%(w/v) CHO] solution. The participating amateur cyclists were of international level. The subjects cycled 4.5 h/day, simulating two intensive competition days. During this time approximately 1 h 20 min of exercise was performed at an intensity of 80% Wma, (maximal working capacity, W) and final exercise to exhaustion was performed at 90% Wmax.. An extensive description of the experiments is presented elsewhere [lo-131. The results of the study show that the subjects ingesting the conventional CHO-rich diet at rest and during exercise were not able to maintain energy balance during days of exhaustive physical work in contrast to when the diet was supplemented with (Table 1). As a result of the supplementation total CHO intake increased significantly, as did the relative contribution of CHO to total daily energy intake. From these observations it becomes apparent that the two main factors influencing energy exchange processes and performance capacity, i.e. energy balance and CHO availability, were significantly different between the two treatments and must have had their influence on metabolic regulation and substrate availability. Although the subjects receiving the conventional CHO-rich diet were in negative energy balance on days 4 and 5, and the amount of CHO oxidized was substantially greater than the amount of CHO intake, especially on day 4, they did not develop hypoglycaemia. A possible explanation may be that the intake of food ad libitum during exercise conserved sufficient liver glycogen and/or supplied adequate amounts of CHO to the blood to avoid a fall in blood glucose. A second explanation may be that highly trained individuals have developed an enhanced capacity to synthesize glucose from lactate, glycerol and alanine during exercise. A contribution of gluconeogenesis to the maintenance of the blood glucose level is supported Table I Summary of observations with respect to nutritional indices and performance time For complete description of the nutritional data see Brouns [ 10-121. Mean values f S.E.M. (for N, n = 13; for, n = 6). CHO intake was determined by a weighed food intake procedure using a computerized food table for analysis. Nitrogen intake was determined by a weighed food intake procedure after previous determination of the nitrogen content of all food items by the chemiluminescence method. Energy balance was determined by indirect calorimetry and weighed food intake after previous determination of energy content of all food items by bomb calorimetry. Nitrogen balance was calculated from total nitrogen intake and nitrogen losses in urine, sweat, faeces and blood. Statistical significance with respect to the initial value on day 3 is indicated by *P<O.O5; **P <O.OI; mp<o.oo1. Statistical significance of with respect to N (each subject is his own control) is indicated by tp<0.05; ttp<o.oi; tttp < 0.00 I. Day Cumulative Diet 3 4 5 6 (day 4 + 5 + 6) CHO intake (energy %) Nitrogen intake (g) Energy balance (MJ) N N N Nitrogen balance (g of N) N Final performance at 90% Wmm, (min) over days 4 and 5 61.8f 1.7 62.9 f I.3 62.4 f I.6 79.7 f I.3**tt 66.8 f 2.7 80.5 f I S"tt 19.3 f 0.9 18.9 f I.2 18.6% 1.1 19.7f 1.0 15.7 f I.5 17.7k 1.4 I. I8 k I.20 4.90 f 0.90 I.93 f I.46 I.39 f 0.68-9.78 f 0.82-0.28 f I.23ttt - 2.56 f I.34-2. I3 f 0.39-8. I2 f 0.88- - 0.54 f I.75ttt - 3.65 f I.08 I. 17f 2.23 N 9.9 f 2.6 22.4 f 7.9t 62.7 2 I.6 67.5 f 2.6 21.5f 1.0 17.9f I.lt-/t 2.65 f 0.92* 2.132 1.32-2.47 f I.44-1.12+ 1.01-58.9 f 2.6 5 I.3 f 2.8-15.26f2.05 I.86 f 2.36-8.69 f 2.50-2.09 f 2.50 Volume 19

Biochemistry of Exercise by the observed increase of hormones that favour gluconeogenesis in the liver and by the observation that blood lactate was significantly increased halfway during exercise and at exhaustion. The fact that mean maximal blood lactate did not increase above 3.8 mmol/l, despite the final exercise intensity of 90% W, indicates that lactate clearance in these subjects must have been substantial. Blood glucose was significantly increased during days 4 and 5 with as a result of the high CHO intake, whereas it was maintained within the normal physiological range with N. The differences between N and were statistically significant, except at the moment of exhaustion on day 4. These differences most probably are due to the intake ad libitum of the CHO beverage started at the onset of exercise, which must have led to a continuous supply of CHO from the gastrointestinal tract to the blood. The intake of 300 g of CHO from the beverage supplemented during the rest days hardly influenced the blood glucose level. Oxidation and/or storage in endogenous energy depots at the same rate as entry in the blood may have been the reason for this. Insulin is not required for glucose uptake in the muscle cell during exercise, because of an insulin-like factor having the same effect on glucose transport through the muscle cell membrane. However, it is known that insulin in the presence of muscle contractions has an additive effect on glucose uptake. Therefore, it may be assumed that during the present study where both blood glucose and insulin were increased with (compared with N), substantial amounts of CHO will have been available for oxidation in the muscle cell and consequently must have induced glycogen sparing (in the liver and the muscle). A statistically significant difference in postexercise muscle glycogen level was observed after exercise on day 5 between N and. Subjects supplemented with showed a significant glycogen sparing, although cycling time to exhaustion during the final 90% Wmm, load lasted significantly longer in this group. In this respect, it is an interesting observation that exhaustion under treatment was not caused by a low level of muscle glycogen. Mean muscle glycogen at exhaustion in the group was greater than 250 pmol/g dry weight. Thus factors other than glycogen must have played a role in inducing the state of exhaustion. The availability of fatty acids is assumed to be of great importance for energy metabolism in the endurance athlete because enhanced fat oxidation may spare endogenous CHO stores. A high CHO intake makes a high lipolytic activity superfluous and a low CHO intake or glycogen depletion enhances the lipolytic rate via a number of hormones and mediating substrates. Therefore, the difference in CHO availability between N and must have had its impact on overall fat metabolism. The hormonal changes in the present study were all in favour of an enhanced lipolytic activity, especially during N treatment where energy balance became significantly negative on both exercise days. Plasma levels of glycerol, fatty acids and ketones increased significantly in this group as a result of the exercise. Since the uptake of free fatty acids (FFA) is related to muscle blood flow and the plasma fatty acid concentration, and observed R values declined as a result of exercise, it can be concluded that fatty acids have contributed substantially to energy exchange with N. The significant increase in plasma ketones may be directly related to the enhanced fat metabolism, since a high plasma fatty acid and a low insulin level enhance the rate of ketogenesis [Z]. With, fat metabolism was suppressed through the high CHO intake and the related metabolic changes. Although glycerol and FFA also increased significantly in this group, the magnitude of this increase was significantly smaller than that observed with N. The fact that a significant increase in lipolysis occurred also with, despite the high blood glucose and insulin level, can only be explained by the mutual action of factors that exert inhibiting and/or stimulating effects on lipolytic activity. The inhibiting effects of increased blood glucose and insulin levels may at the very moment have been overruled by the effect of an increase in catecholamines, glucagon and cortisol. Interestingly it has been observed that the plasma glycerol level at exhaustion on day 5 was not significantly different between N and. This coincides with disappearance of the significant differences between N and treatments in blood glucose and insulin despite the large CHO intake from the supplement in the group. So far it can be concluded that blood-borne fatty acids have contributed significantly to energy exchange despite the high level of CHO intake during exercise (Fig. 2). Protein metabolism is influenced by the concerted action of anabolic and catabolic regulators which are activated or inhibited by physical activity, energy balance, CHO availability and the level of daily protein intake. It is known that protein turnover is quite sensitive to the immediate energy supply. Low-energy intakes lead to a negative nitrogen balance, whereas high-energy intakes have a positive effect on nitrogen balance. Our results 365 1991

Biochemical Society Transactions 366 Fig. 2 Changes in CHO and fat metabolism as measured by substrate levels in blood Blood glucose and FFA are presented as absolute values. Ketones and glycerol are corrected for changes in plasma volume. Statistical significance with respect to the initial value on day 3 is indicated by *P < 0.05; *P < 0.0 I ; * P < 0.00 I. For FFA the initial value is day 4, 7.00 a.m. Statistical significance of with respect to N (each subject is his own control) is indicated by tp C 0.005. -, N; ---, ; a, exercise. Abbreviation: aceto A, aceto acetate. ** t t.. *** 3 4 5 6 Day show that plasma urea increased significantly, as a result of exercise and negative energy balance, in the subjects with N treatment. In contrast, plasma urea remained on a more-or-less stable level with *,* 1 and tended only to rise at the point of exhaustion on the second exercise day. Nitrogen losses were affected by exercise, leading to a negative nitrogen balance in N treatment, also during the recovery day (Table 1). The mean cumulative nitrogen balance (mean f s.e.m.) over 2 exercise days and the following recovery day was -8.69f2.5 g. In the group, there was a tendency to nitrogen sparing despite the significantly longer performance time at 90% Wmm, (-2.09f2.5 g). However, most probably due to the large variations, this effect did not reach significance. This difference in nitrogen balance between the treatments may entirely be explained by the effect of a negative energy balance and 'CHO balance'. From the data obtained it may be concluded that protein degradation and nitrogen loss from the body are increased in athletes during intense sustained exercise, and that this increase can be reduced by measures leading to a maintenance of energy balance and enhancement of CHO intake. Plasma ammonia was not different between the two treatments. The reason for this is not clear. Ammonia may be derived from the deamination of amino acids or from the intracellular adenylate pool. The latter takes place during exercise of high intensity, when immediate energy requirements exceed the amount of energy released from substrate degradation. Rises in plasma ammonia during exercise may be influenced by the availability of local glycogen stores. In our experiments, the subjects cycled until exhaustion. It may thus be that AMP deamination has played a role in ammonia production apart from the involvement of amino acid degradation for reasons of gluconeogenesis. Cycling performance quantified by the time that the subjects were able to perform at 80% and finally 90% W,,,, was substantially effected by the two treatments. The subjects increased their mean final 90% W,,, performance over 2 days from 9.9 min (k2.6 s) to 22.4 min (f7.9 s) when under treatment compared with N treatment. Muscle glycogen depletion with N may have been responsible for this difference. The subjects with N were not able to restore their muscle glycogen levels within 24 h of recovery. Assuming a similar recovery pattern, glycogen resynthesis during the 18 h of rest between days 4 and 5 will have amounted to approximately 75% of the measured value on day 6. This incomplete glycogen recovery may be related to the decreased performance of subjects with N on the second exercise day. In contrast, with, performance on day 5 was improved in five of the six subjects. Of particular Volume 19

Biochemistry of Exercise interest is the observation that subjects in the group were able to perform significantly longer, but became exhausted, without any relation to metabolic changes which normally are related to exhaustion, such as hypoglycaemia, high blood lactate, severe dehydration associated with hyperthermia and glycogen depletion. It may be that with glycogen sparing induced by CHO ingestion a shift occurs from glycogen, as limiting factor, to another unknown factor. It has been suggested that ammonia plays a role in processes of central fatigue and it is known that ammonia may influence a number of steps in the metabolic pathways of energy release [ 13, 141. In conclusion, the most important observations from our work can be summarized as follows: (i) elite athletes have changed eating patterns and consume 30-40% of total energy intake as small snacks in between meals, consisting mainly of energy-dense foodstuffs; (ii) with increasing energy expenditures, there is an increased selection of foods and liquids that are high in energy, sweet, and convenient; (iii) there is an energy threshold of approximately 20 MJ/day, above which athletes involved in prolonged sustained exercise are unable to consume enough normal food to maintain energy balance; (iv) this threshold occurs mainly through a decreased appetite and a limited tolerance of the stomach to a large food intake under exercise circumstances; (v) for the degree of exercise intensity described, the normally recommended CHO intake of 60% energy was not sufficient to achieve complete recovery of muscle glycogen within 24 h; (vi) the supplementation of a CHO beverage, in addition to the normal meals and during exercise, was shown to be an effective measure for the maintenance of energy- and CHO-balance at higher energy expenditure levels; (vii) under the exercise circumstances described, a protein intake of > 1.5 g/k per day was required to maintain nitrogen balance; (viii) when energy balance was negative, 1.7 g of proteidkg per day was insufficient to maintain nitrogen balance; and (ix) an increased CHO intake induced protein sparing. This work was supported by an Isostar Research Grant from Wander Ltd, Bern, Switzerland. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Christensen, E. H. & Hansen, 0. (1939) Scand. Arch. Physiol. 81, 152-159 Newsholme, E. A. (1976) Clin. Endocrinol. Metab. 5, 543-578 McGilvery, R. W. (1973) in Metabolic Adaptions to Prolonged Physical Exercise, pp. 12-20, Birkhauser Verlag Basel Jansson, E. & Kaijser, L. (1984) Acta Physiol. Scand. 122,145-153 Bergstrom, J. & Hultman, E. (1967) Scand. J. Clin. Lab. Invest. 19,218-228 Bergstrom, J., Hermansen, L. & Hultman, E. (1967a) Acta Physiol. Scand. 71,140-150 Christensen, E. H. & Hansen, 0. (1939) Scand. Arch. Physiol. 81, 180-189 Christensen, E. H. & Hansen, 0. (1939) Scand. Arch. Physiol. 81, 160-171 Saris, W. H. M., van Erp-Baart, M. A. & Brouns, F. (1989) Int. J. Sports Med. 10, S26-S31 Brouns, F., Saris, W. H. M. & Stroeken, J. (1989) Int. J. Sports Med. 10, S32-S40 Brouns, F., Saris, W. H. M. & Stroeken, J. (1989) Int. J. Sports Med. 10, S41-S48 Brouns, F., Saris, W. H. M. & Beckers, E. (1989) Int. J. Sports Med. 10, S49462 Brouns, F., Beckers, E., Wagenmakers, A. J. M. & Saris, W. H. M. (1990) Int. J. Sports Med. 11, S78-S84 Wagenmakers, A. J. M., Coakley, J. H. & Edwards, R. H. T. (1990) Int. J. Sports Med. 11, S101-S113 Received 29 November 1990 367 1991