INTRODUCTION

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AT REST, THE END PRODUCT OF energy metabolism of red blood cells (RBC) is lactate from anaerobic glycolysis. This lactate is dissociated into a lactate anion and a proton, which are transported out of the RBC and further into other tissues, such as muscle and liver, to be metabolized. During intense exercise, lactic acid is produced also in skeletal muscles. Lactate and hydrogen ions are released into the circulation by a carrier-mediated system (4), which minimizes the accumulation of lactate and protons in muscles and prolongs the muscle's capacity for anaerobic work. From plasma, lactate may be transported into liver, heart, and noncontractive tissues for oxidation, or it may be transported into RBCs. It has been speculated that in horses during maximal exercise, when the rate of lactate oxidation is exceeded, RBCs act as a sink for lactate (11, 14) and thus contribute to the plasma-muscle concentration difference that is the driving force for lactate efflux from working muscle.

Lactate is transported across the RBC membrane by three distinct pathways. The undissociated lactic acid may diffuse across the cell membranes, whereas the lactate anion needs a carrier protein. On the RBC membrane, two carrier proteins have been identified: the monocarboxylate transporter (MCT), which cotransports a lactate anion and a proton, and the inorganic anion-exchange transporter (band-3 protein), which is an antiport carrier and exchanges lactate for Cl or HCO (10). In animals with a sedentary lifestyle, such as sheep and goats, the main routes for lactate transport are nonionic diffusion and the inorganic anion-exchange system (15). In active animals, such as dogs, MCT represents the main route for lactate transport, and the importance of the other two mechanisms is minor (15).

In the RBCs of another active species, horses, inter-individual variation is large both in total lactate-transport activity and also in the capacity of different lactate-transport mechanisms (17). In 70% of horses studied, the total lactate-transport activity has been high, and in these horses, MCT appeared to be the main carrier. In the other 30%, total lactate-transport activity was low with no or only low activity of MCT detected. In these horses, the main routes for lactate transport were the band-3 protein and nonionic diffusion. Although the transport of lactate into the RBC of humans has been studied extensively (7, 10), to our knowledge, comparisons with other active species or between athletes and sedentary individuals have not been reported.

The aim of the present study was to investigate whether total lactate-ransport activity and the different transport mechanisms in the RBC of three other species (reindeer, humans, and dogs) show similar interindividual variation as found in horses. These four were chosen because athletes can be found among each of these species. Furthermore, individuals from each of these species participate in races that require maximal performance. The second aim of this study was to compare the lactate-transport activity of trained and sedentary individuals within each species. Both sprint- and endurance-trained individuals were included to study the role of high lactate production on the possible training effect. Lactate-transport activity was studied at 10 and 30 mM lactate concentrations, which were selected because 30 mM represents maximal plasma lactate concentrations measured in human athletes, dogs, reindeer, and horses, and 10 mM represents plasma lactate concentrations seen after submaximal exercise bouts (5, 7, 12, 13).

	MATERIAL AND METHODS

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Animals

Reindeer (Rangifer tarandus tarandus). Twenty-one clinically healthy reindeer were used (14 females), ages 4-12 yr, from the experimental herd of the Reindeer Herder Association (Kaamanen, Finland) and seven privately owned racing reindeer, ages 4-10 yr. Samples from the racing reindeer were taken in April, the end of the racing season. All the racing reindeer had been in regular training for several winters and at least for 5 mo before sampling. They all belonged to the group of 24 best racing reindeer in Finland and were thus considered well trained.

Dogs (Canis familiaris). Three groups of privately owned dogs were studied: 14 domestic dogs (2 males, 12 females), 6 greyhounds (2 males, 4 females), and 18 sled dogs (9 males, 9 females). All these dogs were adults, ages 1-12 yr. The breeds of the dogs were smooth fox terrier, springer spaniel, Karelian bearhound, Bernese sennenhund, Leonberger, Bastard, German shepherd, Rottweiler, Labrador retriever, collie, Alaskan husky, Siberian husky, greyhound, saluki, and Afghaner. The sedentary dogs were not regularly trained, but they were walked daily and allowed to move freely in the yard. The greyhounds were trained for racing by their owners. They were trained on a racetrack 1-2 times/wk and otherwise allowed to move freely. The amount of exercise was variable, and none of the dogs was yet in a condition as to compete in an actual race. These dogs were considered moderately trained. From sled dogs, one blood sample was taken before training and another after a 13-wk training program. Every week the duration of training was gradually increased, so that in the first week, the dogs were trained on the treadmill 2 × 40 min at 15 km/h and in the field 3 × 10 km at 15 km/h and in the 13th training wk on the treadmill 3 h at 15 km/h and in the field 2 × 40 km at 15 km/h and 1 × 20 km at 15 km/h. At the end of the training program, the sled dogs were well trained but had not yet reached their maximal performance level.

Horses (Equus caballus). The three groups of horses were comprised of 16 sedentary Standardbred brood mares and 3 stallions (ages 6-18 yr), 8 endurance-trained horses [4 stallions, 2 geldings, and 2 mares (ages 5-10 yr)], and 13 Standardbred trotters [8 stallions, 4 geldings, and 1 mare (ages 4-7 yr)]. The endurance-trained horses were in such a condition as to compete in a 50-km race. The trotters were actively racing and were considered well trained.

Human subjects (Homo sapiens). Three groups of human subjects (ages 18-56 yr) were studied: 8 national-level competing male sprinters, 8 international-level male endurance athletes, and 23 nonathletes (2 males and 21 females). Within the last group, physical activity ranged from low to moderate.

Blood Samples

Methods

Lactate influx was measured at lactate concentrations of 10 and 30 mM. The suspension of RBC was divided into three parts. One portion contained no inhibitors, one contained 5 mM -cyano-4-hydroxycinnamic acid (CHC; Sigma Chemicals, St. Louis, MO), and the last portion contained DIDS (Sigma). The same volume of buffer or stock solutions of CHC and DIDS was added to the RBC suspensions. At the concentrations used, CHC inhibits mainly the MCT, and DIDS inhibits the band-3 protein (2, 10). Lactate influx activity was measured as accumulation of radioactive lactic acid [L-(U-C) lactic acid sodium salt, 5.62 GBq/mmol; Amersham, Buckinghamshire, UK] in RBC. All measurements were made in triplicate; the incubation time was 20 s, temperature was 37°C, and pH 7.4. The radioactive lactic acid in RBCs was measured by a liquid scintillation counter (Rack-Beta Scintillation Counter, model 1217 and Winspectral 1414, Wallac, Turku, Finland). Hemoglobin concentrations, hematocrits, and RBC volumes were measured by an automatic counter (Coulter counter, model T 850; Coulter Electronics, Hialeah, FL). Results are expressed as nanomoles lactate per milligram RBC protein per minute. Protein concentration was estimated from hemoglobin concentration (15). The results were calculated also as nanomoles lactate per milliliter RBCs per minute.

Statistics

	RESULTS

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Figure 1 shows total lactate-transport activity in the four species at 10 and 30 mM lactate concentrations. As previously (16, 17), the horses could be divided into two groups according to their lactate-transport activity: low (LT)- and high-activity (HT) groups. Because of this bimodal distribution, these two groups of horses will be treated separately. At 30 mM lactate concentration (Fig. 1B), lactate-transport activity in reindeer and LT horses was similar and lower (P < 0.001) than in humans, dogs, and HT horses. Activity in dogs was higher (P < 0.001) than in HT horses and humans. No statistical difference was found between HT horses and humans. When results at 10 mM lactate concentration were compared with those at 30 mM lactate, the total lactate-transport activity was lower, but the statistical differences between species remained the same (Fig. 1A). However, in dogs, the activity at 10 mM lactate concentration in relation to that at 30 mM was higher (67%) than in the other species (reindeer 10%, humans 50%, HT horses 46% and LT horses 29%). RBC volumes (MCV) and the lactate-transport activities calculated as nanomoles lactate per milliliter RBC per minute are shown in Table 1. Despite the large interspecies differences in MCV, the interspecies differences in total lactate-transport activities were the same as those described above.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   The total lactate transport activity (nmol lactate/mg protein · min) in red blood cells of reindeer (n = 21), horses (n = 40), humans (n = 31), and dogs (n = 38) at 10 mM (A) and at 30 mM (B) lactate concentration. Because of the bimodal distribution of lactate-transport activity, the horses are divided into 2 groups: horses with high lactate-transport activity (HT) and horses with low lactate-transport activity (LT).

High inhibition by CHC indicates that MCT was the main transporter in HT horses, humans, and dogs (Fig. 2, Table 2). In reindeer and LT horses, neither CHC or DIDS inhibited transport, so the influx occurred mainly by nonionic diffusion. Actually, in reindeer, the inhibitors, CHC and DIDS, did stimulate the influx of lactate. This stimulation was seen at both lactate concentrations, 10 (results not shown) and 30 mM (Table 2), and it also persisted when inhibitor concentrations were lowered to 1 mM CHC and 0.050 mM DIDS (results not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Roles of lactate transporters in red blood cell membranes with inhibitors of monocarboxylate transporter and inorganic anion-exchange transporter. , activity in the presence of 5 mM -cyano-4-hydroxycinnamic acid and 30 mM lactate; , activity in the presence of 0.2 mM DIDS and 30 mM lactate.

In horses, the distribution was bimodal, but in the other species, activity appeared to be normally distributed (Fig. 3); in the domestic dogs, the sprint-trained greyhounds and the endurance-trained sled dogs, lactate-transport activity was similar, but in the sedentary sled dogs, influx activity was lower (P < 0.001) than that in trained sled dogs. A training effect was also seen in the reindeer. In racing reindeer, lactate influx activity was higher (P < 0.001) than in nonracing reindeer.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   The frequency distribution of lactate-transport activity in red blood cells of trained and sedentary individuals. Reindeer: open bars, adult reindeer; crosshatched bars, racing reindeer. Horses: open bars, sedentary horses; filled bars, endurance horses; crosshatched bars, racing horses. Humans: open bars, sedentary individuals; filled bars, endurance-trained athletes; crosshatched bars, sprint-trained athletes. Dogs: open bars, sedentary dogs; hatched bars, sled dogs before training; filled bars, sled dogs after 13-wk training program; crosshatched bars, sprint-trained greyhounds.

	DISCUSSION

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The major finding in this study was the difference in total lactate-transport activity and in the role of the transport mechanisms in the RBC of reindeer, horses, humans, and dogs. This finding confirms species differences found earlier (2, 15). Total lactate-transport activity of these species showed the following order from low to high: reindeer, horses, humans, dogs. At 10 and 30 mM lactate concentrations, the mean lactate-transport activity in the dogs was 56 and 8 times as great as that in the reindeer, respectively. These findings are supported by the Michaelis constant (K_m) values for lactate transport. At pH 7.4, the dog had the lowest K_m (6.6 mM) followed by humans (9.14 and 13.4 mM) and horses (24.4 mM) (3, 4, 15, 17). An attempt to measure the K_m of lactate transport in reindeer RBC failed because of the low activity of the transporters. Interestingly, glucose utilization in RBC follows the same order, from reindeer to dog (9, and see also M. Koskinen, unpublished results). It is obvious that in RBCs, some connection exists between the rate of glucose utilization and the efflux of lactate, which is the end product of glucose metabolism in RBCs, although the differences in lactate-transport activity between species are much greater than the reported differences in glucose consumption.

The role of different transporters was studied with inhibitors, CHC and DIDS. At the concentrations used, CHC inhibits mainly MCT, and DIDS inhibits the band-3 protein (10). The present results show that in dogs and humans, MCT was the main transporter, representing 70-80% of total lactate transport. In reindeer, the total lactate-transport activity was low, and MCT was either inactive or nonexistent. The reindeer is a ruminant, and our result is in accordance with another study showing low lactate-transport activity in the sheep and goat, two other ruminant species (15). In reindeer, the inhibitors of MCT and band-3 protein did not inhibit lactate influx; on the contrary, they stimulated it. This stimulation was seen at moderate, 10 mM, and high, 30 mM, lactate concentrations, and the stimulation persisted also when the inhibitor concentrations were lowered. On the basis of these results, it is not possible to explain this stimulation, but it can be speculated that there are species differences in the binding of inhibitors to the proteins on the RBC membrane. This stimulation is characteristic of reindeer, because it has been shown (M. Koskinen, unpublished results) that in other ruminants such as cow, sheep, and goat, the inhibitors, CHC and DIDS, did not stimulate lactate influx.

The lactate-transport activity in the RBC of horses is interesting, because its distribution differed from that in dogs, humans, and reindeer. According to our earlier study, the transport activity in horses varies interindividually, and horses can be divided into two groups according to their lactate-transport activity: the low- and high-transport activity groups (16, 17). In the present study as well, this bimodal distribution in lactate transport was apparent in horses, whereas in the other species, the activity was normally distributed. In the HT horses it was similar to the activity in dogs and humans, and in all these, MCT was the main transporter. In the LT horses instead, it was as low as in the reindeer. In these animals, either the band-3 protein or nonionic diffusion is the main lactate-transport mechanism, and MCT is either inactive or nonexistent. Skelton et al. (15) have found that MCT is the primary pathway in RBCs of species with an active lifestyle; a low level of MCT correlates with the inactive lifestyles of sedentary species. In our study, the low lactate-transport activity in some of the horses and in reindeer does not support this view. In this study, the LT horses were active and racing for distances of 1,600-2,600 m with fair success. As with horses, also the reindeer, especially the racing reindeer that race for distances of 1,000 and 2,000 m, spend an active life moving about freely and have good endurance and sprint capacity (12, 13). The reindeer results, together with the results from other ruminants (15), indicate that lactate-transport activity is related more to the general physiology of ruminants than to their physical activity. This does not, however, explain the bimodal distribution of the lactate-transport activity in horses.

At rest, the flux of lactate is from RBCs into plasma, but during intense exercise, the flux may be reversed. Individuals from each of the four species studied here, compete in races that require maximal performance and increase plasma lactate concentrations up to 30 mM (5, 7, 11, 13). We have speculated previously that in horses, during maximal exercise, when the rate of lactate oxidation is exeeded, RBCs may act as a sink for lactate (11). Whether this is true for all species is not known, because there are species differences in the amount of lactate taken up by RBCs. Juel at al. (8) have calculated that only 17% of lactate can be found in the RBCs of humans, whereas our earlier study with horses showed that up to one-half of the blood lactate is transported into RBCs (16). Because lactate-transport activity in humans is similar to that in horses, the activity as such does not indicate the importance of RBCs as a lactate sink. Skelton et al. (15) suggested that the high activity of lactate transport in dogs is an indication of a high number of lactate-transport proteins on their RBC membranes. Likewise, the interindividual variation in horses suggests that horses may have different concentrations of lactate-transport proteins on their RBC membranes. However, our recent results do not support this view, because in horses, MCT activity does not correlate with the amount of MCT protein (N. Koho, L. K. Väihkönen, and A. R. Pösö, unpublished results). We have shown in our earlier study that after maximal exercise (trotting races), the horses with high MCT activity have more lactate in their RBCs than the horses with low MCT activity (16). Previously, Räsänen et al. (14) have shown that after exercise, those horses with high individual performance indexes have the highest concentration of RBC lactate. The individual performance index is calculated annually by the Finnish Trotting and Breeding association for 3- to 5-yr-old horses of the same sex and age from performance traits. In the present study, however, there are actively racing horses with reasonably high individual performance indexes in both the low- and the high-MCT activity groups. Thus, on the basis of this finding, MCT activity does not directly correlate with performance capacity in horses. Together, these studies indicate that performance capacity is complex and cannot be predicted with one single parameter.

The second major finding in this study was that the effect of training on lactate-transport activity among the species studied is not similar. A training-induced increase was found in reindeer and in sled dogs, but not in humans and horses. According to our earlier sudy, lactate-transport activity is similar in suckling foals and racing trotters (17), which indicates that in horses, physical activity/training plays a minor role in the regulation of activity of lactate transporters in RBCs. In the present study, the racing trotters and endurance-trained horses showed lactate-transport activity similar to that of the sedentary horses. Among both our racing and sedentary horses, one-third belonged to the low lactate-transport activity group, and two-thirds belonged to the high lactate-transport activity group. Arai et al. (1) have shown that glucose utilization is higher in RBCs of trained race horses than in untrained horses. This finding, together with our present results, indicates that in RBCs, changes in lactate production and in lactate transport do not occur in parallel. In humans as well, the trained athletes had lactate-transport activity similar to that of the sedentary individuals. In dogs, the situation was more complicated. The domestic dogs had lactate-transport activity similar to that of trained greyhounds, which have enormous maximal sprint capacity, whereas in the sled dogs, training increased lactate-transport activity. It is difficult to explain this result, because the amounts of daily exercise of the domestic dogs and of the greyhounds were not recorded, and only the sled dogs participated in a controlled training program. Thus it is possible that a similar effect of training might also be found in other breeds of dogs.

Another species in which training had an effect is reindeer, because in the ordinary reindeer, the lactate-transport activity was lower than in the racing reindeer. Together, these results on dogs and reindeer indicate that the training effect may be found in species with both low and high transport activity. On the basis of the studies on muscle lactate transport (7), it is plausible that the training effect is modified both by the intensity and duration of training. To our knowledge, the effect of training on lactate transport in RBCs has not been previously studied. One can speculate that only training with long duration has an effect on RBC, because the life span of RBCs is 100-120 days and no protein synthesis takes place in the mature erythrocyte. However, the duration of training is not the only determinant. In our study, sprint- and endurance-trained human athletes and horses had been in regular training longer than the life span of RBCs and did not show any training effect, but a similar or even shorter training period caused an increase in the lactate-transport activity in racing reindeer and sled dogs. Also, the intensity of training that causes increases in lactate-transport activity varied, because training effect was seen in sprint-trained racing reindeer and endurance-trained sled dogs. This indicates that high lactate concentration during training sessions is not a prerequisite for an increase in the lactate transport in RBC. Thus further studies on the properties of lactate transporters in each of these species are warranted to clarify the mechanism of the training effect and also the importance of lactate transport in RBCs in exercise performance.

In summary, large differences exist in lactate-transport activity among the four species studied, and in the horses, also interindividually. MCT activity did not show association to the lifestyle of the species or individuals, as shown by the data on the reindeer and the LT horses. The effect of training also differed between species. In horses and humans, training had no effect on lactate-transport activity, but in the reindeer and sled dogs, training-induced increase was evident.

Perspectives

Studies on horses have shown that at high plasma lactate concentration, the activity of MCT may regulate the influx of lactate into RBCs, but whether this has any physiological function or influence on the performance capacity is not known. Although the racing success of the two groups of horses with highly different MCT activity was about the same, it would be interesting to know whether a horse with low MCT activity could ever make it to the very top. Comparison of the studies with human and equine RBCs indicate that in addition to the activity of MCT, other factors influence the amount of lactate transported into RBCs. In humans, 17% of blood lactate is in RBCs, whereas in horses with equally high MCT activity, the percentage may even be 50. The training effect that was seen in dogs and reindeer supports the view that during exercise accumulation of lactate in RBCs may be beneficial, but again, it has to be asked why the effects of training are not apparent in all species. The question is especially interesting for horses, because our unpublished data indicate that in a horse, born with a low MCT activity, the activity will remain low despite training and racing.

Altogether, the results of this study raise new questions on the kinetics of blood lactate, and further studies are needed to clarify the physiological influences of RBCs in lactate metabolism.