264   Relationship of ergometer-specific VO2 max and muscle enzymes to blood lactate during submaximal exercise

ABSTRACT

 This study compared the relationship of maximum oxygen uptake and skeletal muscle enzyme activities to the submaximal exercise intensity eliciting 4 mM blood lactate (OBLA). Twelve subjects performed both cycle (Cy) and treadmill (Tr) submaximal exercise with step-wise increments each fourth minute. Blood lactate concentration and oxygen uptake (W02) were determined during the final minute of each step. Peak V02 during exhaustive exercise was also measured on each ergometer. Biopsies were taken from the gastrocnemius (gast) and vastus lateralis (vl) muscles as representatives of muscles recruited during Tr and Cy exercise, respectively. Citrate synthase (CS), phosphofructokinase (PFK), and lactate dehydrogenase (LDH) activities were assayed. Peak 02 was 10% greater and the V02 at OBLA was 16% greater during Tr compared to Cy exercise. The percent of peak V02 at OBLA was 85% and 79% for Tr and Cy exercise, respectively. The absolute enzyme activities were not different in the two muscles, however the ratio LDH/CS was greater in the v1 than in the gast. The results indicate that the absolute differences between Cy and Tr exercise in peak V02 are not commensurate with the differences in the relative exercise intensity at which OBLA occurs.

INTRODUCTION

 Exercise testing in the laboratory has traditionally included the determination of maximal oxygen uptake (W02 max) as an indication of the capacity for endurance exercise (Saltin and Astrand, 1967). The cycle (Cy) and the treadmill (Tr) are the two ergometers most commonly used for this purpose. Recently, maximal endurance exercise performance has been shown to be closely related to physiological measurements during submaximal exercise. In particular, it has been repeatedly demonstrated that an individual's endurance is correlated more strongly with the blood lactate response to submaximal exercise than with V02 max (Farrell et al, 1979; Kumagai et al, 1982; Hagberg and Coyle, 1983). In light of the increasing popularity of using blood lactate related variables in the evaluation of aerobic fitness, the present study was designed to compare the blood lactate response to continuous, progressive exercise on the Cy and Tr ergometers. Specifically, the exercise intensities, both the absolute oxygen uptake (V02) and V02 relative to V02 max, which corresponded to a blood lactate concentration of 4 mM (OBLA = onset of blood lactate accumulation) on each ergometer, were compared. It was hypothesised that any observed difference in this lactate determined intensity would be related to and commensurate with the difference in V02 max measured between the two ergometers. It has been previously shown that OBLA, in addition to being strongly correlated with marathon running performance (Sjodin and Jacobs, 1981), is also related to several metabolic characteristics of the muscle cell such as fibre type, capillary density, and key enzyme activities (Sj6din and Jacobs, 1981; Sjodin et al, 1981). Thus, it was also speculated that any differences in OBLA between Cy and Tr exercise would be reflected in differences in enzyme activities between the muscles chosen to be representative of the predominant muscle groups recruited during Cy or Tr exercise, i.e. the vastus lateralis and lateral gastrocnemius, respectively.

METHODS 

The details of the experimental protocol and all associated risks or discomforts were explained to volunteers before obtaining their informed consent to participate as subjects. The protocol was approved by the Karolinska Hospital's Human Ethics Committee. The subjects were 12 males with a mean (± SD) age, height and weight of 23 ± 5 years, 180 ± 6 cm, and 75 ± 9 kg, respectively. 

Muscle sampling. 

The subjects visited the laboratory on three occasions. On the first occasion two muscle samples were obtained with the needle biopsy technique (Bergstrom, 1962) from both the vastus lateralis and the lateral head of the gastrocnemius. One tissue sample from each muscle was mounted in an embedding medium and frozen in isopentane cooled with liquid nitrogen for later histochemical analyses. The second sample, for biochemical assays, was frozen directly in liquid nitrogen. The tissues were stored at -800C until analysed. Muscle fibres were classified as slow twitch (ST) or fast twitch (FT) after staining for myofibrillar ATPase activity at a pH of 9.4, following preincubation at pH 10.3 (Guth and Samaha, 1970). Photographs of serial sections stained for NADH-tetrazolium reductase (Novikoff, 1961) were used to calculate the mean fibre area and the relative area occupied by each fibre type (Thorstensson, 1976; Tesch, 1980). The second tissue specimen from each muscle was freeze dried. Remaining visible impurities, connective tissue and blood clottings were dissected out before weighing the tissue on a Cahn® electrobalance. The tissue was then homogenised by sonication in an ice-cooled medium of 0.1 M phosphate buffer, pH 7.3. The activities of phosphofructokinase (PFK, EC 2.7.1.11), lactate dehydrogenase (LDH, EC 1.1.1.27), and citrate synthase (CS, EC 4.1.3.7) were determined with assays based on fluorometric techniques (Essdn et al, 1980). 

Submaximal exercise.

 Since most subjects cannot attain the same value for V02 max during both Cy and Tr exercise, we will refer to the ergometer-specific V02 max as "peak V02 Cycle ergometer or treadmill tests were performed one and two weeks after the biopsies, with the order of testing being counter-balanced among subjects. Submaximal and maximal test protocols were administered for each ergometer. A warmup was performed for 5 minutes on the Tr at an intensity estimated to require between 40 to 50% of peak V02. Both submaximal exercise protocols were continuous with intensity increments every fourth minute of -50 W for the Cy and 0.5 to 2 km.h-1 for the Tr. The initial exercise intensity was 50 W on the Cy and 12 to 14 km.h-1 on the Tr, which was 0.5 to 

1 km.h-1 faster than the warm-up velocity. Exercise was halted when subjects rated their perceived leg exertion during Cy exercise, or whole body exertion during running, as greater than six on the 10-point scale of ratings of perceived exertion (RPE) developed by Borg (1982). It has been previously reported that almost all subjects accumulate blood lactate concentrations greater than 4 mM at this RPE (Jacobs, 1981; Noble et al, 1983). Expired air was collected in Douglas bags during the final minute at each intensity and transferred into a wet spirometer for volume determination. The fractions of CO2 and 02 were analysed with a mass spectrometer (Centronics® MGA200) to enable the calculation Of '02. Blood samples (25 Ml) were taken from the finger-tip while the subject continued to exercise during the final 30 s at each intensity. The blood was deproteinised immediately and stored under refrigeration until assayed for lactate concentration with a fluorometric enzymatic technique (Karlsson et al, 1983). By plotting the relationship of lactate to exercise intensity for each subject, OBLA was interpolated as previously described (Jacobs, 1981). 

Maximal exercise. 

A 30 min uncontrolled recovery period followed each submaximal exercise test after which peak '702 was determined with a continuous, progressive protocol until voluntary exhaustion. For the Tr peak '702 determination, running speed remained constant while slope increased stepwise by 0.5 degrees. 30 s-'. For the Cy peak '02 determination, exercise commenced at 100.250 W and increased stepwise by 50 W every two minutes until voluntary exhaustion. Blood was sampled for lactate determination four minutes after the maximal exercise tests. 

Statistics.

 The BMDP Statistical Software package was used for all calculations (Dixon and Brown, 1977). Student's t-test for paired observations was used to determine the significance of differences between mean values. Statistical significance was set at the 0.05 level. 

RESULTS 

Maximal Exercise

Peak V 2? was higher during Tr exercise (mean ± SD, 66 ± 8 ml.kg' min-') than during Cy exercise (60 ± 6 ml.kg1' minr ) (p = 0.007). There was a moderate but significant correlation between individual peak '02 values on the two ergometers with 10 of the 12 subjects demonstrating a higher peak '02 on the Tr (r = 0.61, p < 0.05) (Fig. 1). During Tr exercise the "levelling-off" criterion for'V02 was observed in nine of the 12 subjects since '702 decreased, remained the same, or increased by less than 2 ml.kgt1 min1 during the last 30 s of exercise in spite of the increased Tr elevations. Using the same criteria "levelling-off" was observed in only seven subjects during the maximal Cy exercise. All subjects stated that leg fatigue limited their ability to continue

Cy exercise. Peak blood lactate levels four minutes after the maximal tests were 9.8 ± 1.6 and 10.3 ± 1.2 mM for Tr and Cy exercise, respectively, and these were not significantly different. Submaximal Exercise. The relationship between blood lactate and steady state V02 is depicted in Figure 2.

Fig. 2: Relationship of blood lactate to oxygen uptake during submaxinal cycle and treadmill exercise.

Using OBLA as a reference point, V02 was 16% higher during Tr than Cy exercise (p < 0.001). The relative utilisation of peak V02 (% peak V02) at OBLA was 85% Tr peak V02 and 79% Cy peak V02 (p < 0.03) (Fig. 3, Table 1).

Fig. 3: Relationship of blood lactate to the relative utilisation ofpea '02 during submaximal cycle and treadmill exercise. The arrows indicate the % peak 1702 at 4 mM lactate.

The Y/02 at OBLA was not significantly correlated to peak '02 on either ergometer. Nonsignificant inter-ergometer correlation coefficients were observed for the V02 at OBLA, the % peak '02 at OBLA, and the absolute exercise intensity at OBLA (i.e. W.kg 1 for the Cy vs. m.s71 for the Tr).

Muscle characteristics. The frequency of ST fibres in the two muscles sampled is shown in Figure 4 and the mean values did not differ significantly.

The mean FT and ST fibre areas were 5511 ± ± 1157Mm2, respectively, in the vastus ± 2135 and 5318 ± 1647 pm2 in the gastrocnemius. ences in mean fibre area between muscles were not significant nor was the percent muscle area occupied by a given fibre type. Not all enzyme assays could be performed for both muscles of four of the subjects due to the small biopsy specimen size and their enzyme data has been excluded from the statistical analyses. The two muscles did not differ in the absolute activity levels of CS, PFK, and LDH, but the ratio of the glycolytic enzymes, LDH or PFK, to the oxidative enzyme marker, CS, did differ significantly. The vastus lateralis demonstrated a higher ratio suggesting a higher glycolytic relative to oxidative activity potential than the gastrocnemius (Fig. 5).

Fig. Activities of citrate synthase (CSJ, phosphofructokinase (PFK), lactate dehydrogenase (LDH), and the LDH/CS ratio in the gestrocnemius and vastus lateralii Staples represent mean and SD. The difference between the ergometers in either the absolute V02 or % peak V02 at OBLA was not significantly related to the absolute difference between ergometers in the measured peak V02k.

DISCUSSION

 The present study has clearly demonstrated that a blood lactate concentration of 4 mM occurs at a higher steady state V02 during Tr running than during Cy exercise. This is consistent with several earlier investigations (Hermansen and Saltin, 1969; Koyal et al, 1976; Kindermann et al, 1980). The 9% greater peak )702 during Tr exercise is similar to previous investigations (Hermansen and Saltin, 1969; Kindermann et al, 1980). This higher Tr peak V02 has been attributed to a larger stroke volume leading to a greater cardiac output when compared to maximal Cy exercise (Hermansen and Saltin, 1969; Hermansen et al, 1970). It was hypothesised that these functional differences would affect OBLA to the same extent that they affect the oxygen transport system. The end result would be a similar % peak V02 corresponding to a given lactate concentration during both forms of exercise. If such were the case the higher lactate level at a given absolute V02 during Cy exercise could possibly be explained by the assumed smaller muscle mass being required to perform the same work as during Tr exercise. This, however, was not the case. The relative difference between ergometers in the V02 at OBLA was almost twice as great as the difference in peak V02 (16% vs. 9%)0 similar to what was reported by Kindermann et al (1980). The end result was the significantly higher % peak V02 at OBLA during Tr exercise. In order to evaluate the relationship of some metabolic characteristics of the exercising muscle to the inter-ergometer difference observed in the blood lactate responses the lateral head of the gastrocnemius was chosen to be representative of running, and the vastus lateralis to be representative of cycling. The choice may be questioned since the vastus lateralis is recruited also during running. However, muscle substrate depletion studies on biopsies taken from various leg muscles after Tr running indicate a far greater recruitment of the gastrocnemius (Costill et al, 1974). It should be emphasised that the gastrocnemius and the vastus lateralis are only considered to be representative of the predominant muscle groups involved in each type of exercise. Although they did not present any experimental evidence, Kindermann et al (1980) suggested that a lower oxidative capacity of the exercising musculature during Cy exercise could account for the earlier onset of lactate accumulation during this form of exercise. This suggestion is supported in the present study where the activities of both glycolytic and oxidative "marker" enzymes have been assayed. Although the absolute enzyme activities did not differ between muscles, the ratio of the glycolytic to oxidative activities suggested a relatively greater oxidative potential in the gastrocnemius than in the vastus lateralis. Green et al (1981) also reported higher LDH activities in the vastus lateralis than in the gastrocnemius, although oxidative enzyme activities were similar in the two muscles. The theoretical metabolic consequence is a greater capacity to oxidise pyruvate through oxidative phosphorylation and consequently a delayed production and accumulation of lactate in muscle and blood. This ratio has been shown previously to be directly related to the exercise intensity at OBLA (Sjodin et al, 1981). It still remains to be demonstrated that the higher blood lactate concentrations at a given submaximal V02 during Cy exercise are reflective of greater intramuscular lactate production when compared to the same V02 during Tr exercise. An alternative explanation of the "delayed" accumulation of blood lactate during a specific form of exercise may revolve around the rate of lactate clearance from muscle and blood (Donovan and Brooks, 1983). It may be speculated that Tr running is associated with a larger active muscle mass, greater capillarisation and blood flow in the active muscles, and a metabolic profile in the exercising musculature which favours lactate oxidation and/or gluconeogenesis from lactate to a greater extent than the musculature involved during Cy exercise. All of these factors could directly affect the rate of lactate clearance from muscle tissue and blood. In conclusion, the steady state V02 at OBLA was significantly higher during Tr than during Cy exercise. This difference was greater than could be accounted for by the difference between exercise modes in peak V02. Consequently, OBLA occurred at a higher % peak °02 during Tr running than during Cy exercise. There are indications that this difference may be due, at least partially, to differences in the metabolic profile of the exercising musculature, as reflected by the ratio of key oxidative to glycolytic enzyme activities, in addition to the established differences in certain cardiovascular variables associated with Tr and Cy exercise.