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. 2002 Jun 1;541(Pt 2):575–580. doi: 10.1113/jphysiol.2002.019216

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

Hans N Rasmussen *, Gerrit van Hall †, Ulla F Rasmussen *
PMCID: PMC2290332  PMID: 12042361

Abstract

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate.

In skeletal muscle, lactate formation appears to occur under all conditions, even at rest. Lactate may be released into the circulation and taken up from the blood by the liver, heart and skeletal muscles. At increased blood levels, it is taken up by working as well as resting skeletal muscles (Richter et al. 1988). The lactate taken up by the muscles may be oxidized to carbon dioxide and water or used for glycogenesis. In both cases pyruvate is the first product. For recent reviews on the dynamic lactate metabolism in muscles, see Brooks (1998) and van Hall (2000).

The formation of pyruvate from lactate is catalysed by lactate dehydrogenase (LDH), which is present in the sarcoplasm at very high levels. Some LDH activity has, however, been reported in the mitochondrial fraction from muscle homogenates (e.g. Brandt et al. 1987; Szczesna-Kaczmarek, 1990; Brooks et al. 1999). This has led to the proposal of an intracellular lactate shuttle, which involves transport of lactate into the mitochondrial matrix followed by oxidation to pyruvate, catalysed by LDH (Brooks et al. 1999). The model is proposed to explain the oxidation of lactate in muscle. However, the direction of the LDH reaction is determined by the lactate/pyruvate ratio multiplied by the NAD/NADH ratio. Lactate oxidation only occurs if this mass action ratio is larger than the equilibrium constant. The likely steady-state NAD/NADH ratio in the cytoplasm is of the order of 1000 and in the mitochondria less than 10 (e.g. Newsholme & Leech, 1983). The lactate/pyruvate ratio that causes lactate oxidation, therefore, is ∼100 times higher in the mitochondria than in the cytoplasm. Accordingly, lactate oxidation is basically much more likely in the cytoplasm than in the mitochondria. It might even be anticipated that within the mitochondria pyruvate would become reduced and NADH oxidized, if LDH was present; this would deprive the Krebs cycle and the respiratory chain of their main substrates. Thus the model has major consequences for the concept of intercellular metabolism, and its key point, the existence of a mitochondrial LDH, represents a crucial problem.

The present study addressed the question of a possible mitochondrial LDH, and of lactate as a substrate for mitochondrial metabolism. Mitochondria were isolated in high yields with a method that used about 100 mg skeletal muscle and in which the preparation was routinely surveyed by sampling all fractions and assaying them for marker enzymes and cytochrome c (Rasmussen et al. 1997). These preparations were well suited for the study as the mitochondria were obtained at high purity and integrity (Rasmussen & Rasmussen, 1997, 2000).

METHODS

Mitochondrial preparation

Experiments were approved by the ethics committees of the municipalities of Copenhagen and Frederiksberg. Mice were kept with free access to food and water. Part of the vastus lateralis muscle (∼100 mg) was sampled immediately after the mice were killed by stunning followed by decapitation. Human mitochondria were prepared from needle biopsy samples taken from vastus lateralis of young, healthy male subjects. The present human data on the LDH distribution were obtained by supplementary measurements in a study that included screening of metabolic parameters of mitochondria isolated from muscle biopsies. The study was performed according to the Declaration of Helsinki and the volunteers gave written informed consent.

Figure 1 shows a summary of the preparation method, which has been described in detail previously (Rasmussen et al. 1997). The figure also shows the dilution of the protease that occurs in the preparation procedure. The present small-scale method differs in several respects from the usual methods for isolation of mitochondria; probably most important is the homogenization. It is carried out in a large volume with a Potter homogenizer operated in a special set-up, which ensures that no grinding occurs and that the shearing effect is restricted to the Couette flow due to rotation of the pestle. The homogenization is carefully observed, and it causes complete disintegration of the muscle tissue, as it is extended to about 10 min. This period of operation does not affect the integrity of the isolated mitochondria.

Figure 1. Summary of the preparation method.

Figure 1

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F, dilution factor of the protease medium. Triton X-100 (0.1 %) was added to the fractions before enzyme assays and the P1 fraction was obtained by homogenization in the presence of Triton.

The preparation medium was a traditional salt medium containing KCl (100 mm), Tris (50 mm), MgSO4 (5 mm) and EDTA (1 mm); pH 7.40. In the initial steps of the preparation, it was supplemented with ATP (1 mm) and bovine serum albumin (0.5 % w/v) and, for the protease treatment, furthermore with Novo Subtilisin A (2 mg ml−1, ∼30 Anson units g−1). The final pellet was suspended in 225 mm mannitol and 75 mm sucrose.

Enzyme and cytochrome assays

Citrate synthase, a marker of the mitochondrial matrix, was assayed according to Shepherd & Garland (1969). LDH was assayed by oxidation of NADH with pyruvate (Vassault, 1983). The cytoplasmic marker, pyruvate kinase, was assayed as described by Fujii & Miwa (1983). LDH is used as an indicator enzyme in this assay. It is important that sufficient activity is added (see Errata to the reference) and that preparations devoid of ammonium sulphate are used, as this compound is inhibitory to pyruvate kinase.

The fractions (Fig. 1) were sampled quantitatively and kept strictly at 0 °C. Triton X-100 (0.1 %) was added to liberate the enzymes. The assays were typically performed ∼3 h after start of the preparation. It was possible to preserve the activities by freezing the samples in liquid nitrogen, storing them at −80 °C, and thawing them at 0 °C. This was used in experiments where the enzymes were assayed at different times after sampling of the fractions. The activities of pyruvate kinase and citrate synthase did not decline in the fractions, whereas the LDH activity declined at rates corresponding to the concentration of the protease. The ratio between LDH and pyruvate kinase activities in the fractions decreased with time, and an estimate of the degree of LDH degradation could be obtained from a time plot of this ratio. The S3 fraction, in which the protease was diluted ∼30 000 times, showed no decline of LDH activity.

The detection limit of the enzyme assays was determined by the dilution of the samples and the stability of the spectrophotometer (a modified Beckman DB instrument). The reactions were recorded for ∼10 min and it was estimated that a rate of absorbance change of 0.0005 min−1 could be detected with confidence. In the case of the mitochondrial fraction, this corresponded to the presence of 0.05 % of the tissue activities of pyruvate kinase and LDH.

It was obvious that the homogenate was not sufficiently homogeneous to allow reproducible sampling of aliquots for assays. All structures causing inhomogeneity were sedimented in the first pellet, which was homogenized vigorously in the presence of Triton to obtain the homogeneous P1 fraction. As the fractions were sampled quantitatively, the homogenate activity was equal to the sum of activities in the P1 and S1 fractions, and the tissue activity equal to the sum of the homogenate activity and the activity of the protease fraction (Rasmussen et al. 2001b). No correction for LDH degradation in the protease fraction was made in the calculation of the tissue LDH activity.

The cytochrome c conservation is the ratio between the yields of cytochrome c and citrate synthase in the mitochondrial fraction. It was estimated from low-temperature spectra of the fractions as described by Rasmussen & Rasmussen (1997). In situ, all cytochrome c and all citrate synthase probably reside in the mitochondria. The cytochrome c conservation, therefore, equals the cytochrome c per mitochondrial quantity in the preparation relative to that in the tissue, or the percentage of the native cytochrome c that is recovered in the isolated mitochondria.

Respiration measurements

Mitochondrial respiration was measured at 25 °C in the 36.5 μl vessel described previously (Rasmussen & Rasmussen, 1993). The assay medium contained: mannitol (225 mm), sucrose (75 mm), Tris (20 mm), phosphate (10 mm) and EDTA (0.5 mm); pH 7.35. The oxygen concentration of this medium was 468 μM O-atoms (25 °C, 101.3 kPa, 100 % humidity).

Protein was determined by the method of Schaffner & Weissmann (1973) with bovine serum albumin as standard.

RESULTS

Table 1 shows the distribution of the mitochondrial matrix marker citrate synthase, the cytoplasmic marker pyruvate kinase, and LDH in the fractions of human and mouse preparations. The protease fraction was always enriched with pyruvate kinase relative to citrate synthase. This indicates that the unhomogenized tissue leaked cytoplasmic components faster than mitochondria. Only little pyruvate kinase was present in the P1 fraction, indicating effective disruption of the muscle cells during homogenization.

Table 1.

Distribution of citrate synthase (CS), pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the fractions of mitochondrial preparations from human and mouse vastus lateralis

Human preparations (n = 4) Mouse preparations (n = 4)
Fraction (see Fig. 1) CS PK LDH CS PK LDH
Protease 9 ± 4 14 ± 5 0.4 ± 0.4 2 ± 0.7 6 ± 2 0.0 ± 0.1
P1 30 ± 3 3 ± 1 5 ± 1 15 ± 5 3 ± 1 3 ± 1
S1 70 ± 3 97 ± 1 95 ± 1 85 ± 5 97 ± 1 97 ± 1
S2 15 ± 2 97 ± 5 94 ± 5 24 ± 2 98 ± 5 92 ± 4
S3 10 ± 4 0.6 ± 0.2 0.8 ± 0.3 3 ± 2 0.5 ± 0.2 0.6 ± 0.2
Mitochondrial 39 ± 5 0.01 ± 0.02 a 0.02 ± 0.02 a 50 ± 3 0.12 ± 0.01 a 0.03 ± 0.03 a
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Data are means ± s.d. in per cent of the homogenate activity and in per cent of the tissue activity

a

The homogenate activities (means ± s.d. in μmol min−1 (g tissue wet wt)−1, 25°C) of the human preparations were: CS, 22 ± 5; PK, 343 ± 42; LDH, 169 ± 32; and of the mouse preparations: CS, 33 ± 6; PK, 379 ± 50; LDH, 360 ± 39.

The distribution of LDH was very similar to that of pyruvate kinase, except in the protease fraction. In these experiments the LDH assays were performed ∼3 h after start of the preparation and, at that time, practically all LDH activity in the protease fraction was degraded. However, the degradation of activity was much slower in the fractions S1, S2 and P1, and it affected the observed distribution of LDH among the fractions very little. It was estimated that ∼75 % of the native LDH activity was present when the pellet from the first high-speed centrifugation was suspended in fresh medium, and further degradation of LDH activity was stopped at the highly increased dilution of the protease.

The mitochondrial fractions showed LDH activities that were below the estimated detection limit of 0.05 % of the tissue content and not significantly different from zero in the t test, i.e. the mitochondrial fractions contained no LDH activity.

The cytochrome c conservation of the human preparations was 83 ± 7 % and of the mouse preparations 67 ± 9 % (means ± s.d.). A more energetic homogenization was used for the mouse tissue, which contained considerable amounts of fasciae. This might have caused the lower cytochrome c conservation.

Figure 2 shows two respiratory experiments with mitochondria from mouse vastus lateralis muscle. Experiment A demonstrates at first the absence of endogenous respiration and the very low state 3 rate with malate as the only substrate. Addition of lactate caused no increase of the rate of respiration, indicating that lactate was not respired and that pyruvate was not formed from lactate. A hardly detectable increase of respiratory rate was observed after addition of NAD. This might be due to the presence of traces of extramitochondrial LDH activity in the mitochondrial preparation. Finally, addition of supernatant from a mouse muscle homogenate caused a substantial rate of respiration, demonstrating that it was possible to establish the LDH-mediated respiration in this system.

Figure 2. Oxygen electrode traces from two experiments with mouse vastus lateralis mitochondria.

Figure 2

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The protein concentration was 195 μg protein ml−1 and both experiments were started in aerobic medium. Rates are marked below the curves in μmol O-atoms min−1 (g protein)−1. A, lactate respiration in the presence of malate and ADP (ADP, 1 mm; malate, 4 mm; lactate, 9 mm; NAD, 0.4 mm). LDH was the cytosolic fraction from mouse muscle homogenized in standard preparation medium. B, two state 4-3-4 cycles with pyruvate + malate (9 + 4 mm) as substrates. Each ADP addition was 343 μM.

With four mouse preparations, the increase of respiration due to lactate was 0.5 ± 1.0 μmol O-atoms min−1 (g protein)−1 and the response to NAD was 3 ± 3 μmol O-atoms min−1 (g protein)−1 (means ± s.d.). The human preparations showed similar non-significant respiratory changes.

Experiment B (Fig. 2) is a normal state 4-3-4 experiment with pyruvate + malate as substrates. This type of experiment is indicative of the quality of the mitochondria, and data from a number of human and mouse preparations are shown in Table 2. The high respiratory control ratios indicate high integrity of the mitochondria, and the high specific state 3 rates indicate both high integrity and high purity. Furthermore, lactate exerted no significant effect upon the rate of pyruvate + malate respiration. This indicates that lactate did not interfere with the transport of pyruvate into the mitochondria.

Table 2.

Respiratory data of mitochondria from human and mouse vastus lateralis

Human Mouse
Pyruvate + malate respiration:
 State 3 rate (μmol O-atoms min−1 (g protein)−1) 395 ± 27 (18) 578 ± 107 (8)
 Respiratory control ratio 8.7 ± 1.4 (18) 11.4 ± 1.1 (8)
Ratio of state 3 rate to
 state 3 rate of pyruvate + malate + lactate (%): 103.2 ± 6.2 (4) 96.5 ± 4.4 (3)
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Data are means ± s.d. (n). The respiratory control ratio is the ratio between a state 3 rate with pyruvate + malate and a subsequent state 4 rate. The measurements were made at 25 °C. Additional data for human preparations are given in Rasmussen et al. (2001a).

DISCUSSION

The major findings of the present study are that the mitochondrial fraction from human and mouse skeletal muscle contained no LDH activity and that lactate did not cause oxygen uptake, even in the presence of malate, and neither affected mitochondrial respiration of pyruvate + malate.

The detection limit of the respiratory measurements was similar to that of the enzyme assays, and the two types of data agree. As described above, the non-significant increase of respiratory rate due to lactate + NAD was 3.5 μmol O-atoms min−1 (g protein)−1. This would correspond to the presence of ∼0.03 % of the tissue LDH activity in the mitochondrial fraction. The calculation was based on the measured value of 10 mg mitochondrial protein (g tissue wet wt)−1 and the assumed ratio of five between the rates of NADH oxidation and NAD reduction in the LDH reaction. If equal rates were assumed, the figure was five times lower.

These observations seem to exclude that LDH is a constituent of skeletal muscle mitochondria in the sense that the enzyme is located in or behind the outer mitochondrial membrane. This is in accordance with the generally accepted use of LDH as a cytoplasmic marker enzyme, but in obvious conflict with observations that have led to the suggestion of the existence of a mitochondrial LDH in muscles. Such observations were made by Brandt et al. (1987), working with rat tissues including heart muscle, by Szczesna-Kaczmarek (1990), working with rat skeletal muscle, and by Brooks et al. (1999), working with liver, heart and skeletal muscle from rats. The observations by Szczesna-Kaczmarek (1990) were later ascribed to contamination of the preparation (Popinigis et al. 1991).

The classical tissue fractionation was perfected in the 1950s and 1960s, in particular through the contributions of De Duve and co-workers (for reviews, see De Duve & Berthet, 1954 (techniques) and De Duve, 1971). Experience shows that no fraction contains a given type of organelle in pure form. Differential centrifugation offers only a limited fractionation efficiency and artificial redistributions may occur, for instance by adsorption of soluble enzymes to particles. These methodological reservations also apply when the techniques are used for isolation of mitochondria. The mitochondrial fraction is usually obtained by combining one low-speed centrifugation, aiming at the removal of unbroken cells and nuclei, with several high-speed centrifugations in which the mitochondria (and other particles) are sedimented and ‘washed’. However, these washings only remove slower sedimenting contaminants from the mitochondrial fraction. The contamination with faster sedimenting particles is essentially controlled by the low-speed centrifugation, which often is insufficiently specified, and by the decantation, which may be subject to a large personal factor.

The present preparation method differs in several respects from those of Brandt et al. (1987), Szczesna-Kaczmarek (1990) and Brooks et al. (1999), and that might explain the different results obtained. Our medium-to-tissue ratio was much higher than in conventional preparation methods and the degree of cell disruption during homogenization was probably also higher. Both of these features might have improved fractionation. When taking the differences in experimental temperature into account, the present specific activity of pyruvate + malate state 3 respiration was ∼1.5 times that in the study of Brooks et al. (1999) and ∼5 times that of Szczesna-Kaczmarek (1990). This probably indicates a very significant difference in purity of the preparations studied. Finally, an ionic isolation medium was used in contrast to the sugar media of lower ionic strength used in the other studies. It is a general observation (De Duve & Berthet, 1954) that adsorption phenomena are favoured in sugar media, and Lluis (1984) demonstrated adsorption at low ionic strength of cytosolic LDH to particles in a crude mitochondrial fraction from rabbit skeletal muscle.

A crucial question is how large a part of the tissue LDH is recovered in the mitochondrial fraction. This is not reported in the publications on a mitochondrial LDH mentioned above. However, in the case of heart mitochondria, Brandt et al. (1987) reported the specific activities of the mitochondrial and cytosolic fractions, and from those it may be calculated that about 1 % of the tissue LDH was recovered in the mitochondrial fraction. No calculations can be made from the data of Szczesna-Kaczmarek (1990), but the respiratory data of Brooks et al. (1999) probably indicate that a few per cent of the muscle LDH activity was recovered in the mitochondrial fraction. Thus the LDH activity observed in the mitochondrial fraction in these studies represents only a small part of the tissue activity. It is most difficult to prove unambiguously that the presence of such a small part is due to LDH as a mitochondrial constituent and not to contamination. The mitochondrial subfractionation may suffice as an example of these difficulties. Brandt et al. (1987) reported that LDH is located in the mitochondrial intermembrane space and Brooks et al. (1999) reported that it is located in this space and in the mitochondrial matrix + inner membrane compartment. These assignments are based on the distribution of LDH in three fractions obtained in a standard procedure for subfractionation of mitochondria, namely the pellet after centrifugation of the digitonin-treated mitochondrial fraction, the pellet after stronger centrifugation of the supernatant, and the final supernatant. However, LDH is bound to show up in one or more of these three fractions, whether it is a constituent of the mitochondria or a contamination. The result of the experiment may be interesting, but it does not prove the existence of a mitochondrial LDH.

Enzyme assays as well as respiratory experiments indicated that the present mitochondrial fractions contained no LDH activity. The specific state 3 rates and the respiratory control ratios are generally used for evaluation of mitochondrial integrity. Preparational damage of the inner mitochondrial membrane causes lowering of the specific state 3 rates and, therefore, also of the respiratory control ratios. The present human quadriceps preparation showed specific state 3 rates that were higher than the highest literature values, in the case of pyruvate + malate respiration by a factor of 2.5 and in the case of other metabolic systems by a factor of 1.5 (Rasmussen & Rasmussen, 2000). Thus the mitochondrial inner membrane did not appear substantially damaged and this notion is also supported by the reproducibility of the preparations (Table 2).

A possible mitochondrial LDH, located in the intermembrane space, might be assumed to disappear either by leakage through the outer membrane or by proteolysis due to penetration of the protease into the intermembrane space. In both cases, highly increased permeability, perhaps even rupture, of the outer membrane should have occurred. Cytochrome c is located in the intermembrane space and readily lost from the mitochondria if the outer membrane becomes permeable to molecules of about 13 kDa. This occurs in vivo at the onset of apoptosis (see e.g. Crompton (2000) for a review). The loss of cytochrome c from isolated mitochondria may be evaluated by the cytochrome c conservation. Mitochondria, prepared from frozen mouse muscle, showed cytochrome c conservations well below 20 % (authors’ unpublished observations) and the cytochrome c conservation of pigeon breast muscle mitochondria was highly influenced by the way in which the homogenization was performed (Rasmussen et al. 1997). The preparations used in the present study showed cytochrome c conservations of 67 % (mouse) and 83 % (human). It may therefore be concluded that the outer mitochondrial membrane was not permeabilized or damaged to an extent that could account for the complete loss of a possible mitochondrial LDH.

In conclusion, the absence of LDH activity from the mitochondria cannot be ascribed to preparational damage; the absence indicates that LDH is not a mitochondrial enzyme in skeletal muscles. This rules out the basis for an intracellular lactate shuttle operating in this tissue.

Acknowledgments

The expert technical assistance of Mrs I.-L. Føhns and Mrs H. Lauritzen is gratefully acknowledged.

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