PERSPECTIVES FOR PROGRESS

200th Anniversary of Lactate Research in Muscle

L. Bruce Gladden
Department of Kinesiology, Auburn University, Auburn, AL, United States
GLADDEN, L.B. 200th anniversary of lactate research in muscle. Exerc. Sport Sci. Rev., Vol. 36, No. 3, pp. 109Y115, 2008.
This year, 2008, marks the bicentennial of research into lactate metabolism in muscle. Berzelius linked lactate accumulation to exercise in
1807/1808 when he noted the presence of lactate in the muscles of hunted stags. Today, the exact mechanism of intramuscular lactate
oxidation and the relationship of lactate dehydrogenase to mitochondria remain unresolved as animated debate surrounds the intracellular
lactate shuttle. Key Words: intracellular lactate shuttle, lactate dehydrogenase, lactic dehydrogenase, isozymes, history

into Laj anions and protons (H+) at the physiological pH

levels found in muscle and blood; therefore, it will generally
be referred to as Laj in this review.
Scheele also discovered chlorine, manganese, arsenic acid,
and silicon fluoride along with numerous other compounds
(28[p461]). However, he is best recognized for his work on
O2 in which he first demonstrated that air is composed of
two components, only one of which will support combustion
(28[p461]). Scheele discovered O2 as early as 1772, more
than a year before Joseph Priestley (1733Y1804). Unfortunately, his experiments (A Chemical Treatise on Air and
Fire) were not published until 1777, probably because of
procrastination by his friend and advocate, Torbern Bergman
(1735Y1784), who wrote an introduction to the book
(28[pp49,461]). As a result, he shares credit for the discovery
of O2 with Priestley.

INTRODUCTION
Discovery of Lactate
Lactate/lactic acid was discovered in sour milk by a
Swedish apothecary assistant, Carl Wilhelm Scheele
(1742Y1786) in 1780 (2[pp6Y8]). Benninga (2[pp7Y8])
provided a modern translation of Scheeles isolation technique; it is quoted partially here: Sour whey is evaporated to
one eighth of its volume, the curd [precipitated milk
proteins] is then removed by filtration. The filtrate is
saturated with milk of lime [calcium hydroxide], filtered
again and diluted with three times its volume of water (2).
After a series of additional steps, Scheele concluded that
(modern translation): The lactic acid produced is as pure as
can be obtained by chemistry (2). The new acid was named
Mjolksyra, meaning acid of milk (2[p8]). Lactate (Laj)
occurs as the acid ingredient of sour dairy products,
fermented fruits and vegetables, and sausages (2[p1]) and as
a flavor modifier or enhancer in carbonated soft drinks. It has
also been used in tanning leather, acid dyeing of wool, and as
a pharmaceutical in the form of calcium lactate; its esters
have been used as lacquer solvents, and small amounts have
been used in plastics. Benningas book (2) details the history
of Laj and its large scale production for industrial purposes
from a biotechnology perspective, offering a viewpoint that
is completely outside the mind-set of most biological
scientists. Note that lactic acid is more than 99% dissociated

LACTATE AND EXERCISE

Although Scheele is remembered as the discoverer of Laj,
it was Jons Jacob Berzelius (1779Y1848), another Swede,
who indelibly joined Laj to the study of exercise when he
reported the presence of Laj in the muscles of hunted stags
in either 1807 (19[p41]) or 1808 (20[pxxvii]). Berzelius
apparently convinced himself that the amount of Laj in a
muscle was proportional to the amount of exercise that the
muscle had performed (19[p41]). Mentioned less often is the
fact that Berzelius also discovered pyruvic acid (28[p56]).
Accordingly, it is fitting that we pause at the bicentennial of
research into lactate metabolism in muscle for a brief historic
overview and discussion of current controversies. Presently,
there are at least three contentious areas in the physiological/
biochemical study of Laj: 1) the role of Laj as either a
causative or a preventive agent of muscle fatigue, 2) the role
of Laj in the acidosis of intense exercise, and 3) the

Address for correspondence: L. Bruce Gladden, Ph.D., FACSM, Department of

Kinesiology, 2050 Memorial Coliseum, Auburn University, Auburn, AL 36849-5323
(E-mail: gladdlb@auburn.edu).
Accepted for publication: March 13, 2008
Associate Editor: Stephen M. Roth, Ph.D., FACSM
0091-6331/3603/109Y115
Exercise and Sport Sciences Reviews
Copyright * 2008 by the American College of Sports Medicine

109
Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

intracellular pathway of Laj metabolism in muscle. This

review focuses on the third controversy. Laj metabolism in
muscle during and after exercise is of special importance
because skeletal muscle is not only the most important source
of Laj but is also the primary consumer of Laj. For example,
during recovery from intense exercise, oxidation is the major
pathway for Laj removal, particularly in skeletal muscles.

LACTATE DEHYDROGENASE: DISCOVERY

AND ISOZYMES
The discord surrounding intracellular Laj metabolism is
not only a story about Laj but also about the enzyme
that catalyzes its formation, lactate dehydrogenase (LDH).
Berzelius has a distant relationship to this side of the tale as
well because he was the first (in 1836) to propose the term
catalysis that he used to explain the action of compounds that
facilitated chemical reactions without undergoing any
change themselves (19[pp30Y31]). A long period followed
during which scientists studied the ability of ferments,
tissues, or tissue extracts of various organisms to promote
reactions. Along the way, the ferments were divided into two
groups: 1) those that were disorganized or soluble and 2)
those that were organized or insoluble (i.e., they were made
up of intact microorganisms). In 1877, Willy Kuhne
(1837Y1900) offered the name enzymes (Greek, in
yeast) for the unorganized ferments (28[pp300Y301]).
Twenty years later, Eduard Buchner (1860Y1917) ground
yeast cells with sand at a controlled temperature to derive an
extract that was free of yeast cells but still able to ferment
sucrose to ethanol, demonstrating conclusively that cells
were not essential for fermentation (28[pp87Y88]). Subsequently in 1926, American James Batcheller Sumner
(1877Y1955) first isolated the crystalline form of an enzyme,
urease, from jack beans (28[p498Y499]).
Perhaps not surprisingly, it was Otto Meyerhof (1884Y
1951) (28[p368]) in 1919, studying skeletal muscle, who first
demonstrated the action of LDH. After perhaps hundreds of
investigations on the topic of LDH, pure LDH in crystalline
form was isolated from the hearts of bullocks. Beginning in
the 1950s, several investigators provided evidence that
preparations of LDH were heterogeneous. This work culminated in 1959 when Markert and Mller (17), using the
superior separation method of starch gel electrophoresis
combined with histochemical staining, clearly demonstrated
five different forms of the LDH enzyme. They (17) also first
proposed the term isozymes to designate the multiple
molecular forms of an enzyme. In 1962, Kaplans group (6)
hypothesized the model of tetrameric organization of the
LDH isozymes that still applies today: there are two
monomer subunits, H (heart) and M (muscle), which can
be combined in groups of four to yield five different
molecular species; HHHH, HHHM, HHMM, HMMM,
and MMMM. Modern nomenclature designates the monomers as A (muscle) and B (heart) yielding LDH1(B4), LDH2
(B3A1), LDH3(B2A2), LDH4(B1A3), and LDH5(A4).
Kaplans laboratory (6) went on to propose that the different
properties of the isozymes were physiologically important in
the regulation of cellular metabolism. Specifically, it was
110 Exercise and Sport Sciences Reviews

proposed that the sensitivity of the heart forms (H4, H3M1)

to pyruvate inhibition directed pyruvate toward oxidation in
an aerobic environment such as the heart, whereas the
relative insensitivity of the muscle forms (H1M3, M4) to
pyruvate inhibition facilitated the anaerobic breakdown of
carbohydrates to lactate in exercising muscles (6).
This scheme has been supported by circumstantial evidence. For example, muscle capacity for Laj utilization is
correlated with both the oxidative capacity and the content
of the heart-type LDH. As reviewed by Van Hall (27), there
is also evidence that oxidative muscle fibers have approximately half of the total LDH activity of glycolytic fibers, and
of that activity, a greater relative amount is in the heart
forms (H4 and H3M1). Also, endurance training results in a
lower total LDH content and a decrease in the muscle forms
(H1M3, M4) of LDH (27). However, Newsholme (21) has
questioned the potential role of LDH isozyme profile in Laj
production versus utilization on several counts: 1) the LDH
reaction is near-equilibrium, suggesting that pyruvate inhibition would have a negligible effect on flux through the
reaction, 2) the differences between function of the isozymes
may be less in vivo than in vitro because of higher temperature
and other factors in the intracellular milieu, 3) the pyruvate
inhibition may not apply at the high concentrations of the
LDH enzyme found in vivo, and 4) the reported inhibition
may be due to traces of the enol form of pyruvate that are
likely more prevalent in vitro than in vivo. Further, Van Hall
(27) notes the following: 1) that the absolute amount of the
heart forms of LDH remains fairly constant among fiber types
and with endurance training and 2) that the concentrations
of pyruvate and Laj required for LDH inhibition in vitro are
several times greater than the highest concentrations
observed in vivo. It is also possible that the binding of LDH
to cellular proteins may change its kinetic parameters. My
conclusion is that the exact regulatory role of the LDH
isozymes remains unknown.
LDH: CELLULAR LOCATION
Lactate dehydrogenase has long been considered a soluble
glycolytic enzyme found in the cytoplasm of cells, predominantly in the I band of striated muscle (18, 27). There is also
significant evidence that LDH binds reversibly to cytoskeletal proteins including actin, tubulin, and troponin among
others (see (18) for references). Furthermore, it seems clear
that an LDH fraction is localized to the sarcoplasmic
reticulum (SR), particularly in glycolytic skeletal muscle
(e.g., (1)). In agreement with the studies previously cited, it
is commonly reported that glycolytic activity is found
exclusively in the supernatant fraction of tissue homogenates, a fraction that contains essentially no mitochondria.
The story of the location of LDH, and thereby of
intracellular Laj metabolism, became more provocative
with a 1971 publication of Baba and Sharma (1). They (1)
cited several studies that reported LDH activity in the
mitochondrial fractions of tissues as well as numerous
histochemical studies that demonstrated LDH activity in
mitochondria of various tissues. Their own results (1),
obtained with a combination of histochemical and electron
www.acsm-essr.org

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

microscopy techniques in skeletal muscle, indicated the

presence of mainly heart-type isozymes in mitochondria and
cytoplasm and primarily muscle-type isozymes in association
with the SR. They conjectured that heart forms of LDH
might serve a role similar to that of glycerol phosphate
dehydrogenase and malate dehydrogenase. However, they
concluded that Permeability of the mitochondria to lactate
has not been well-demonstrated, and the lactate shuttle remains a pure speculation ((1); emphasis is mine).
Subsequently, via a variety of techniques, LDH was found
in association with mitochondria by Skilleter and Kun (25),
Deimann et al. (8), Kline et al. (16), Brandt et al. (3), and
Chretien et al. (7) among others. In 1972, Skilleter and Kun
(25) also reported the oxidation of Laj by isolated liver
mitochondria, although, perplexingly, the process seemed to
require energy. Szczesna-Kaczmarek (26) became the first
investigator to report direct mitochondrial oxidation of Laj
in skeletal muscle in 1990.

THE INTRACELLULAR LACTATE SHUTTLE

Figure 1. State 3 lactate and pyruvate oxidation by isolated rat skeletal

muscle mitochondria. Concentration of both substrates was set at
10 mM, and 2.5 mM malate was included in the medium. Compare
with values reported by Yoshida et al. (30) in Figure 2 who contend that
the results in this figure are likely due to contamination. Based on data
from Table 1 of Brooks et al. (5).

Finally, in 1999, Brooks et al. (5) fully elucidated an

intracellular lactate shuttle that had only been inferred by
Baba and Sharma (1). A central tenet of this intracellular
shuttle was that Laj is an inevitable product of glycolysis,
particularly during rapid glycolysis, because LDH has the
highest Vmax of any enzyme in the glycolytic pathway, and the
Keq for pyruvate to Laj is far in the direction of Laj and
nicotinamide adenine dinucleotide (NAD+) (4,5). Given this
information, Brooks et al. (4) questioned how it would be
possible for Laj to be converted back to pyruvate in the
cytosol, thus permitting oxidation of Laj by well-perfused
tissues, a universally accepted phenomenon that provides the
foundation for the cell-to-cell lactate shuttle theory (9,10,27).
One simple explanation would have been that LDH catalyzes a
near-equilibrium reaction, allowing small changes in substrate
and product concentrations to immediately direct the reaction in
the opposite direction. However, a different explanation was
proffered, and in recent years, the Brooks laboratory (4,5,11,13)
has reported evidence of the following key components of an
intracellular lactate shuttle in skeletal muscle: 1) direct uptake
and oxidation of Laj by isolated mitochondria without prior
extramitochondrial conversion of Laj to pyruvate (Fig. 1), 2)
presence of an intramitochondrial pool of LDH, and 3)
presence of the Laj transporter, monocarboxylate transporter
1 (MCT1), in mitochondria, presumably in the inner
mitochondrial membrane.
Operation of an intracellular lactate shuttle such as
described by the Brooks group (5) entails constant production of Laj in the cytosol, with the rate of production
increasing with elevated glycolytic activity. Because of its
higher concentration relative to pyruvate, Laj would be
the primary monocarboxylate diffusing to mitochondria. In
the original version of this shuttle, this Laj would then be
transported across the inner mitochondrial membrane by
MCT1. Once inside the mitochondrial matrix, mitochondrial LDH would catalyze the conversion of Laj back to
pyruvate that would be oxidized through the PDH reaction
to acetyl coenzyme A (CoA). The acetyl CoA would then

enter the tricarboxylic acid cycle. An important point is that

this intracellular lactate shuttle would not only deliver
substrate in the form of Laj for conversion to pyruvate, it
would also deliver reducing equivalents (reduced form of
NAD+ (NADH)), thus supplanting or supplementing the
role of the malate-aspartate and glycerol-phosphate shuttles
to varying degrees, depending on the rate of Laj formation
and its rate of transport into mitochondria (9).
Is Brooks correct? Can mitochondria oxidize Laj directly?
After Brooks proposal of the intracellular lactate shuttle,
Rasmussen et al. (23) and Sahlin et al. (24) found no
evidence that mitochondria can use Laj as a substrate
without prior conversion to pyruvate in the cytoplasm. A
preliminary report by Willis et al. (29) also found insignificant activity of the proposed intracellular lactate shuttle in
mitochondria isolated from rat skeletal muscle (Types I and
IIb) and liver. Ponsot et al. (22) reported no sign of direct
mitochondrial Laj oxidation in skinned fibers from heart
muscle, glycolytic skeletal muscle, or oxidative skeletal
muscle. Finally, Yoshida et al. (30) from the Bonen
laboratory have also recently found minimal direct oxidation
of Laj by either subsarcolemmal or intermyofibrillar mitochondria from either red (oxidative) or white (glycolytic)
skeletal muscle (Fig. 2).
A central point in this debate is the exact location of LDH.
Although several investigators have reported that LDH is
associated with mitochondria (as previously cited), these
reports warrant further scrutiny. First, some researchers
(23,24,30) note that the amount of LDH associated with
mitochondria is quite small and conclude that the mitochondria isolated by Brooks et al. (5) as well as others are
contaminated with cytoplasmic LDH. In fact, Chretien and
colleagues (7), who found LDH in mitochondria-enriched
preparations of human skeletal muscle, considered the LDH
to be a contaminant from the cytoplasm. On the contrary,
Hashimoto and Brooks (11) point to the difference in
mitochondrial isolation techniques and argue that others most
likely lost LDH that was associated with mitochondria.

Volume 36

Number 3

July 2008

Lactate Metabolism

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

111

Figure 2. Lactate and pyruvate oxidation in intermyofibrillar (Panel A) and subsarcolemmal (Panel C) mitochondria from rat red and white muscle at
different concentrations (0.18, 1.8, and 10 mM). Lactate oxidation is also shown on a 30-fold more sensitive scale in both red and white muscle
intermyofibrillar (Panel B) and subsarcolemmal (Panel D) mitochondria. Note negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar
mitochondria obtained from both red and white rat skeletal muscle. Compare with values reported by Brooks et al. (5) in Figure 1. Hashimoto and Brooks
(11) contend that these results are likely due to loss of mitochondrial LDH in the isolation procedures. (Reprinted from Yoshida, Y., G.P. Holloway,
V. Ljubicic, H. Hatta, L.L. Spriet, D.A. Hood, and A. Bonen. Negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar mitochondria
obtained from red and white rat skeletal muscle. J. Physiol. 582:1317Y1335, 2007. Copyright * 2007 Blackwell Publishing. Used with permission.)

Clearly, it would be helpful if someone were to carefully

compare LDH activity and Laj oxidation in mitochondria
isolated by the methods of Brooks and coworkers as compared
with mitochondria isolated by the techniques of other
investigators who do not find mitochondrial Laj oxidation.
Another critical obstacle to the notion of Laj oxidation
within the mitochondrial matrix is the possible violation of
the first law of thermodynamics as described by Sahlin et al.
(24). Separate pathways for cytosolic pyruvate and NADH
entry into the mitochondrial matrix provide for a large
oxidation/reduction potential gradient spanning the inner
mitochondrial membrane. Crucial for maintenance of this
gradient is the existence of a nonequilibrium step within
both the glycerol phosphate and malate-aspartate shuttles.
Neither empirical evidence nor theoretical conjecture has
been presented in support of a nonequilibrium step in the
intracellular lactate shuttle hypothesis.
LDH IN THE MITOCHONDRIAL
INTERMEMBRANE SPACE?
In my opinion, the weight of the evidence is against the
presence of LDH in the mitochondrial matrix and, therefore,
the oxidation of Laj within the mitochondrial matrix.
112 Exercise and Sport Sciences Reviews

However, LDH may be present in the intermembrane space

of mitochondria, perhaps attached (loosely?) to the inner
mitochondrial membrane. Baba and Sharma (1), as previously noted, found LDH associated with mitochondria but,
at the same time, questioned the permeability of mitochondria to lactate, leaving the intermembrane space as a likely
location in the heart and skeletal muscle. Skilleter and Kun
(25) undertook submitochondrial fractionation and arrived
at the conclusion that LDH in intact mitochondria is
probably on the outer side of the inner membrane in liver.
Deimann et al. (8) used scanning transmission electron
microscopy and found the reaction product for LDH clearly
identified in the intermembranous space of mitochondria in
rabbit glycolytic skeletal muscle. Using proteolytic disruption
of isolated liver mitochondria, Kline et al. (16) concluded
that LDH is mainly in the outer membrane and periplasmic
space. Brandt et al. (3) used digitonin fractionation of
mitochondria isolated from rat heart, kidney, liver, and
lymphocytes; they reported that the mitochondrial LDH is
located primarily in the periplasmic space. Periplasmic space
and intermembrane space are equivalent terms.
Recent observations of Hashimoto et al. (12) from the
Brooks laboratory provided additional detail for the possibility of LDH in association with the inner mitochondrial
membrane. Using the techniques of confocal laser scanning
www.acsm-essr.org

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

microscopy and immunoblotting after immunoprecipitation

in L6 skeletal muscle cells, Hashimoto et al. (12) found
evidence suggesting that LDH, MCT1, the single-span
transmembrane glycoprotein CD147, and cytochrome oxidase are colocalized in the inner mitochondrial membrane,
with the LDH enzyme apparently residing on the outer
surface of the inner membrane. They (12) have called this
a lactate oxidation complex and have enumerated their
experimental evidence supporting the existence of this
complex elsewhere (11). Potential location of LDH loosely
attached to the outside of the inner mitochondrial membrane rekindles the possibility that such LDH might be
susceptible to loss during the isolation of mitochondria from
muscle tissue. However, it seems unlikely that LDH
(molecular weight of 134,000) can pass through an intact

outer mitochondrial membrane because it is impermeable to

molecules larger than 5000 daltons, whereas NAD+/NADH
at a molecular weight of approximately 664 moves through
readily. It also seems unlikely that LDH within the
mitochondrial intermembrane space would be susceptible to
destruction by proteases used in mitochondrial isolation
(trypsin, molecular weight ,23,000; nagarse, molecular
weight ,27,000).

INTRACELLULAR LACTATE SHUTTLE

REMAINS VIABLE
Despite the serious reservations previously outlined, it
remains quite possible that an intracellular lactate shuttle

Figure 3. Illustration of the essential elements of possible intracellular (intramuscular) lactate shuttles, one with lactate dehydrogenase (LDH) in a lactate
oxidation complex (12) on the inner mitochondrial membrane and one without LDH attached to the inner membrane or even in the intermembrane space.
A high activity of cytosolic LDH is considered to guarantee lactate (Laj) formation in the cytoplasm under virtually all conditions but especially during
exercise. Although Laj could be formed throughout the cytosol, two particular locations are illustrated V one in association with the Na+/K+-ATPase
pump in the sarcolemma; and the other, the Ca2+-ATPase in the SR membrane. The sarcolemma is illustrated by the thick double lines at the top of the
cartoon, whereas the inner and outer mitochondrial membranes are dramatically enlarged to demonstrate possible Laj pathways. The dashed red and
black lines connecting Pyrj and Laj in the upper left quadrant indicate the near-equilibrium nature of the LDH reaction and the movement of Laj and
Pyrj mass from a site of net formation to a site of net removal. The gaps in the outer mitochondrial membrane illustrate that it is freely permeable to most
small molecules (but probably not permeable to LDH, trypsin, or nagarse; see text). Laj is shown in bold and red and larger than pyruvate (Pyrj) to
indicate that Laj is typically present in much higher concentration than Pyrj. Whether Laj were converted back to Pyrj outside the intermembrane
space, inside the space, or via a mitochondrial LDH, the resulting NADH + H+ would be shuttled across the inner mitochondrial membrane via the malateaspartate and glycerol phosphate shuttles. Please note that illustration of these traditional shuttles on the inner mitochondrial membrane is not intended
to imply that their components are limited to this location; the reactions that are outside the inner membrane can occur throughout the cytosol. Pyrj
could be transported across the inner mitochondrial membrane by either a pyruvate carrier, (MPC, arguably more likely) or a monocarboxylate transporter
(MCT1), both of which have been identified in the inner membrane. The MCT1/MPC in the lactate oxidation complex is not meant to imply that
Hashimoto et al. (12) reported the presence of MPC there; their evidence supports MCT1. To avoid confusion, arrows indicating possible diffusion routes
of NAD+ and NADH + H+ are not shown. COX indicates cytochrome oxidase; cLDH, cytosolic lactate dehydrogenase; CD147, single-span transmembrane
glycoprotein; ETC II and III, electron transport chain complexes II and III; Gly, glycogen; Glu, glucose; Inner, inner mitochondrial membrane; Laj, lactate;
MCT1, monocarboxylate transporter 1; mLDH, mitochondrial LDH; MPC, mitochondrial pyruvate carrier; NADH-dh, NADH dehydrogenase complex I;
Outer, outer mitochondrial membrane; Pyrj, pyruvate. [Adapted from Hashimoto, T., R. Hussien, and G.A. Brooks. Colocalization of MCT1, CD147, and
LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex. Am. J. Physiol. Endocrinol. Metab.
290:E1237YE1244, 2006. Copyright * 2006 The American Physiological Society. Used with permission.]
Volume 36

Number 3

July 2008

Lactate Metabolism

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

113

operates, albeit without intramatrix lactate metabolism

(Fig. 3). It is reasonable to speculate that pyruvate and
NADH concentrations are lowest adjacent to the inner
mitochondrial membrane where the pyruvate carrier and the
NADH shuttles (malate-aspartate and glycerol phosphate)
are moving pyruvate and NADH equivalents, respectively,
into the mitochondria. In other words, actively oxidizing
mitochondria would create sinks for the utilization of
pyruvate and NADH and, therefore, their uptake from
adjacent cytosolic locations. At the same time, sites of
cellular glycolysis would create driving concentrations of
Laj because the primary end product of glycolysis would be
La j due to the high activity of LDH as described
earlier. This situation would lead to the highest Laj
production and concentration at cytosolic locations remote
from mitochondria. Then, because of the relatively higher
[Laj] as compared with [pyruvatej], Laj would be the
primary species diffusing to areas near mitochondria. The
[Laj] is typically approximately 10Y200 times greater than
[pyruvatej] in skeletal muscle. Adjacent to mitochondria, or
if Hashimoto et al. (12) are correct, in the intermembrane
space, Laj and NAD+ would be converted back to pyruvate
and NADH via LDH for uptake into the mitochondria. Such
a scheme, arguably analogous to the phosphocreatine shuttle,
would be in accord with near equilibrium of the LDH
reaction throughout the cytosol and would accommodate
ready Laj production with subsequent oxidation and less

transport of Laj out of the cell. I should note clearly that

specific location of LDH, either in the intermembrane space
or attached to the inner membrane, might not be a
requirement for operation of an intracellular lactate shuttle.
Finally, despite the evidence for the presence of MCT1 in
a lactate oxidation complex (12), it is quite possible,
perhaps probable, that pyruvate is transported across the
inner mitochondrial membrane via the mitochondrial
pyruvate carrier (MPC (14)) that has a very high affinity
for pyruvate.
COMPARTMENTATION OF METABOLISM
The model previously described is consistent with the
compartmentation of metabolism as described in several
studies. James and colleagues (15) proposed that the Na+/K+ATPase pump derives its energy heavily from glycolysis that
is closely associated with the pump, an idea that has recently
been supported in studies of mechanically skinned skeletal
muscle fibers. There is also considerable evidence for a
functional compartmentation of glycolysis with the SR.
Figure 4 provides visual circumstantial evidence for glycolytic
compartmentation with the SR. Is it possible that Laj
derived from glycolysis associated with SR Ca2+ pumping
is shunted toward intermyofibrillar mitochondria, whereas
Laj derived from glycolysis affiliated with Na+/K+-ATPase

Figure 4. Transmission electron microscopy (TEM) image of a human skeletal muscle fiber (original magnification 20,000; scale bar = 2 Km). The 60nm-thick section has been cut precisely on the surface of the myofibril, enabling visualization of the triads situated in the I band on each side of the z line,
with paired long mitochondria located transversely at the I band level wrapped around the contractile apparatus and in contact with the SR but clearly
separated from the t tubules. Traces of longitudinal tubules of SR can be seen as strings from the terminal cisternae toward the fenestrated collar at the
level of the M band. The abundant glycogen granules are visualized as black dots. Although some glycogen granules are obviously near mitochondria in
the micrograph, the density of granules surrounding the SR is suggestive of glycolytic compartmentation with the SR. Human muscle fibers were fixed
with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 24 h then rinsed four times in 0.1 M sodium cacodylate buffer, postfixed with
1% osmium tetroxide (OsO4) and 1.5% potassium ferrocyanide (K4Fe(CN)6) in 0.1 M sodium cacodylate buffer for 90 min, rinsed two times in 0.1 M
sodium cacodylate buffer, dehydrated through a graded series of alcohol, infiltrated with graded mixtures of propylene oxide and Epon, and embedded in
100% Epon. Ultra-thin sections (60 nm) were cut using a Leica Supernova ultramicrotome and contrasted with uranyl acetate and lead citrate. The
sections were examined and photographed in a Philips EM 208 electron microscope and a Megaview III FW camera. Images were taken at 20,000
magnification with a precalibrated transmission electron microscope. Figure courtesy of Niels Krtenblad and Joachim Nielsen, University of Southern
Denmark, Odense, Denmark. Not previously published.

114 Exercise and Sport Sciences Reviews

www.acsm-essr.org

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

activity is shunted toward subsarcolemmal mitochondria?

Perhaps future studies will provide evidence in this regard.

CONCLUSIONS
In conclusion, research into muscle Laj metabolism
remains a hotbed of activity 200 yr after the report of
elevated Laj in exercised muscle. A major source of
contention surrounds the exact pathway(s) of intracellular
Laj oxidation. The model of interest is the intracellular
lactate shuttle. The key questions are as follows: 1) How can
Laj be tracked from one intracellular location to another? 2)
Where is LDH precisely located inside muscle cells? 3) Does
the intracellular lactate shuttle model require fixed locations
of LDH to operate? Future studies may provide conclusive
answers to these queries.
Acknowledgments
The author wishes to thank Matthew L. Goodwin, Andres Hernandez, and
James E. Harris for their criticism and discussion of the manuscript drafts.

References
1. Baba, N., and H.M. Sharma. Histochemistry of lactic dehydrogenase in
heart and pectoralis muscles of rat. J. Cell. Biol. 51:621Y635, 1971.
2. Benninga, H. A History of Lactic Acid Making: A Chapter in the History
of Biotechnology. Boston, MA: Kluwer Academic Publishers, 1990,
pp. 1Y61.
3. Brandt, R.B., J.E. Laux, S.E. Spainhour, and E.S. Kline. Lactate
dehydrogenase in rat mitochondria. Arch. Biochem. Biophys. 259:
412Y422, 1987.
4. Brooks, G.A., M.A. Brown, C.E. Butz, J.P. Sicurello, and H.
Dubouchaud. Cardiac and skeletal muscle mitochondria have a
monocarboxylate transporter MCT1. J. Appl. Physiol. 87:1713Y1718,
1999.
5. Brooks, G.A., H. Dubouchaud, M. Brown, J.P. Sicurello, and C.E. Butz.
Role of mitochondrial lactate dehydrogenase and lactate oxidation in
the intracellular lactate shuttle. Proc. Natl. Acad. Sci. U.S.A. 96:
1129Y1134, 1999.
6. Cahn, R.D., N.O. Kaplan, L. Levine, and E. Zwilling. Nature and
development of lactic dehydrogenases. Science. 136:926Y969, 1962.
7. Chretien, D., M. Pourrier, T. Bourgeron, M. Sene, A. Rotig, A.
Munnich, and P. Rustin. An improved spectrophotometric assay of
pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle. Clin. Chim. Acta.
240:129Y136, 1995.
8. Deimann, W., R. Freeman, and H.D. Fahimi. Improved contrast in
cytochemistry of dehydrogenases by scanning transmission electron
microscopy. J. Histochem. Cytochem. 29:678Y681, 1981.
9. Gladden, L.B. Lactate metabolism: a new paradigm for the third
millennium. J. Physiol. 558(Pt 1):5Y30, 2004.
10. Gladden, L.B. A lactatic perspective on metabolism. Med. Sci. Sports.
Exerc. 40:477Y485, 2008.

Volume 36

Number 3

July 2008

11. Hashimoto, T., and G.A. Brooks. Mitochondrial lactate oxidation

complex and an adaptive role for lactate production. Med. Sci. Sports
Exerc. 40:486Y494, 2008.
12. Hashimoto, T., R. Hussien, and G.A. Brooks. Colocalization of MCT1,
CD147, and LDH in mitochondrial inner membrane of L6 muscle cells:
evidence of a mitochondrial lactate oxidation complex. Am. J. Physiol.
Endocrinol. Metab. 290:E1237Y1244, 2006.
13. Hashimoto, T., S. Masuda, S. Taguchi, and G.A. Brooks. Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat
plantaris muscle. J. Physiol. 567(Pt 1):121Y129, 2005.
14. Hildyard, J.C.W., C. Ammala, I.D. Dukes, S.A. Thomson, and A.P.
Halestrap. Identification and characterisation of a new class of highly
specific and potent inhibitors of the mitochondrial pyruvate carrier.
Biochim. Biophys. Acta. 1707:221Y230, 2005.
15. James, J.H., F.A. Luchette, F.D. McCarter, and J.E. Fischer. Lactate is an
unreliable indicator of tissue hypoxia in injury or sepsis. Lancet.
354:505Y508, 1999.
16. Kline, E.S., R.B. Brandt, J.E. Laux, S.E. Spainhour, E.S. Higgins, K.S.
Rogers, S.B. Tinsley, and M. G. Waters. Localization of L-lactate
dehydrogenase in mitochondria. Arch. Biochem. Biophys. 246:673Y680,
1986.
17. Markert, C.L., and F. Mller. Multiple forms of enzymes: tissue,
ontogenetic, and species specific patterns. Proc. Natl. Acad. Sci. U. S. A.
45:753Y763, 1959.
18. Nakae, Y., and P.J. Stoward. Effects of tissue protectants on the kinetics
of lactate dehydrogenase in cells. J. Histochem. Cytochem. 45:1417Y1425,
1997.
19. Needham, D.M. Machina Carnis: The Biochemistry of Muscular Contraction in Its Historical Development. Cambridge, UK: Cambridge University
Press, 1971, 782.
20. Needham, J. Introduction. In: The Chemistry of Life; Eight Lectures on the
History of Biochemistry, J. Needham (Ed.). Cambridge, UK: University
Press, 1970, pp. viiYxxx.
21. Newsholme, E.A. Enzymes, energy and endurance. Some provocative
thoughts. In: Principles of Exercise Biochemistry, J.R. Poortmans (Ed.).
Basel, Switzerland: Karger, 2004, pp. 1Y35.
22. Ponsot, E., J. Zoll, B. NGuessan, F. Ribera, E. Lampert, R. Richard, V.
Veksler, R. Ventura-Clapier, and B. Mettauer. Mitochondrial tissue
specificity of substrates utilization in rat cardiac and skeletal muscles.
J. Cell. Physiol. 203:479Y486, 2005.
23. Rasmussen, H.N., G. van Hall, and U.F. Rasmussen. Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus
lateralis muscle. J. Physiol. 541(Pt 2):575Y580, 2002.
24. Sahlin, K., M. Fernstrom, M. Svensson, and M. Tonkonogi. No
evidence of an intracellular lactate shuttle in rat skeletal muscle.
J. Physiol. 541(Pt 2):569Y574, 2002.
25. Skilleter, D.N., and E. Kun. The oxidation of L-lactate by liver
mitochondria. Arch. Biochem. Biophys. 152:92Y104, 1972.
26. Szczesna-Kaczmarek, A. L-lactate oxidation by skeletal muscle mitochondria. Int. J. Biochem. 22:617Y620, 1990.
27. Van Hall, G. Lactate as a fuel for mitochondrial respiration. Acta.
Physiol. Scand. 168:643Y656, 2000.
28. A Biographical Dictionary of Scientists. 3rd ed. T.I. Williams (Ed.). New
York, NY: John Wiley and Sons, 1982.
29. Willis, W.T., A. Thompson, J.I. Messer, and J.S. Thresher. Vmax of
mitochondrial electron shuttles in rat skeletal muscle and liver
(Abstract). Med. Sci. Sports. Exerc. 35:S396, 2003.
30. Yoshida, Y., G.P. Holloway, V. Ljubicic, H. Hatta, L.L. Spriet, D.A.
Hood, and A. Bonen. Negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar mitochondria obtained from red and white
rat skeletal muscle. J. Physiol. 582(Pt 3):1317Y1335, 2007.

Lactate Metabolism

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Copyright @ 2008 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

115