Download ATP production in brain and liver mitochondria of Fischer

innovative methodology
Am J Physiol Regul Integr Comp Physiol 285: R1259–R1267, 2003.
First published July 10, 2003; 10.1152/ajpregu.00264.2003.
Method for measuring ATP production in isolated mitochondria:
ATP production in brain and liver mitochondria of Fischer-344 rats
with age and caloric restriction
Barry Drew and Christiaan Leeuwenburgh
University of Florida, Biochemistry of Aging Laboratory, Gainesville, Florida 32611
Submitted 13 May 2003; accepted in final form 7 July 2003
firefly luciferase; bioluminescence assay; ATP production;
aging; caloric restriction
useful energy in the form of ATP. Cellular function is
maintained through the hydrolysis of ATP to ADP. The
catalytic sites on the F1 head of the ATP synthase
complex, which projects onto the protochemically negative matrix side (N-phase) of the membrane, contain
three nucleotide binding sites that undergo a multiphase, catalytic cycle to produce ATP (43) from ADP
and phosphate (i.e., oxidative phosphorylation). Because ATP is tightly bound to the F1 head in the
mitochondrial membrane, a large amount of free energy is released during the rapid hydrolysis of ATP to
ADP or AMP. The large free energy released during the
hydrolysis of ATP is available for immediate use (i.e.,
muscle contraction, chemiosmotic homeostasis, and
normal cellular function) or can be stored for future
cellular activity, such as protein synthesis, genomic
replication, and cell growth (37, 43).
To study ATP content and production from isolated
mitochondria, we used a commercially available ATP
determination kit. The bioluminescence assay is based
on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. Upon addition, ATP
combines with luciferin to form luciferyl adenylate and
inorganic pyrophosphate (PPi) on the surface of the
luciferase enzyme as shown in reaction 1
luciferase
luciferin ⫹ ATP ™™™™™3
luciferyl adenylate ⫹ PPi
While bound to the enzyme, luciferyl adenylate combines with O2 to form oxyluciferin and AMP (5)
through a series of enzymatic redox reactions. As oxyluciferin and AMP are released from the enzyme’s
surface, a quantum yield of light is emitted in proportion to the ATP concentration as shown in reaction 2
luciferyl adenylate ⫹ O2 3 oxyluciferin
⫹ AMP ⫹ h␯
produce ⬃90% of the required energy
necessary for cellular function (46). The inner membrane of the mitochondria contains a complex series of
enzymes and other proteins that are involved in the
catabolism of food molecules to liberate metabolically
MITOCHONDRIA
Address for reprint requests and other correspondence: C. Leeuwenburgh, Univ. of Florida, Biochemistry of Aging Laboratory, P.O.
Box 118206, Gainesville, FL 32611 (E-mail: [email protected]).
http://www.ajpregu.org
(reaction 1)
(reaction 2)
The light emission (h␯) can then be recorded and
quantified using a chemiluminometer.
This study introduces a quick and reproducible
methodology for measuring ATP in isolated mitochonThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6119/03 $5.00 Copyright © 2003 the American Physiological Society
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Drew, Barry and Christiaan Leeuwenburgh. Method
for measuring ATP production in isolated mitochondria: ATP
production in brain and liver mitochondria of Fischer-344
rats with age and caloric restriction. Am J Physiol Regul
Integr Comp Physiol 285: R1259–R1267, 2003. First published July 10, 2003; 10.1152/ajpregu.00264.2003.—The production of ATP is vital for muscle contraction, chemiosmotic
homeostasis, and normal cellular function. Many studies
have measured ATP content or qualitative changes in ATP
production, but few have quantified ATP production in vivo
in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology,
physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially
available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production
in isolated mitochondria. The bioluminescence assay is based
on the reaction of ATP with recombinant firefly luciferase
and its substrate luciferin. The stabilities of the reaction
mixture as well as relevant ATP standards were quantified.
The luminescent signals of the reaction mixture and a 0.5 ␮M
ATP standard decreased linearly at rates of 2.16 and 1.39%
decay/min, respectively. For a 25 ␮M ATP standard, the
luminescent signal underwent a logarithmic decay, due to
intrinsic deviations from the Beer-Lambert law. Moreover, to
test the functionality of isolated mitochondria, they were
incubated with 1 and 5 mM oligomycin, an inhibitor of
oxidative phosphorylation. The rate of ATP production in the
mitochondria declined by 34 and 83%, respectively. Due to
the sensitivity and stability of the assay and methodology, we
were able to quantitatively measure in vivo the effects of age
and caloric restriction on the ATP content and production in
isolated mitochondria from the brain and liver of young and
old Fischer-344 rats. In both tissues, neither age nor caloric
restriction had any significant effect on the ATP content or
the rate of ATP production. This study introduces a highly
sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.
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ATP PRODUCTION IN MITOCHONDRIA WITH AGE AND CALORIC RESTRICTION
dria. The effects of age and caloric restriction on the
amount of ATP present as well as the rate of ATP
production on isolated mitochondria in the brain and
liver have yet to be studied. Hence, we determined in
vivo whether ATP content and production change with
age and lifelong caloric restriction in the brain and
liver. During aging, the production of reactive oxygen
species in the mitochondria may lead to a reduction in
the activity of the respiratory chain complexes, and
hence a decline in ATP production (2, 20, 26, 47). In
contrast, caloric restriction may be able to reduce the
age-related increase in oxidative damage to the respiratory complexes.
METHODS
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Chemicals. The ATP determination kit (A-22066) by Molecular Probes (Eugene, OR) containes d-luciferin, luciferase
[40 ␮l of a 5 mg/ml solution in 25 mM Tris 䡠 acetate, pH 7.8,
0.2 M ammonium sulfate, 15% (vol/vol) glycerol, and 30%
(vol/vol) ethylene glycol], dithiothreitol (DTT), ATP, and a
reaction buffer (10 ml of 500 mM tricine buffer, pH 7.8, 100
mM MgSO4, 2 mM EDTA, and 2 mM sodium azide). The
reagents and reaction mixture were combined according to
the protocol by Molecular Probes; however, the volume of
each assay was increased to 1,000 ␮l to account for the
⬃100-fold stoichiometric increase in the quantum yield due
to the higher optical density (OD) readings for the production
of ATP relative to the content. In addition, 1 mM pyruvate
and 1 mM malate were added to the reaction mixture as
substrates for oxidative phosphorylation (14, 48).
Animals. Young ad libitum-fed (young, n ⫽ 8, 12–13 mo),
old ad libitum-fed (old, n ⫽ 6, 26–28 mo), and old caloricrestricted (CR, n ⫽ 6, 27–28 mo) male Fischer 344 rats
[National Institute of Aging (NIA) colony, Harlan SpragueDawley, Indianapolis, IN] were used. Caloric restriction was
started at 3.5 mo of age (10% restriction), increased to 25%
restriction at 3.75 mo, and maintained at 40% restriction
from 4 mo throughout the individual animal’s life. The rats
were housed one per cage in a temperature-controlled (18–
22°C) and light-controlled environment with a 12:12-h lightdark cycle. Following 1 wk of acclimation after arrival from
the NIA, one animal was randomly killed daily, after being
anesthetized with an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). All treatment of animals
throughout this study conformed fully to the “Guiding Principles for Research Involving Animals and Human Beings” of
the American Physiological Society. In addition, protocols
received local institutional animal care committee approval.
Mitochondrial isolation and protein concentration. Intact
mitochondria from the liver and frontal brain cortexes were
immediately isolated using differential centrifugation. The
tissue from the liver was homogenized (on ice) in 1:10 wt/vol
of ice-cold buffer A [0.220 M mannitol, 0.070 M sucrose, 0.5
mM EGTA, 2 mM HEPES (pH 7.4) and 0.1% fatty acid free
BSA] using a Potter-Elvehjem glass-glass homogenizer. The
homogenate from the liver was centrifuged (Eppendorf
5810R, Brinkmann Instruments) at 1,600 g (4°C) for 10 min,
and the resulting supernatant was centrifuged at 18,000 g
(4°C) for 10 min. The pellet was resuspended in buffer A and
centrifuged at 18,000 g (4°C) for an additional 10 min. The
tissue from the frontal cortex of the brain was homogenized
as before but in ice-cold suspension (1:10 wt/vol) buffer B [20
mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1
mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.1 mM PMSF]
(38). The homogenate from the frontal cortex was centrifuged
at 750 g at 4°C for 5 min. The supernatant was then centrifuged at 8,000 g for 20 min at 4°C. The pellets from the liver
and frontal cortex were resuspended in their respective buffers, and the freshly isolated mitochondria were used immediately for the analysis of ATP content and rate of ATP
production. Mitochondrial protein concentration was determined using the Bradford method (4).
Standard curve for ATP determination. For all experiments, ATP standard curves were run in the range of 0.5 to
50 ␮M (0.5, 1.0, 5.0, 10.0, 25.0, and 50.0 ␮M, respectively).
The standard curve was linear up to a concentration of 25.0
␮M; however, the curve started to deviate from linearity as
the concentration approached 50.0 ␮M. Hence a second-order
polynomial trend line was used to fit the data due to apparent
deviations from the Beer-Lambert law. In all experiments,
the correlation coefficient was 0.990 or higher.
Stability of ATP. The stability of the reaction mixture as
well as two relevant ATP standards, which gave luminescent
(OD) readings comparable in magnitude to median luminescent readings for ATP content, 0.5 ␮M, and rate of production, 25.0 ␮M in a 10 ␮l mitochondria sample, were measured
(see ATP content and production methodology). Measurements were recorded every 5 s for a total analysis of 5 min.
Inhibition of ATP production. To test the functionality of
the ATP determination assay, 0, 1, and 5 mM oligomycin
were added and incubated for 10 min at 37°C to freshly
isolated mitochondria. The inhibition in the rate of ATP
production was determined by comparing the oligomycintreated mitochondria to an equal portion of freshly isolated
mitochondria from the same tissue.
ATP content and production methodology. All solutions
were placed in a heating block and maintained at a temperature of 28°C that was, kinetically, the optimum temperature
(Molecular Probes) for the reaction mixture. Analyses began
on the Turner Designs TD 20/20 (Sunnyvale, CA) luminometer using a blank (deionized H2O) to correct for instrumental drift and possible physical interferences within the
polypropylene cuvettes (12 ⫻ 50 mm, Turner Designs,
Sunnyvale, CA). Next, the luminescent signals for six ATP
standards ranging from 0.5 to 50 ␮M (0.5, 1.0, 5.0, 10.0, 25.0,
and 50.0 ␮M, respectively) were measured. Initially, for each
standard, background measurements of the reaction mixture
(990 ␮l) were recorded. Then, 10 ␮l of each standard was
added to each of the reaction mixtures, and the luminescence
was recorded. After the standards were completed, the luminescence of 990 ␮l of the reaction mixture was recorded. To
this mixture, 10 ␮l of isolation buffer and 10 ␮l of 2.5 mM
ADP were added and recorded so that this background associated with the ADP solution could be subtracted from the
measurements of the mitochondria used to determine the
maximum ATP production. Measurements were obtained
every 5 s for a total analysis time of 30 s (a total of 6
measurements) for each mixture or subsequent addition.
Because all of these measurements were stable over the
entire analysis, the six ADP background measurements were
averaged, and the mean was used in the calculations. Because the sensitivity of the standards and reaction mixture
decreases over prolonged time, all of the standards were
analyzed during isolation of the mitochondria to maximize
the luminescent signal.
The measurements for ATP content and rate of production
were made immediately after isolation of the mitochondria to
improve the accuracy of the measurements and reduce any
inherent errors associated with the luminescent decay or
reduced viability of the isolated mitochondria. The mitochondria were kept on ice to ensure this added stability and were
not incubated at 28°C (consistent with the standards) or
ATP PRODUCTION IN MITOCHONDRIA WITH AGE AND CALORIC RESTRICTION
RESULTS
Reaction mixture and standards stability. Wibom et
al. (48) studied the stability of mitochondrial preparations in pyruvate and malate and found a linear decrease in activity (i.e., mitochondrial degradation) of
11 ⫾ 2%/h over a 5-h period. While the activities of the
mitochondrial preparations were not measured, the
overall stability of the mitochondria to produce ATP
was apparent in this study as the readings tended to
decrease for subsequent measurements. As a result, we
measured the decline in the sensitivity of the reaction
mixture as well as two relevant standards with time.
For the reaction mixture (Fig. 1A), the linear decay was
4.88 ⫻ 10⫺3 U/min or a 2.16% decay/min. Because the
OD values for the reaction mixture are much smaller
than the ATP OD readings, the inherent decay is
negligible. For a 0.5 ␮M ATP standard (Fig. 1B), i.e.,
OD readings consistent in magnitude with the luminescent readings for the ATP content present in 10 ␮l
AJP-Regul Integr Comp Physiol • VOL
Fig. 1. Luminescent decay and time of the reaction mixture. A: the
reaction mixture (see METHODS for components) showed a very stable
signal (0.230 ⫾ 0.019 U, 75% of data points within 10% of mean)
during a 5-min analysis measuring data every 5 s. The linear decay
was 4.88 ⫻ 10⫺3 U/min or 2.16% decay/min. B: 0.5 ␮M ATP standard
(readings consistent with ATP content in 10 ␮l mitochondrial sample
before normalization to the protein concentration) showed a linear
decay of 5.70 U/min or 1.39% decay/min. C: 25 ␮M ATP standard
(readings consistent with rate of ATP production in 10 ␮l mitochondrial sample before normalization to the protein concentration)
showed a logarithmic decay defined by equation: y ⫽ ⫺533.91 ln(x) ⫹
13,512, where r2 ⫽ 0.993. Hence after the 1st min, the luminescent
signal decreased by 16.2%, but slows to 22.4% for the entire 5-min
run.
of mitochondrial sample before normalization to the
protein concentration, the luminescent signal underwent a linear decay (r2 ⫽ 0.988) for the entire 5 min of
analysis. The signal decayed at a rate of 5.70 U/min or
1.39% decay/min. Because the amount of ATP present
in the tissue of interest is analyzed over a 30-s interval,
this small linear decay can also be considered negligible. For a 25-␮M ATP standard (Fig. 1C), OD readings
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biological temperature (⬃37°C); however, this should have
no effect on the accuracy because thermodynamic laws of
heat transfer dictate that the small volume (10 ␮l) of cold
mitochondria will have virtually no effect on the overall
temperature of the larger assay volume of the reaction mixture (990 ␮l), which is kept at 28°C. Like the standards, the
background of the reaction mixtures (990 ␮l) was recorded
before the addition of mitochondria. Afterward, 10 ␮l of
freshly isolated mitochondria from the frontal cortex of the
brain or liver were added to the reaction mixture to determine the ATP content. The analyses were carried out and
recorded similarly to the aforementioned standards. The
luminescent signal decreased rapidly during each successive
reading as the ATP was quickly converted through a series of
enzymatic redox reactions to AMP through the release of the
two terminal phosphate groups (see reactions 1 and 2). Thus
only the first reading was used to calculate the ATP content
in the mitochondria because it corresponded to the maximum
amount of ATP present [and hence yielded the highest OD
reading on the luminometer of the 6 measurements]. Immediately after the ATP content measurements, 10 ␮l of 2.5 mM
ADP was added to the cuvette containing the reaction mixture and mitochondria to determine the rate of ATP production. ADP (2.5 mM) was used because it corresponds to a
physiological concentration found in myocytes (33). The
amount of ATP produced within the mitochondria is directly
proportional to the ADP available, so any change in ADP
concentration should directly affect the ATP concentration.
Hence, it is supportive that ATP content and rate of production varied in unison, because production and consumption
are regulated by ATP-to-ADP ratio (11). The system was
monitored as previously mentioned, and the final reading
was used to determine the rate. The values for ATP content
and rate of production were normalized to the mitochondrial
protein concentration present in each 10-␮l aliquot. All mitochondrial samples were performed in triplicate, and an
average of these results was used in the quantification of the
ATP content and rate of production.
Statistical analysis. Unpaired t-tests were used for comparisons between groups, Pearson product-moment correlations were performed between dependent variables, and the
intra- and interassay coefficients of variation were calculated
using a statistical package from Prism (San Diego, CA). A P
value of ⬍0.05 was considered significant.
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consistent with the luminescent readings for rate of
ATP production in 10 ␮l mitochondrial sample before
normalization to the protein concentration, the luminescent signal underwent a logarithmic decay defined
by Eq. 1
⫺533.91 ln(x) ⫹ 13,512
(1)
Fig. 2. Inhibition of the F0F1-ATPase enzyme by oligomycin. To
confirm the functionality of the ATP assay, 0, 1, and 5 mM oligomycin, an inhibitor of oxidative phosphorylation, were incubated with
the isolated mitochondria for 10 min at 37°C. The 1 and 5 mM
oligomycin were able to inhibit the production of ATP by 34%
(*** P ⫽ 0.0001) and 83% (*** P ⬍ 0.0001), respectively, compared
with control.
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where r2 ⫽ 0.993.
Hence during a 30-s analysis on the rate of ATP
production, the luminescent signal corresponding to
this high concentration would decay by 13.4%.
Inhibition of the F0F1-ATPase enzyme by oligomycin.
To confirm the functionality of the ATP assay, 1 and 5
mM oligomycin, an inhibitor of oxidative phosphorylation, were incubated with a portion of the isolated
mitochondria for 10 min at 37°C and compared with a
control (equal portion of freshly isolated mitochondria
from the same sample). The 1 and 5 mM oligomycin
were able to significantly inhibit the production of
ATP by 34 and 83% (1 mM, P ⫽ 0.0001; 5 mM, P ⬍
0.0001), respectively (Fig. 2). All tissues were run in
triplicate (n ⫽ 4), and the average of these results was
used to determine the extent of inhibition to the production of ATP.
Effects of age and caloric restriction on body and
tissue mass and ATP content and rate of production in
liver and brain. As expected, body mass decreased by
15% with age (young vs. old, P ⫽ 0.002) and an additional 33% through lifelong caloric restriction (old vs.
calorie restricted, P ⬍ 0.0001) (Table 1). In the brain
and liver, there was no significant change in mass due
to age. In striking contrast, caloric restriction led to a
41% (old vs. calorie restricted, P ⬍ 0.0001) reduction in
the liver mass while having no effect on brain weight
(Table 1).
Previously, we showed that with age, ATP content
and production decreased by ⬃50% in isolated rat
mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria
(11). However to date, no one has quantitatively measured in vivo the effects of age and caloric restriction on
the ATP content or on the rate of ATP production in the
isolated mitochondria of the brain or liver of 12- to
13-mo-old ad libitum-fed, 26- to 28-mo-old ad libitumfed, and 27- to 28-mo-old lifelong caloric-restricted
rats. Consequently, measurements were made on two
separate tissues: brain and liver (Table 2). In the brain,
the ATP content was unaffected by age or caloric restriction (Fig. 3A). Moreover, the rate of ATP production was not different due to age and caloric restriction
(Fig. 3B). In the liver, the results showed a similar
trend: neither age nor caloric restriction had any significant effect on the ATP content (Fig. 3C) or on the
rate of ATP production (Fig. 3D). However, the lifelong
calorie-restricted rats tended to have a higher production rate of ATP in the liver, but these changes where
not statistically significant (Table 2).
Recently, we determined the rate of ATP production
in skeletal muscle and the heart (complete data published in Ref. 11) . The rate of skeletal muscle ATP
production was 7.29 ⫾ 1.01 nmol ATP 䡠 mg protein⫺1 䡠
min⫺1 in 12-mo-old ad libitum-fed animals compared
with 3.17 ⫾ 0.99 nmol ATP 䡠 mg protein⫺1 䡠 min⫺1 in
the 26-mo-old animals. In the heart mitochondria
there were no significant changes with age: 18.31 ⫾
1.46 (12 mo old) vs. 17.92 ⫾ 2.02 nmol ATP 䡠 mg
protein⫺1 䡠 min⫺1 in 26-mo-old rats. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP
content or rate of production in skeletal muscle and
had no effect on the heart. The production of ATP in
skeletal and heart muscle mitochondria was 2.77 ⫾
0.53 and 18.86 ⫾ 1.84 nmol ATP 䡠 mg protein⫺1 䡠 min⫺1
for 26-mo-old caloric-restricted rats (11). When the rate
of ATP production of the brain and the liver (Table 2)
are compared with that of the heart and skeletal muscle (gastrocnemius), the data show that the heart has
the greatest rate of production: heart ⬎ liver ⬎ brain ⬎
gastrocnemius muscle.
Intra- and interassay coefficients of variation to test
precision and variability. To test the precision and
variability of the aforementioned methodology to measure ATP content and production in isolated mitochondria, we determined the intra- and interassay variation coefficients. The intra-assay variation coefficient,
a measure of the precision of the assay, was determined by measuring the average of the triplicate readings for each sample (Table 3). The SD of each triplicate reading was divided by its average and multiplied
by 100 to create a percentage (%CV). The calculated
%CV was then averaged for each animal (n ⫽ 20 total
animals) to determine the intra-assay variation coefficient for isolated mitochondria from the brain and
liver. For both tissues, intra-assay %CV for ATP content and production was between 19.0 and 19.7%, respectively. To obtain the interassay variation coefficient, the variability of the assay from one animal to
the next, the average quantitative value for ATP content and production for each tissue was divided into
one of three variables (young, old, and caloric restricted) (Table 3). The SD of each group of variables
was divided by the respective average from that group
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Table 1. Body mass and brain and liver weight in young and old ad libitum-fed and old caloric-restricted
male Fischer 344 rats
Body mass, g
Brain, g
Liver, g
Young
Old
CR
P Value
434.8 ⫾ 4.5
2.03 ⫾ 0.04
14.80 ⫾ 0.41
369.5 ⫾ 20.5*
2.09 ⫾ 0.02
14.04 ⫾ 0.79
247.8 ⫾ 1.9†
2.06 ⫾ 0.03
8.79 ⫾ 0.25†
*P ⫽ 0.002; †p ⬍ 0.0001
NS
†P ⬍ 0.0001
Value are means ⫾ SE. Young ad libitum-fed (Young, n ⫽ 8, 12–13 mo); old ad libitum-fed (Old, n ⫽ 6, 26–28 mo); and old caloric-restricted
(CR, n ⫽ 6, 27–28 mo) male Fischer 344 rats (National Institute of Aging colony, Harlan Sprague Dawley, Indianapolis, IN) were used.
* Young vs. Old; † Old vs. CR. NS, nonsignificant.
DISCUSSION
It has been proposed that as we age, an accumulation of mitochondrial damage from reactive oxygen
species leads to a degradation in the efficiency of the
respiratory chain and hence a decline in ATP production (8–10, 47). Moreover, most studies have focused on
the activities of mitochondrial matrix enzymes and
respiratory enzyme complexes to determine mitochondrial viability and function. Few studies have determined the actual rate of ATP production in isolated
mitochondria in vivo. The following ATP determination
assay, as well as the preceding methodology, presents a
highly sensitive, reproducible method to quantitatively
measure in vivo ATP content and rate of ATP production in isolated mitochondria. A limitation with this
method hinges on the assumption that the intact mitochondria isolated for these studies are all functional
and that any inert, damaged, or unhealthy mitochondria have not been isolated. Recently, we used this
method to measure the ATP content and production in
both the heart and skeletal muscle of aging and caloricrestricted rats (11) and found a 50% decline in ATP
content and the rate of ATP production in skeletal
muscle, but not in the heart.
In this paper we describe the stability, inhibition
using oligomyocin, and ATP content and production in
the liver and the cerebral cortex with age and calorie
restriction. This step-by-step approach should allow
the easy use for the protocol to measure ATP production in vivo and demonstrate the high sensitivity of this
assay. Other studies show that the sensitivity of the
luciferin/luciferase reaction has been used to detect
trace levels of ATP attributed to low-level bacterial
contamination (19). Its sensitivity was apparent in our
study showing its ability to detect a 5 nmol ATP standard (Fig. 1B; 10 ␮l of 500 nmol diluted to 1,000 ␮l total
volume). Moreover, when the diluted standard was
diluted another 500-fold, there still was a linear response to the reaction between ATP and luciferin/
luciferase (see reactions 1 and 2). Hence, it is evident
that the sensitivity of the aforementioned assay can
detect levels of ATP as low as 10 pmol.
Beyond the sensitivity of the assay, the ATP determination kit was shown to be stable. The luminescent
signal of the reaction mixture showed a slight decay of
2.16%/min (Fig. 1A). However, because the quantum
signal of the reaction mixture is on the order of ⬃10–
100 times less than the yield observed for the ATP
content and ⬃100–1,000 times less than that observed
for the rate of ATP production, the decay rate seen for
the reaction mixture is negligible. For a 0.5 ␮mol ATP
standard (Fig. 1B), which corresponds to the linear
response observed for the ATP content, the linear decay was 1.39%/min. The ATP content was taken as the
highest OD reading (usually the 1st or 2nd reading).
Thus any decay beyond the first reading would have no
effect on its value and would be negligible to the quantitation of the ATP content.
Following the first reading, five more subsequent
readings are taken for a total analysis time of 30 s (a
total of 6 measurements). The final reading taken at
30 s is utilized in the determination of the rate of ATP
synthesis by factoring out the contribution of the signal
from the ATP content, the signal from the 2.5 mM ADP
(33) in isolation buffer and reaction mixture alone, as
well as the decay of the bioluminescent signal associated with it. Hence the determination of the rate of
ATP production is exclusive of these aforementioned
parameters and is indicative of the production of ATP.
The decay rate of a 25.0 ␮mol ATP standard (Fig. 1C)
yields a quantum signal that is parallel to the intensity
Table 2. ATP content and rate of production in liver and brain
Brain
ATP content
ATP production
Liver
Young
Old
CR
Young
Old
CR
10.67 ⫾ 2.03
12.40 ⫾ 3.77
12.33 ⫾ 1.80
14.66 ⫾ 2.04
14.27 ⫾ 2.51
12.25 ⫾ 2.88
0.14 ⫾ 0.04
15.61 ⫾ 2.39
0.14 ⫾ 0.04
17.56 ⫾ 2.23
0.17 ⫾ 0.03
19.93 ⫾ 3.69
Value are means ⫾ SE. ATP content (nmol/mg protein) and ATP production (nmol/mg protein/min) were determined.
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and multiplied by 100 to create a percentage (%CV).
Hence three variables yielded three individual %CV.
These three %CV were then averaged to give the interassay variation coefficient for isolated mitochondria
from the brain and liver from one animal to the next.
For both tissues, interassay %CV for ATP content and
production yielded values of 53.5 and 49.6%, respectively.
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ATP PRODUCTION IN MITOCHONDRIA WITH AGE AND CALORIC RESTRICTION
Fig. 3. Effects of age and caloric restriction (CR) on the
content of ATP present and rate of ATP production. In
the brain (A and B), neither age nor CR had any effect
on the amount of ATP present (A) or on the rate of ATP
production (B). In the liver (C and D), the amount of
ATP present (C) as well as the rate of ATP production
(D) were also unaffected by age and CR.
Table 3. Intra-assay (precision) and interassay
(variability) %CV
Intra-assay %CV
Brain
Liver
Both Tissues
Interassay %CV
ATP
content
ATP
production
ATP
content
ATP
production
9.6
28.3
19.0
19.8
19.6
19.7
44.2
62.7
53.5
59.2
39.9
49.6
%CV, coefficient of variation.
AJP-Regul Integr Comp Physiol • VOL
inhibiting the proton pump of the mitochondrial inner
membrane, thus impeding the production of ATP. Various concentrations of oligomycin were able to significantly inhibit the production of ATP by 34% (1 mM)
and 83% (5 mM) (Fig. 2). Hence this demonstrates the
resilience of the F0F1-ATPase enzyme to function even
after incubation with a relatively high concentration of
oligomycin, an inhibitor of oxidative phosphorylation.
To determine the precision and variability of the
assay, the intra- and interassay coefficients of variation were determined (Table 3). The intra-assay
%CV, an indicator of precision from one measurement to the next on the same tissue, yielded values of
19.0% for the ATP content and 19.7% for the rate of
ATP production for both tissues, brain and liver.
While values of 10% are considered ideal, the intraassay %CV determined for this methodology is good
and is indicative of the reason measurements were
run in triplicate and then averaged. The interassay
%CV, an indication of variability of the assay from
one animal to the next, gave values of 53.5 and 49.6%
for the measurements of ATP content and rate of
ATP production, respectively. This interassay %CV
shows the inherent variation seen in vivo for biological markers as shown by others (3, 17, 23).
Neurological diseases and diseases associated with
age, such as Alzheimer’s (25, 46), Parkinson’s (1, 16,
34–36, 39, 49), and Huntington’s (6, 22), have all been
partly attributed to decreases in the function of respiratory chain complexes and the assumption that there
is a lack of ATP production and content. A deficiency in
key energy-metabolizing enzymes, such as cytochrome
c oxidase (complex IV), could lead to a reduction in
energy stores and thereby contribute to the neurodegenerative process. Moreover, liver diseases, such as
Wilson disease, are associated with frequent, diverse,
and early deletions of mitochondrial DNA (mtDNA),
which could lead to altered energy production, because
respiratory complexes contain subunits encoded in
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observed for the production of ATP in isolated mitochondria. It is assumed that the decay rate defined by
Eq. 1 is concurrent in the analysis of ATP synthesis in
isolated mitochondria and digresses from linearity due
to the high concentration. Consequently, this is where
the intrinsic deviations from the Beer-Lambert law
take effect.
This decay is probably due to combination of different factors such as deviations in absorbance coefficients at higher concentrations due to electrostatic
interactions of luciferyl adenylate on the luciferase
substrate (see reaction 1) in close proximity, scattering
of light due to the relatively high concentration of ATP
in the sample, fluorescence or phosphorescence of the
sample during the reaction (see reactions 1 and 2),
changes in the refractive index due to analyte concentration, and/or shifts in chemical equilibrium as a
function of increased concentration of ATP. Applying
Eq. 1 to a 30-s analysis, the luminescent sample would
decay by 13.4%. While the average values are given for
the rate of ATP production as well as the SD about the
mean, it can be assumed that there is an intrinsic
variation contemporaneous with the decay rate seen in
the higher concentration standards.
We tested oligomycin, a strong inhibitor of oxidative
phosphorylation, in its ability to attach to and inhibit
the F1-ATPase complex. Oligomycin functions by binding to the stalk region of the F0F1-ATPase enzyme and
ATP PRODUCTION IN MITOCHONDRIA WITH AGE AND CALORIC RESTRICTION
AJP-Regul Integr Comp Physiol • VOL
of different ages (2, 6, 12, 18, 24, and 26 mo). All
cytochrome c oxidase subunits showed an age-related
increase from 2-mo-old rats up to 24 mo with a small
decrease at the oldest age (26 mo). The same pattern of
age-dependent changes was observed for ␥-ATP synthase, while the ␣- and ␤-subunits increased progressively up to 26 mo. Thus, while mtDNA deletions
increase with age, they appear to have no significant
effect on capacity for oxidative phosphorylation of distinct brain regions, and there are no previous studies
that suggest a failure in energy production with age in
the brain. In our study using very old Fischer-344 rats
(27–28 mo old; mean life span is 24 mo old) we found no
decline in ATP production in the brain and liver, suggesting that the selective deletions and changes to
respiratory complexes that may occur with age have no
overall effect on the capacity of these tissues to produce
energy under basal conditions.
Caloric restriction is one of the only proven, preventative methods for inhibiting the effects of age and
extending life span. Moreover, caloric restriction has
been shown to decrease the production of reactive oxygen species and reduce oxidative stress with increased age (15, 20, 21, 31, 40, 41). Hence, an assumption could be that lifelong caloric restriction should
lead to a higher concentration as well as a higher rate
of production of ATP within the isolated mitochondria
compared with ad libitum-fed animals. This experimental model would examine the hypothesis that the
rate of aging may depend on the production of cellular
ATP. However, in the cerebral cortex of the brain, no
change was observed with age or with caloric restriction on the amount of ATP present or on the rate of
production of ATP in the brain. In the liver, neither
age nor caloric restriction had any significant effect
on the amount of ATP present or on the rate of ATP
production.
Recently, others have investigated the effects of
long-term caloric restriction on extracts from brain
mitochondrial fractions and mitochondria isolated
from the heart and skeletal muscle. Olgun et al. (29)
found no significant difference in the protein levels of
complexes I and III between ad libitum-fed and longterm caloric-restricted mice (alternative day feeding).
Complex IV activity increased by 8% with caloric restriction, but this change was not statistically significant. Furthermore, recent studies by Sreekumar et al.
(42) and Drew et al. (11) found that caloric restriction
did not have any major impact on ATP production
and/or mitochondrial function in muscle and heart.
In conclusion, this study demonstrates that the following methodology provides a sensitive means to
quantitatively measure ATP content in addition to the
rate of synthesis in isolated mitochondria. Moreover,
age and caloric restriction had no major effects on
energy production in the mitochondria isolated from
the brain and the liver. The relationship between mitochondrial complex damage-inhibition and ATP synthesis has been recently reviewed by Rossignol et al.
(32). They describe three “mitochondrial threshold effects.” The first shows that, in most cases, phenotypic
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mtDNA (24, 50). The occurrence of specific base pair
deletions are consistent with liver mitochondrial respiratory function decline with age (50). Surprisingly,
little is known as to what the effects of a decrease in
specific respiratory chain complex activities are on
ATP production in vivo in isolated mitochondria from
disease pathologies and in the normal aging brain.
Previously, Davey et al. (7, 8) showed that titration
of complex I, III, and IV activities with specific inhibitors generated threshold curves that showed the extent to which a complex activity could be inhibited
before causing impairment of mitochondrial energy
metabolism. Using rat brain mitochondria of synaptic
origin, the activities of complexes I, III, and IV of the
respiratory chain were decreased by 25, 80, and 70%,
respectively, before major changes in the rates of oxygen consumption and ATP synthesis were observed (7,
8). Respiratory chain complexes from mitochondria of
nonsynaptic origin showed similar resilience to inhibitors (72, 70, and 60%), respectively, before ATP production was reduced (7, 8). From our data and others
(7, 8), it appears that electron transport chain complexes are more resilient to reactive oxygen species
than previously thought and that any inhibition in
ATP production would be a result of a severe impairment to one or more of these complexes (7, 8, 30, 34,
36). Therefore, acute reduction in ATP content and rate
of production in mitochondria is only due to severe
damage to the respiratory complexes, possibly leading
to the aforementioned age-related disease states (7, 8,
24, 45, 50).
In vivo, in the brains of normally aging humans and
rats there is a disparity of results regarding a correlation between respiratory chain complex activities and
energy production. Ojaimi et al. (28) show that there is
a significant decrease in cytochrome c oxidase activity
in several brain regions (frontal cortex, superior temporal cortex, cerebellum, and putamen) with age in
humans. However, this study is far from conclusive,
because only 12 human subjects were studied, while
there were only 2 younger subjects used for statistical
comparison (26 and 36 yr of age). Furthermore, a study
conducted by Hansford’s group (12) suggests that there
is little evidence of a decrease in respiratory enzyme
complex activities. In young 6-mo-old Wistar rats, deletion-containing mtDNA were present at low levels
(⬃0.0003%) in the striatum, hippocampus, cerebral
cortex, and cerebellum, and mtDNA levels increased
25-, 7-, 3-, and 2-fold in these brain regions of 22- to
23-mo-old rats, respectively. Importantly, the activities
of mitochondrial respiratory chain complexes I, III, IV,
and V, the mitochondrial ATPase, each of which contains subunits encoded in mtDNA, showed no agerelated decrements in activity in any of the brain
regions (12). In contrast, another study shows that
there is a moderate decline in activities of mitochondrial enzymes NADH-cytochrome c reductase, cytochrome oxidase, and citrate synthase, by ⬃14–58% in
72-wk-old mice (26). Nicoletti et al. (27) examined
contents of subunits cytochrome c oxidase and of subunits of F0F1 ATP synthase from cerebral cortex of rats
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ATP PRODUCTION IN MITOCHONDRIA WITH AGE AND CALORIC RESTRICTION
We thank Dr. C. Selman, S. Phaneuf, and A. Hiona for input and
for critical reading of the manuscript.
DISCLOSURES
The National Institute of Aging supported this research (Grants
AG-17994, AG-10485, and AG-21042), as well as American Heart
Association, Florida-Puerto Rico Affiliate Grants 30334B and
0225194B.
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