The role of mineralized tissue in the buffering of lactic acid during anoxia and exercise in the leopard frog Rana pipiens
Brown University, Department of Molecular Pharmacology, Physiology and Biotechnology, Box G, Providence, RI 02912, USA
* Author for correspondence (e-mail: Daniel_E_Warren{at}brown.edu)
Accepted 10 January 2005
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Summary |
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Key words: anoxia, bone, buffering, Ca2+, exercise, lactic acid, leopard frog, Rana pipiens
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Introduction |
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Reptile bone buffers lactic acid in several ways. It functions as a lactate
`sink' because it has low endogenous lactic acid production and, therefore,
lactic acid produced by other tissues and distributed to the extracellular
fluid can accumulate in the bone. When lactate accumulates to concentrations
greater than calculated from the tissue water, the bone is said to sequester
lactate, which is most likely by complexing to calcium in the bone. Vertebrate
bone also contains significant quantities of calcium carbonate, which when
liberated, chemically buffer protons generated by glycolysis. The resultant
carbon dioxide from this chemical reaction diffuses into the surrounding water
while the remaining calcium accumulates in the extracellular fluid and can
reach high concentrations, especially at 3°C after 3 months of anoxic
submergence in painted turtles (100 mequiv l-1;
Jackson, 2002
).
Anuran amphibians also possess significant calcium carbonate deposits in
their endolymphatic system (Whiteside,
1922). Although restricted to the inner ear in most vertebrates,
this system is large and extends down the length of the vertebral column in
anuran amphibians. The exact function of the large endolymphatic system in
frogs is not known. Proposed hypotheses include the protection of the spinal
ganglia, as an endolymph reservoir when pressures in the auditory labyrinth
are high, or as an aid in sound transmission
(Simkiss, 1967
). After he had
observed that Ca2+ excretion increased during environmental
hypercapnia, Simkiss (1968
)
proposed that the frog endolymphatic system helps maintain acid-base
homeostasis by releasing CaCO3. This hypothesis was further
supported in a subsequent study (Tufts and
Toews, 1985
) in which hypercapnia increased plasma Ca2+
concentrations in toads. These investigators estimated that half of the
observed compensatory HCO3- response must have come from
internal CaCO3 stores because the levels could not be accounted for
solely by uptake across the skin.
Our purpose was to determine whether the skeleton and endolymphatic system
of the leopard frog Rana pipiens play roles in buffering lactic acid
accumulated during anoxic submergence and strenuous exercise. There are no
previous reports of lactate accumulation in mineralized tissue as a result of
anoxia in an amphibian, or following exhaustive exercise in any vertebrate.
Although the leopard frog is not likely to experience anoxia in its natural
environment, it is very likely to accumulate extreme levels of lactic acid due
to lesser degrees of oxygen lack during winter hibernation
(Donohoe and Boutilier, 1999).
Therefore, to study the frogs' response to anoxia is useful for determining a
role for mineralized tissue responses in buffering lactic acid, which we
assessed by measuring lactate accumulation in the mineralized tissues and
comparing it to the lactate concentrations in other tissues of the frog. We
assessed the skeletal contribution as sources of chemical buffering by
measuring the concentrations of their major ionic constituents in plasma.
Because it was easy to dissect rapidly, we sampled the entire frog auditory
capsule, a part of the skull that encloses the membranous labyrinth and a
portion of the endolymphatic sac (Fig.
1; Whiteside,
1922
). The mineral compositions of femur and auditory capsule were
analyzed for comparisons with bone of other vertebrates. We also incubated
frog femur in 30 mmol l-1 lactate solutions at pH 8.0 and 7.3 for 6
and 24 h to ascertain the capacity for lactate uptake in frog bone.
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Materials and methods |
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Determination of bone and auditory capsule Ca2+, Mg2+, Na+, K+ and Pi concentrations
The ionic compositions of frog bone and auditory capsule were determined.
Frogs were pithed and their femurs (N=1x5 animals) dissected
from skeletal muscle. Auditory capsules (N=2x5 animals), formed
by the prootic and exoccipital bones of the skull, were sampled by bisecting
the skull mid-sagitally and dissecting them from surrounding muscle and the
squamosal bone on the lateral edge of the skull. Femur and auditory capsules
were dried for 2-3 days at 87°C and ground to a powder under liquid
nitrogen (Spex 6700, Freezer Mill, Metuchen, NJ, USA). The powder was placed
in porcelain vials and heated to 475°C for 2 days to burn away all organic
material. The resultant ash was dissolved in 12 vol. 2 mol l-1 HCl
and analyzed for Na+ and K+ using flame photometry (IL
model 943; Lexington, MA, USA). The HCl was further diluted with 5 vol. of
deionized water (60 vol. total) and analyzed for Mg2+. A portion of
this solution was further diluted with 30 vol. of deionized water (1800 vol.
total) and analyzed for Ca2+ and inorganic phosphate
(Pi; see below for the details of the analysis). Atomic absorption
spectrophotometry (Perkin-Elmer model 280, Boston, MA, USA) was used to
measure Ca2+ and Mg2+ levels and a standard kit for
Pi (Kit 670 Sigma, St Louis, MO, USA).
Determination of bone and auditory capsule CO32-
A known amount (0.02-0.1 g) of bone or auditory capsule powder (obtained as
described above) was introduced into a flask containing 15 ml 2 mol
l-1 HCl through which humidified nitrogen gas was passed (240
ml min-1; Jackson et al.,
1999
). Carbon dioxide, generated as the result of
CO32- titration, was then carried by the nitrogen gas
through a drying column (Drierite, Xenia, OH, USA) and then through a
CO2 analyzer (AEI, model CD-3A, Pittsburgh, PA, USA). The output
from the carbon dioxide analyzer was recorded on a laptop computer using a
data acquisition system (BIOPAC MP100, Goleta, CA, USA) and analyzed using
software (Acqknowledge, BIOPAC, Goleta, CA, USA). The volume of CO2
generated was calculated by the following:
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All volumes were corrected to STPD and converted to mmoles
CO2, assuming the constant 22.26 ml per mmole CO2
(Cameron, 1989). One mole of
CO2 was assumed to be derived from one mole of
CO32-.
Anoxia and exercise experimental protocol
The anoxia and exercise experiments were carried out at 15°C. Frogs
were randomly chosen as controls, anoxic or exercised.
Control frogs (N=6) were placed individually into darkened, 1-liter containers (diameter=11 cm, height=12 cm) containing 0.5 l aerated water. The next day, 5 ml of a buffered 0.1 g ml-1 MS222 solution (buffered to pH 7.0 with 1 mol l-1 NaOH) were added to one of the containers. After 20 min, theanesthetized animal was removed, pithed, weighed and sampled as described below. The remaining frogs were similarly sampled in turn. Control frogs were treated in this manner because preliminary experiments in which we pithed frogs without anesthesia showed significant lactate accumulation in their tissues and it was our intention to sample the tissues with tissue lactate contents as low as possible.
Anoxic frogs (N=6) were placed in a water-filled 15 cmx21
cmx43 cm (WxHxL) aquarium vigorously bubbled with nitrogen
gas for at least 1 h prior, in order to displace any dissolved oxygen. A
plastic mesh screen was placed just below the surface of the water to prevent
access to air. An additional acrylic lid covered all but a small part of the
water's surface to allow for nitrogen gas to escape. Vigorous bubbling of the
nitrogen gas was continued throughout the submergence. After 2.5 h, the
animals were removed, pithed, weighed and sampled as described below. Anoxic
frogs were not anesthetized because the animals were quiescent, but still
alive, by the end of anoxic submergence and did not struggle during the
pithing. The quiescence has been observed in previous studies of anoxia in
frogs (Wegener and Krause,
1993).
Exercised frogs (N=6) were placed in a 15 cmx21 cmx43 cm (WxHxL) aquarium with 2-3 cm deep water and chased with a metal rod until they were incapable of further burst swimming (10-19 min). The animals were removed, pithed, weighed and sampled as described below. Exercised frogs were not anesthetized because doing so would have required an additional 20 min after the exercise period ended and would have prevented us from examining the immediate post-exercise condition of the animal. The animals struggled little, if at all, during the pithing because they were exhausted from the exercise and so additional lactate production after the exercise period was unlikely.
Tissue sampling
All sampling was performed in a cold-room at 3°C to minimize changes in
tissue metabolites during dissection. After pithing, a mid-line incision on
the ventral side of the frog was made and blood (0.1-0.4 ml) was sampled
via cardiac puncture and placed on ice. Heart, liver and
gastrocnemius muscle were quickly sampled and flash-frozen in clamps cooled
using liquid nitrogen. The femur (1 per animal) was cleared of skeletal
muscle, its shaft cut lengthwise and the marrow removed. The bone fragments
were quickly freeze clamped. Auditory capsules (2 per animal) were sampled by
completely bisecting the skull mid-sagitally and cutting them free of
surrounding muscle and bone (orbital and maxilla), and were freeze clamped.
The remaining carcass was clamped and frozen by immersion in liquid
nitrogen.
The blood samples were kept on ice for 1-2 h until they could be centrifuged for 3 min at 9300 g. Previous experience in our laboratory has shown that the lactate and ionic composition of the plasma do not change when treated in this manner. The plasma was transferred to another vial and stored at -25°C until analyzed for lactate (Kit 735-10; Trinity Biotech, St Louis, MO, USA), Ca2+ and Mg2+ (atomic absorption spectrophotometry; Perkin-Elmer model 280), Na+ and K+ (flame photometry; IL model 946, Lexington, MA, USA) and Pi (Kit 670, Sigma). The frozen tissues were kept on dry ice for 1-2 h before they could be transferred to a deep freeze, where they were stored at -75°C until analyzed for lactate.
Lactate analyses of tissues and plasma
Frozen heart, liver and gastrocnemius muscle (200 mg) were homogenized
in 1 ml ice-cold 0.6 mol l-1 perchloric acid using a
Mini-Beadbeater 3110BX (Biospec, Bartlesville, OK, USA) using 1 mm glass beads
for 3 min. The frozen carcass was homogenized in 4 vol. of ice-cold 0.6 mol
l-1 perchloric acid using a Virtis Super 30 homogenizer (Gardiner,
NY, USA). Frozen femur and auditory capsules were ground to a powder under
liquid nitrogen (Spex 6700, Freezer Mill). The powder was incubated in 5 vol.
of ice-cold 0.6 mol l-1 perchloric acid for 2 h on ice, vortexing
every 20 min. Samples of all tissue homogenates were centrifuged at 9300
g for 3 min. [Lactate] was measured in the resultant
supernatants and plasma using an enzymatic assay (Kit 735-10, Trinity
Biotech).
Bone incubations
Twelve leopard frogs were euthanized with intraperitoneal injections of
Beuthanasia®-D Special (Schering-Plough, Millsborough, DE, USA) and their
femurs removed, cleaned of soft tissue including marrow, and frozen at
-25°C until used in the incubations. One femur from each of the 12 animals
was incubated in a beaker containing a solution of 0.8% NaCl, 10 mmol
l-1 TES and 30 mmol l-1 lactate at pH 8.0, and the other
femurs in an identical solution at pH 7.3. The volume of solution in each
beaker was 80 ml. Six femurs were sampled from each beaker after 6 h and 24 h,
frozen in liquid nitrogen and stored at -25°C until analyzed for lactate
as described above.
Statistical analyses
Differences in the composition of femur and auditory capsule were
determined using t-tests. Two-way multivariate analysis of variance
(MANOVA) was used to determine whether tissue type and treatment affected
lactate concentration. Least-squares mean (LSM) tests were used to determine
whether treatment affected the plasma concentrations of Ca2+,
Mg2+, Na+, K+ and Pi, and whether
time and pH affected lactate uptake into bone in the incubation experiment.
Student's t-tests were used to elucidate differences revealed by the
MANOVA and LSM tests. All statistical analyses were performed using JMP 4.0
(SAS Institute, Cary, NC, USA).
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Results |
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Auditory capsule contained more water and less organic matter than femur. Femur ash contained slightly more Pi and more Mg2+ than auditory capsule. There was no difference in the amount of Na+ between the two tissues. Auditory capsule ash contained more Ca2+ and K+ than femur ash. Dry auditory capsule contained almost twice the CO32- of dry femur.
Lactate distribution in normoxic controls
Tissue lactate concentrations from anesthetized, but otherwise undisturbed,
animals are shown in Fig. 2 and
were low in all tissues examined (means ranged 0.6-1.8 mmol
kg-1).
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Lactate distribution during anoxia
Anoxic submergence at 15°C for 2.5 h significantly increased lactate
concentrations above control levels in all tissues, including femur and
auditory capsule (Fig. 2).
Femur and auditory capsule lactate concentrations (15.0 and 13.2 mmol
kg-1, respectively) were similar to those in carcass (13.5 mmol
kg-1). Gastrocnemius muscle accumulated the most lactate (24.7 mmol
kg-1) and liver the least (9.0 mmol kg-1), both of which
were significantly different from the other tissues. Plasma and heart
accumulated similar lactate concentrations (20.4 and 18.4 mmol
kg-1, respectively), which were significantly greater than all
other tissues except gastrocnemius muscle.
Lactate distribution during exhaustive exercise
The mean time to exhaustion was 13.1±1.6 min (range=10-19 min) at
15°C. Lactate concentrations were significantly elevated relative to
controls in all tissues, including femur and auditory capsule
(Fig. 2). Femur lactate
concentrations were similar to those in liver and auditory capsule and were
significantly lower than the other tissues. Auditory capsule lactate
concentrations were significantly less than in plasma, gastrocnemius and
heart, but similar to all other tissues. Gastrocnemius muscle and plasma
accumulated the most lactate (13.9 and 14.0 mmol kg-1,
respectively) and femur and liver the least (5.3 and 4.6 mmol kg-1,
respectively). The lactate concentration in plasma did not differ from that in
heart (10.6 mmol kg-1).
Plasma ion changes during anoxia and exhaustive exercise
Plasma ion concentrations during anoxia and exhaustive exercise are
summarized in Figs 3 and
4. Plasma [Ca2+]
increased significantly relative to controls after 2.5 h anoxia, but was
unaffected by exhaustive exercise. Plasma [Mg2+] increased in both
anoxic and exercise groups by 35% and 17%, respectively, and was significantly
different across all groups. Plasma [Pi] and [K+]
increased significantly in both anoxic and exercised frogs but did not differ
between the two groups. Plasma [Na+] was unaffected by anoxia or
exercise.
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Bone incubations
The results of the bone incubations are presented in
Fig. 5 and show that frog bone
sequestered lactate at pH 8.0 and 7.3. There was no significant interaction
between pH and time (P=0.07), but time and pH, by themselves, had
significant effects. Lactate concentrations of the solutions fell during
incubation from 29.7 to 27.3 mmol l-1 at pH 7.3 and from 29.3 to
27.9 mmol l-1 at pH 8.0.
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Discussion |
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The composition of frog bone in this study is similar to that in a previous
analysis and to most other vertebrates
(Biltz and Pellegrino, 1969).
Frog femur contains less CO32- than bone and shell of
the extremely anoxia-tolerant painted turtle Chrysemys picta bellii
(Jackson et al., 2000
), and
osteoderms of the broad-nosed caiman
(Jackson et al., 2003
).
Auditory capsule has a CaCO3 composition that is similar to that in
the long bone and shell of the painted turtle Chrysemys picta bellii
(Jackson et al., 2000
) and
greater than caiman osteoderm (Jackson et
al., 2003
).
The large CaCO3 deposits in our analysis of auditory capsule are
also consistent with the earliest descriptions of the endolymphatic lime
deposits (Dempster, 1930;
Whiteside, 1922
) and indicate
that we succeeded in sampling them with our protocol. This verification is
important because (1) our sampling included the dermal bone encapsulating a
portion of the endolymphatic lime sac, and (2) we used the same sampling
technique in our lactate analyses after anoxia and exercise. The sampled
structure had a milky appearance that was consistent with earlier descriptions
of the endolymphatic system.
Both exercise and anoxia increased lactate concentrations in all tissues
examined to levels observed in previous studies
(Andersen and Wang, 2003;
Armentrout and Rose, 1971
;
Bennett and Licht, 1974
;
D'Eon et al., 1978
;
Donohoe and Boutilier, 1999
;
Hutchison and Turney, 1975
;
Warburton et al., 1989
; Wasser
et al., 1993
,
1991
;
Wegener and Krause, 1993
).
With the exception of gastrocnemius and heart, plasma had a higher lactate
concentration than the other tissues examined, as has also been observed in
cold hypoxic frogs (Donohoe and Boutilier,
1999
) and cold anoxic turtles
(Jackson et al., 1996
). High
lactate levels in gastrocnemius and heart can be attributed to their intense
activity during the exercise and during the initial period in the anoxia
chamber. The frogs became quiescent about 30 min into the 2 h anoxia bout.
Higher extracellular than intracellular lactate concentrations have been
reported during cold hypoxia in frogs by Donohoe and Boutilier
(1999), who suggested that
lactate is transported from locally anaerobic tissues, where it is produced,
to locally aerobic tissues, where it can be oxidized or converted to glucose.
In the anoxic frogs with no aerobic tissues, we propose that exporting lactate
from the cells to the extracellular fluid (ECF) may better exploit the buffer
capacity of the mineralized tissues, the skeleton and endolymphatic
system.
Three pieces of evidence revealed by the present study suggest a
potentially important role for the skeleton in buffering lactic acid after
exercise and anoxia. First, the skeleton and auditory capsule accumulated
significant amounts of lactate after both exercise and anoxia. The
post-exercise lactate accumulation in frog bone is notable because it is the
first of its kind to be reported and it occurred in as few as 10 min, a rate
not seen before in vivo in any vertebrate. In the case of anoxia, the
mineralized tissues accumulated more lactate than the liver and the lactate
concentration in femur (15.0 mmol l-1) was greater than what could
be accounted for based on its water composition (54%) and the plasma lactate
concentration (20.4 mmol l-1), indicating that lactate was
chemically bound to mineralized bone, most likely complexed with
Ca2+, as previously suggested to occur in painted turtle shell
(Jackson, 2000). Although the
post-exercise femurs and auditory capsules and the anoxic auditory capsules
did not accumulate more lactate than predicted from their water contents,
sequestration cannot be ruled out. This is especially true in the
post-exercise femurs, which accumulated exactly what might be predicted from
the water content and plasma [lactate]. The prediction is likely to be an
overestimate because it assumes that all water is extracellular and in
equilibrium with plasma.
Second, our in vitro incubation of frog femur demonstrates that
significant sequestration of lactate is possible. These results reveal the
accumulation that is possible at equilibrium, although the in vivo
kinetics of exchange in these experiments did not permit full equilibration to
occur. The in vitro bone lactate levels reached in the frog are
similar to measurements on painted turtle (D.C.J., unpublished observation)
and greater than published uptakes in caiman osteoderms
(Jackson et al., 2003) and
crayfish carapace (Jackson et al.,
2001
).
The third piece of evidence that frog bone contributes to the buffering of
lactic acid is that plasma [Ca2+] was elevated after anoxic
submergence. We interpret this as the result of CaCO3 release from
mineralized tissues. It is unlikely that the changes in plasma
[Ca2+] were caused by hemoconcentration because plasma
[Na+] did not change. It is notable that an earlier study of anoxia
in bullfrogs did not observe an increase in plasma [Ca2+]
(Warburton et al., 1989).
However, the frogs in that study were paralyzed with succinylcholine, and
lactate levels were only half of what we observed, suggesting that the
severity of the acidosis may have been insufficient to demineralize bone.
The increase in plasma [Pi], during both anoxia and exercise is
most likely derived from active muscle as a result of creatine phosphate
hydrolysis during burst swimming and anoxic submergence
(Wegener and Krause, 1993),
rather than from bone. In vitro studies show that
CO32- and not Pi is the principle buffer
anion released from both mouse calvariae
(Bushinsky et al., 2002
) and
turtle shell powder (Jackson et al.,
1999
) when incubated in acid solutions.
The quantitative importance of the frog's skeleton in buffering lactic
acidosis can be estimated by summing the fractions of the total lactate load
that accumulated in the skeleton and that was buffered by CaCO3
released from the skeleton and/or endolymphatic system. The basis for this
calculation is the assumption that each mole of lactate accumulated in frog
bone is accompanied by a proton, as is the case in turtle bone
(Jackson et al., 1999). We
assume that for each mole of Ca2+ released from the skeleton, a
mole of CO32-, which buffers 2 moles of H+
derived from glycolyis, is also released. If a frog's wet skeletal mass is 16%
of total body mass (D. E. Warren and D. C. Jackson, unpublished), then the
percentage of the total lactate load contained within the skeleton at the end
of anoxia and exercise is 18% and 9%, respectively. Unfortunately, a similar
calculation cannot be made for the endolymphatic system because we did not
sample it entirely, although we assume it to be much smaller than the
skeleton. If we assume that all the released Ca2+ distributes
throughout the extracellular fluid and that the extracellular fluid volume is
26% of body mass (Thorson,
1964
), then 3% of the total lactate load was buffered by
CO32- released from the skeletal or endolymphatic
systems during anoxia. There is no evidence that the skeletal and
endolymphatic systems release chemical buffers after exercise because plasma
[Ca2+] was not elevated. After summation, we estimate that 21% and
9% of the total lactate loads produced during anoxia and exercise,
respectively, are buffered by mineralized tissue.
These estimates have several important implications that require more
investigation. Although the quantitative contribution to lactic acid buffering
is modest in exercise, the rapidity with which these mineralized tissues were
recruited has not been observed previously. In addition, the amount and
integrity of a frog's mineralized tissues may determine the severity of a
lactic acidosis that an animal can tolerate. This might help to explain why
frogs, although considered hypoxia tolerant, are anoxia intolerant. Under
hypoxic conditions, lactic acid is produced in locally anaerobic tissue and
moves into the extracellular fluid where it distributes to locally aerobic
tissues to be oxidized, resulting in slower lactate accumulation and glycogen
depletion rates (Donohoe and Boutilier,
1999). Although the extracellular distribution of lactate enables
exploitation of the skeletal and endolymphatic buffers during anoxia, only a
modest fraction of the lactate load is buffered, primarily because of the
structures' small size relative to body mass. Therefore, only the lower rates
of lactate accumulation that occur during hypoxia are sustainable while the
higher rates during anoxia overwhelm the limited buffering capacity of the
frog.
One intriguing conservation implication is that if mineralized tissues
proved vulnerable to demineralization in an acidic environment, then the
tolerance of frogs to anoxia or hypoxia and, possibly, anaerobic performance,
could be compromised. Indeed, acidification of frog environments has been
implicated as a cause of amphibian decline and low environmental pH has been
shown to increase mortality rates in this species
(Brodkin et al., 2003;
Simon et al., 2002
).
In conclusion, the leopard frog skeletal and endolymphatic systems contribute buffering during exercise by rapidly functioning as a sink for a small amount of the lactate load. Their buffering roles are more important during anoxia when these structures, especially bone, sequester a larger fraction of the lactate load as well as release chemical buffers. Future studies should investigate whether these systems are significant contributors to lactic acid buffering under the more ecologically relevant conditions of long-term hypoxia at cold temperatures, and determine how pH affects bone and lime sac demineralization in amphibians. Additional work should examine the relative contributions of skeleton and endolymphatic lime deposits to overall chemical buffer release. A better assessment of the size of the endolymphatic lime sacs is needed to determine their exact contribution to lactic acid buffering.
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Acknowledgments |
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References |
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