Lactate sequestration by osteoderms of the broad-nose caiman, Caiman latirostris, following capture and forced submergence
1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown
University, Providence, RI 02912, USA
2 Departamento de Zoologia, Centro de Aqüicultura, UNESP, Caixa Postal
199, 13506-970 Rio Claro, SP, Brazil
* Author for correspondence (e-mail: donald_jackson{at}brown.edu)
Accepted 15 July 2003
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Summary |
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Key words: acid-base, acidosis, bone buffering, bone minerals, crocodilian, Caiman latirostris, osteoderm, dermal bone, diving, lactic acid
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Introduction |
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A second way that bone buffers extracellular acid, by the uptake and
buffering of lactic acid within the bone itself, has recently been described
in the freshwater turtle Chrysemys picta bellii
(Jackson, 1997). This animal is
capable of tolerating large burdens of lactic acid during prolonged anoxic
submergence by exploiting the large buffering capacity of its shell and
skeleton, which together account for 35-40% of the turtle's body mass. Besides
releasing significant amounts of carbonate buffers into the extracellular
fluid, the turtle also accumulates high concentrations of lactate within its
shell. Experiments on powdered shell in vitro demonstrated that this
mechanism is acid-base relevant and is equivalent to the movement into the
bone of lactic acid. In extreme experimental cases, when circulating lactate
is very high, the lactate uptake mechanism can account for 40-45% of the total
lactic acid buffering in the body. When the buffer release mechanism is
considered as well, the shell and bone account for 70-75% of the total
buffering, indicating that the buffering functions of the shell are crucial
for the turtle's long-term anoxic tolerance.
Although the buffer release mechanism is generally regarded to be a
universal property of bone, the general occurrence of the lactate uptake
mechanism is uncertain. Lactate uptake was recently demonstrated in the
carapace of the crayfish Austropotamobius pallipes during emersion
hypoxia and lactic acidosis (Jackson et
al., 2001) and has also recently been observed in the shell of the
pulmonate snail Helix aspersa following 24 h of anoxia (D.C.J.,
unpublished observations). These invertebrate exoskeletons, unlike vertebrate
bone, are not vascularized and are composed of calcium carbonate.
The objective of the present study was to investigate the occurrence of lactate accumulation during hypoxic acidosis in the mineralized tissues of the broad-nose caiman Caiman latirostris. A particular object of our study was the dermal bone, or osteoderms, of this animal. We hypothesized that this bone, which, like the turtle's shell, forms an overlying armor on the dorsal surface of the animal, although only partial in the case of the caiman, may serve as a site for lactic acid buffering. To test this, we collected and analyzed osteoderms and blood after capture and before and after a period of enforced submergence.
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Materials and methods |
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Capture and surgical preparation
Caimans were captured with a noose from their holding tank and, after tying
their mouths closed with an elastic band, were brought into the laboratory and
restrained on an operating board with rubber bands. A damp cloth covered their
heads. The femoral arterial was dissected out and catheterized occlusively
under local anesthesia (lidocaine) with either PE 60 or PE 90 catheter tubing.
The wound was closed and the catheter was secured by suturing to the skin on
the dorsal surface. At the conclusion of surgery, a blood sample was taken and
a single osteoderm was taken from the mid-dorsal region, also using local
anesthesia. During this entire procedure, the animals remained perfectly still
with no apparent reaction to any of the procedures. The blood was centrifuged,
and plasma and the osteoderm were stored frozen for later lactate analysis. In
some experiments, blood pH and plasma total CO2 were measured
immediately.
Experimental protocol
The caiman was placed in a plastic container (50 cmx40 cmx70
cm) with a small amount of water and allowed to recover overnight. The next
morning, a blood sample was taken with minimal disturbance to the animal and
the caiman was then restrained and a second dermal bone sample taken with
local anesthesia within about 5 min of capture. Blood pH and total plasma
CO2 were analyzed, and bone and remaining plasma were stored frozen
for later analysis.
The following morning (2 days after surgery), a blood sample was taken and analyzed for pH, total CO2, PO (partial pressure of oxygen) and hematocrit, and a plasma sample was saved for lactate analysis. The caiman was then submerged without access to air for 30-60 min. At the end of the submergence period, before the resumption of breathing, blood samples were taken and analyzed as described above. The water level was then lowered to permit breathing and additional blood samples were taken for analysis at 30 min, 60 min and 120 min of recovery. After the final blood sample, the caiman was killed with an overdose of anesthesia (thiopentothal), and multiple samples of dermal bone were collected from the mid-dorsal trunk region and from the dorsal surface of the neck. In addition, in six animals, a section of femur was also removed. One of the animals served as a control and was not submerged. After its final blood sample, it was anesthetized and bone samples from a minimally disturbed animal were obtained. All bone samples were immediately frozen and were analyzed later for lactate, total CO2, % water, % ash and ash mineral composition (Na+, K+, Ca2+, Mg2+ and inorganic P).
Blood analysis
Blood pH and PO were measured using Radiometer BMS Mk 2
thermostatted near the animals' body temperatures of 26-28°C. Plasma total
CO2 concentration was measured with a Cameron chamber
(Cameron, 1971).
PCO2 was calculated using the
Henderson-Hasselbalch equation. Hematocrit was measured in capillary tubes
centrifuged (10 000 g) for 6 min with a Fanem (São
Carlos, Brazil) hematocrit centrifuge.
Bone sample preparation and analysis
Bone samples (dermal bone and femur) were prepared by removing all skin,
muscle and connective tissue with a razor blade. Samples for water content
analysis were immediately weighed and dried to constant mass in an oven.
Samples for determination of solute composition were re-stored frozen until
analysis. In preparation for analysis, samples were ground to powder at liquid
N2 temperature using a Freezer Mill (Certiprep Spex 6700) and dried
in an oven. For lactate analysis, weighed aliquot parts of dry powder were
incubated at room temperature in 0.73 mol l-1 trichloroacetic acid
(10 ml g-1) to extract the lactate. Lactate was analyzed in bone
extracts and in plasma (with suitable dilution where necessary) using lactic
oxidase/peroxidase colorimetric assay (Sigma Procedure 735). Absorbances were
read at 540 nm using a spectrophotometer (Model Ultrospec 2000; Pharmacia
Biotech, Cambridge, UK).
For total CO2 determination, weighed aliquot parts of dermal
bone powder (0.4 g) were incubated, with vigorous stirring, in 15 ml of 2
mol l-1 HCl solution. To prevent loss of CO2, the powder
was added to the acid by rotating a side bulb containing the powder as
previously described (Jackson et al.,
1999
). The evolved CO2 was taken up by a measured flow
of CO2-free gas through the incubation flask and, after drying with
Drierite, into a CO2 analyzer (Model CD-3A; Applied
Electrochemistry, Pittsburgh, PA, USA). The output from the analyzer was
recorded and processed on a computer using Biopac Acknowledge MP100 hardware
and software (Santa Barbara, CA, USA). Results are expressed as mmol
CO2 g-1 dry mass). The analyzer was calibrated with a
precision gas, the composition of which was verified by analysis with a
Scholander 0.5 cc analyzer (Scholander,
1947
).
For mineral determination, weighed aliquot parts of dry dermal bone powder were ashed in a muffle furnace at 450°C. Given the known water content of the bone, the resultant mass change permitted calculation of % ash mass and % organic mass of the original fresh bone. Ash samples were dissolved in 2 mol l-1 HCl (12 ml g-1), and sodium and potassium were analyzed on this dilution using a flame photometer (Model 943; Instrumentation Laboratory, Lexington, MA, USA). The solution was diluted further to 60-fold with deionized water, and magnesium was measured using an atomic absorption spectrophotometer (Model 280; Perkin-Elmer, Norwalk, CT, USA). Finally, the solution was diluted to 1800-fold and analyzed for calcium using the atomic absorption spectrophotometer and for inorganic phosphorus using the Fiske and SubbaRow method (Sigma kit 670-A) and a spectrophotometer (Model 601; Milton Roy Spectronic, Rochester, NY, USA).
In vitro lactate uptake by dermal bone
To test for equilibrated bone lactate uptake, four dermal bone samples from
each of five caimans were first incubated overnight in buffered saline
solution without lactate (125 mmol l-1 NaCl, 10 mmol l-1
Na2HPO4, titrated to pH 7.8 with 1.0 mol l-1
NaOH). One sample from each animal was removed the following morning and
stored frozen. The remaining samples were then placed in a high lactate
solution (29 mmol l-1 lactic acid, 95 mmol l-1 NaCl, 10
mmol l-1 Na2HPO4, titrated to pH 7.0 with
NaOH). Additional samples from each animal were taken after 2 h, 8 h and 24 h
incubation in this solution. During incubation, the bone pieces rested on a
plastic screen submerged in the solution, which was continuously mixed with a
magnetic stirrer and held at room temperature (25°C). They were
processed and analyzed for lactate concentration as described above. Based on
the results of this trial, a second incubation of four bone samples, depleted
of lactate as before, was carried out for 48 h and analyzed for lactate.
Statistics
Values are expressed as means ± S.E.M. except where
noted. Comparison between groups was made using t-tests, either
paired t-tests for comparing control and recovery lactate levels in
dermal bone or standard t-tests for comparing recovery lactate
concentrations in dermal bone and femur. Blood gas and acid-base changes
during and after submergence were tested by one-way analysis of variance
(ANOVA) using the pre-dive values as controls. Differences were considered
significant at P<0.05.
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Results |
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Because of the unpredictability of the response of the caimans to forced submergence, blood acid-base changes were not uniform in this study. One of the eight animals did not recover from submergence, although its lactate level was not elevated as much as that of four of the others, and no data from this animal are reported. In two other animals, plasma lactate levels did not rise above 9 µmol ml-1 throughout the experiment and their dermal bone lactate levels were correspondingly low (mean, 8.0 µmol g-1 wet mass). The remaining five animals responded similarly, with large increases in plasma lactate that reached peak values during the recovery period. The plasma and dermal bone lactate data from these five animals are shown in Fig. 1. Plasma lactate rose only moderately during submergence and then peaked at 31.2±1.9 µmol ml-1 1 h after the resumption of breathing. Bone lactate concentrations at the end of recovery were increased to 14.8 µmol g-1 wet mass, which was significantly (P<0.02) higher than the control values of the previous day. The increase after diving was also statistically significant if the two animals with low post-diving lactate are included in the analysis. Femur lactate was measured on four animals with high plasma lactate levels and on two animals with low plasma lactate levels. The mean value in the high lactate animals was 9.2±0.7 µmol g-1 wet mass, which is significantly lower (P<0.02) than the dermal bone lactate from the same animals (13.0±0.9 µmol g-1 wet mass). The femur lactate concentrations of the two animals with low plasma lactate were 1.9 µmol g-1 wet mass and 1.2 µmol g-1 wet mass.
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Blood gas and acid-base data
Of the five animals depicted in Fig.
1, we obtained complete blood data on only three. The results from
these animals are summarized in Table
1. The results from the other two caimans were similar but
incomplete. During the dive, the caimans developed a combined respiratory and
lactic acidosis, but the fall in pH was minimized by the relatively small rise
in lactate. Following the dive, lactate rose significantly, worsening the
lactic acidosis, but PCO2 fell as the animals
ventilated their lungs. Plasma [HCO3-] fell predictably
as lactate rose during and following submergence, and, like lactate, recovered
slightly by 120 min.
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In vitro bone equilibration with lactate
Dermal bone was almost 80% equilibrated with the bathing solution lactate
at 8 h and was fully equilibrated by 24
h(Fig. 2). The final
concentration of lactate in bone was 13.9 µmol g-1 wet mass,
approximately 50% of the solution lactate (29 mmol l-1).
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Dermal bone composition
The composition of the osteoderms (Table
2) was generally similar to that of vertebrate bone, with the
predominant components being calcium and phosphorus.
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Discussion |
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Caiman bone mass is significantly less than in the turtle. Based on
allometric estimates (Calder,
1984), caiman skeleton is
12% of its body mass and we
estimate that osteoderms probably account for no more than 2% of body mass,
although no thorough determination was attempted in this study. The smaller
bone mass proportionately reduces the potential importance of the structure as
a lactate repository, and we estimate, based on measured lactate
concentrations in plasma and bone, that bone lactate accounted for only 5-10%
of total lactate in the caiman. Even this relatively small amount, however,
relieves the body fluids of an additional acid load and exploits the large
buffering capacity of bone.
The caiman bone also had a relatively short time available for lactate
exchange with the circulating blood. Assuming that blood flow to bone was
limited during submergence, only the 2 h recovery period was available for
lactate uptake. Nonetheless, this relatively short period was apparently long
enough to permit full equilibration with the extracellular fluid. This is
based on the in vitro incubation data, which demonstrate that
osteoderm lactate concentration at equilibrium (expressed as µmol
g-1 wet mass) was about 50% of the bathing solution concentration
(expressed as µmol ml-1). This was the approximate ratio of bone
to plasma lactate concentrations at the end of the recovery period. It should
not be assumed that at equilibrium the concentrations expressed in these units
will be equal. Measured water content of osteoderms was only 30% of bone mass,
so equilibrium of dissolved lactate within only the aqueous phase would
predict a much lower bone value when expressed per mass of wet bone. As in an
earlier analysis of turtle shell and long bone
(Jackson et al., 2000), we
assume that much of the bone lactate exists in combination with calcium. A
rapid equilibration between blood and osteoderms may also explain the
relatively high bone lactate values in the 24 h pre-dive control samples.
Because these samples were obtained after capturing and restraining the
animals from their experimental tanks, circulating lactate levels probably
increased, although we did not measure plasma lactate after this procedure to
verify this.
The slow equilibration with lactate of osteoderms incubated in
vitro can be explained by the absence of blood perfusion in the in
vitro bone. In the animal, osteoderms are perfused by superficial
arteries that run along their ventral surfaces, and a complex internal network
of canals arising from these arteries indicates `a rich vascular supply in the
living animal' (Seidel, 1979).
Presumably, this blood supply enabled lactic acid to rapidly enter these
structures and exploit their buffering potential. The lower lactate in femur
may be due to lesser perfusion of these bones and slower equilibration.
Our observation that lactate accumulates in a variety of mineralized
tissues - vertebrate endochondrial and dermal bone, snail shell and crayfish
carapace - suggests that a common chemical process is responsible. Our working
hypothesis is that lactate equilibrates with the extracellular space of the
mineralized structure by diffusion, which may or may not be facilitated by a
carrier, and then further equilibrates with Ca2+ to form a
calcium-lactate complex as has previously been described in the plasma of
turtles (Jackson and Heisler,
1982). Based on in vitro studies of turtle shell
(Jackson et al., 1999
), we also
believe that CaCO3 is the source of the Ca2+ and that
the carbonate fraction buffers the associated proton of lactic acid.
Interestingly, the crayfish carapace, although largely composed of
CaCO3, does not accumulate lactate any more effectively than does
vertebrate bone, despite the relatively small fraction of Ca2+
associated with carbonate in the vertebrate bone.
The role of osteoderms in crocodilians as sources of buffering in acute
acid-base disturbances adds an additional possible function for these
structures. Seidel (1979)
suggested heat exchange as a primary function, although he also recognized
their use as protective armor and as anchors for insertions of epaxial
muscles. The role in heat exchange would also be favored by the generous blood
supply and would require that this flow could be altered to meet changing
demands for heat loss or heat conservation.
The mineral composition of the osteoderms generally resembles that of
vertebrate bone, with the predominant elements, calcium and phosphorus, in a
ratio of 1.75. This is slightly above the ratio typical of mammalian
hydroxyapatite (1.67) but may be due to a higher CO2 content of
dermal bone than has been reported for mammalian bone
(Biltz and Pellegrino, 1969).
CO2 presumably exists in the bone as carbonate, associated
primarily with calcium. This additional calcium can help account for the
higher calcium:phosphorus ratio.
The blood data collected on the caimans conform with the classic diving
reflex (Scholander, 1940) and
specifically with studies on enforced submergence in another crocodilian, the
American alligator Alligator mississippiensis
(Andersen, 1961
). A hallmark of
the diving reflex is the retention of lactate in muscle until resumption of
breathing, when a sharp increase in circulating lactate occurs. The increased
lactate coincides with hyperventilation, which lowers blood
PCO2 and limits the lactic acid-induced
acidemia. Our study exhibited these properties, although the duration of the
diving period was short compared with earlier studies on the alligator due to
the onset of struggling by the caimans. When voluntarily submerged and
quiescent, these animals can remained submerged for much longer periods.
We believe that the responses we induced by capturing and submerging the caimans in this study are very relevant to the normal behavior of these animals, however. Caimans capture their prey and overcome them by a violent underwater struggle that must produce severe lactic acidosis. The contribution made by osteoderms and skeleton to buffer and sequester some of this lactate may contribute to their success in these struggles and to their recovery from them.
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Acknowledgments |
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References |
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