1 Nephrology Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York 14642; and 2 Enrico Fermi Institute, Department of Physics, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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During an acute fall in systemic pH due
to a decrease in the concentration of serum bicarbonate
([HCO
ion microprobe; calcium; proton
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INTRODUCTION |
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DURING IN VITRO (6, 7, 17,
20) and in vivo (37, 49) metabolic acidosis, bone
is thought to buffer some of the additional hydrogen ions, resulting in
an increase in the medium or systemic pH (respectively). In a classic
study, Swann and Pitts (49) infused specific amounts of
acid into dogs and demonstrated that ~60% of the additional hydrogen
ions are buffered outside of the extracellular fluid, presumably by
soft tissues and/or bone (3, 37). An in vitro
model of metabolic acidosis, produced by a decrement in medium
HCO-glucuronidase
activity and a decrease in osteoblastic collagen synthesis (9,
31, 35), whereas metabolic alkalosis decreases osteoclastic
activity and increases osteoblastic activity (10). In
addition, acidosis inhibits the stimulation of some, but not all, early
immediate response genes (31) and reversibly inhibits expression of certain extracellular matrix genes (29).
This cell-mediated resorption appears to result from increased
prostaglandin E2 synthesis that stimulates osteoclastic
resorption and suppresses osteoblastic function (22, 34,
43).
Using in vitro models, we have shown that in response to acute
metabolic acidosis there is an influx of hydrogen ions into the bone
mineral, buffering the medium acidity (6, 7, 17, 20). In
vitro metabolic acidosis causes the release of mineral sodium and
potassium (13) and a depletion of mineral carbonate (18). The changes in surface sodium and potassium occur
during models of acute metabolic (decreased HCO
Approximately one-third of bone carbonate is located on the hydration
shell of apatite, where it is readily accessible to the systemic
circulation or culture medium, whereas the remainder of bone carbonate
is less accessible to the circulation (41). There also
appears to be more carbonate on the rapidly growing surface of bone
(44, 46). These observations suggest that the surface and
interior of the bone would respond differently to an increased
concentration of hydrogen ions, with depletion of carbonate
preferentially occurring on the surface of the bone. To test this
hypothesis, we utilized a high-resolution scanning ion microprobe with
secondary ion mass spectroscopy (SIMS) to localize changes in bone
carbonate, as measured by HCO
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METHODS |
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Organ culture of bone. Neonatal (4- to 6-day-old) CD-1 mice (Charles River, Wilmington, MA) were killed, their calvariae were removed by dissection, the adherent cartilaginous material was trimmed, and the periosteum was left intact (7, 10, 13-26, 29-31, 34, 35, 48). Exactly 2.8 ml of Dulbecco's modified Eagle's medium (M. A. Bioproducts, Walkersville, MD) containing heat-inactivated horse serum (15%), heparin sodium (10 U/ml), and penicillin potassium (100 U/ml) were preincubated at a PCO2 of 40 Torr at 37°C for 3 h in 35-mm dishes. We have found that 3 h are sufficient for PCO2 equilibration between the incubator and the medium (17). After preincubation, 1 ml of medium was removed to determine initial medium pH, PCO2, and total calcium concentration, and two calvariae were placed in each dish on a stainless steel wire grid. Total bone content in each culture was controlled by using pups that were the same age and size, by using a standardized dissection procedure, and by placing two bones in each dish. Experimental and control cultures were performed in parallel and in random order.
Experimental groups.
Calvariae were incubated for 3 h in either control (Ctl) or acidic
(Acid) medium. In the Ctl group, the calvariae were cultured in basal
medium (Table 1). In the Acid group, the
calvariae were cultured in medium in which the pH was lowered by the
addition of 10 µl of 2.4 M HCl/ml of medium to lower
[HCO80°C) and then lyophilized while frozen until dry (at least 16 h) (11, 13-15, 20, 25, 26). The frontal and
parietal bones of some calvariae were split in half to reveal the
interior of the bone for cross-sectional analysis. All bones were then mounted on aluminum supports with conductive glue and coated with a
thin layer (~5 nm) of gold. This layer, which is rapidly sputtered away by the ion probe from the area being scanned, prevents
artifact-inducing electrical charging.
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Scanning ion microprobe. The scanning ion microprobe utilized for these studies was conceptualized and built at the University of Chicago and employs a 40-keV gallium beam focused to a spot 40 nm in diameter (11, 13-15, 20, 25, 26). The beam is scanned across a sample surface in a controlled sequence, resulting in the emission of secondary electrons, ions, and neutral atoms. These secondary particles originate within, and consequently carry information about, the most superficial 1-2 nm of the sample. The charged secondary particles can be collected to generate images of the surface topography of a sample similar to those obtained using a scanning electron microscope. The particles can also be collected and analyzed by SIMS, a technique that separates the sputtered ions according to their mass-to-charge ratio.
In this study, the spectrometer was rapidly and sequentially retuned to filter several chosen ion species. At the same time, the probe was quickly scanned over a square area so that the measured signals are secondary ion intensities averaged over the entire field of view. Using this "peak-switching" technique, the relative concentrations of several elements can be acquired simultaneously from one area and at one sample depth. We also used SIMS in the mass analysis mode, in which the spectrometer mass tuning is systematically varied (as in a conventional mass spectrometer) to yield mass spectra. If the spectra data are corrected by element-dependent sensitivity factors, they provide quantitative relative abundance measurements for a given area of sample. For SIMS analysis, the secondary ions emerging from the sample are transported through a high-transmission optical system containing an electrostatic energy analyzer and a magnetic sector mass spectrometer (mass resolution of ~0.07 atomic mass units measured at 40 atomic mass units). The secondary ions are accelerated to 5,000-eV energy for mass separation and detection by a secondary electron detector operated in pulse mode (each collected ion yields 1 digital pulse). Mass spectra are accumulated with a multichannel scaler, which counts each detected ion by ramping the magnetic field of the mass spectrometer to scan a preselected mass region of the spectrum while the probe is scanning an area of arbitrary dimensions. The choice of the scanned area determines the depth over which the target composition is sampled for a given probe current and time. Elemental maps are constructed by recording the individual detected pulses in a 512 × 512 array of computer memory, each element of the array corresponding to a position of the probe on the sample. For the most abundant elements (Na, K, and Ca), counting rates as high as 4.0 × 105 cps/pA of primary current were observed (where cps is counts/s). In such cases, statistically significant elemental images could be obtained in scan times of <10 s with probe currents of a few tens of picoamperes. We selected, at random, four calvariae from each group. For each calvaria, we measured the concentration of HCOConventional measurements.
Medium pH and PCO2 were determined with a
blood-gas analyzer (Radiometer model ABL 30) and calcium by an
electrode (Nova Biomedical, Waltham, MA). Medium
[HCO [Ca]i), where Vm is the medium volume
(1.8 ml), and [Ca]f and [Ca]i are the final
and initial medium calcium concentrations, respectively. Net
hydrogen ion flux (JH) was calculated as
Vm (Sf
Si), where S is the amount of
hydrogen ions that must be added to the culture medium to achieve the
final (f) and initial (i) hydrogen ion concentration [H+]. Values of S were derived from
the empirical buffer curve of the medium, which was constructed by
varying medium [H+] at a PCO2 of
40 Torr, at 37°C, by addition of appropriate concentration of HCl or
NaOH. The observed data were fitted best by a second-order polynomial
regression (S = 0.00008 × [H+]3
0.02192 × [H+]2 + 2.14311 × [H+]
47.05462); addition of higher order terms did not
significantly improve the fit (17). For both calcium and
hydrogen ions, a positive flux value indicates movement of the ion from
the bone into the medium and a negative value indicates movement from
the medium into the bone.
Statistics. All regressions and tests of significance, using analysis of variance or t-tests as appropriate, were calculated using the BMDP statistical program on a digital computer. Mean ion concentrations and flux values were expressed as means ± SE. P < 0.05 was considered significant.
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RESULTS |
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Medium ion concentrations and fluxes.
Compared with Ctl medium, the initial and final medium pH and
[HCO0.840,
n = 24, P < 0.001; data not shown) and
directly with JH (r = 0.862, n = 24, P < 0.001; data not shown). JH was correlated inversely with
JCa [r =
0.704,
n = 24, P < 0.001, JCa = (
0.0417 × JH) + 63.6] (Fig.
1).
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HCO
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Phosphorus. The surface of calvariae incubated in Ctl medium contains significantly less total phosphorus (PO2 + PO3) relative to C2 and significantly less (PO2 + PO3) relative to CN than the cross section of calvariae incubated in Ctl medium (Fig. 2A, representative spectra of Ctl surface; Fig. 3A, representative spectra of Ctl cross section; Fig. 4B, compiled data). Compared with incubation in Ctl medium, incubation in Acid medium led to a marked fall in both (PO2 + PO3) relative to C2 and (PO2 + PO3) to CN in the cross section of calvariae [Fig. 3 (Control vs. Acid) and Fig. 4B]. Compared with incubation in the Ctl medium, incubation in Acid medium did not alter either (PO2 + PO3) relative to C2 or (PO2 + PO3) relative to CN on the surface of the calvariae. Thus the Acid medium induces a fall in mineral phosphorus on the cross section, but not on the surface, of calvariae.
HCO
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DISCUSSION |
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During acute metabolic acidosis, the mineral phases of bone
mitigate the decrement in pH (8, 12). In cultured bone, we have previously shown that a reduction of medium pH is associated with
an influx of hydrogen ions into the bone (6, 7, 17), an
efflux of sodium and potassium from bone (13, 14, 20, 25,
26), and a loss of mineral carbonate (18, 19). The sodium and potassium exchange for hydrogen and the carbonate binds hydrogen, all of which leads to significant neutralization of the
increased acidity. In the present study, we utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to test
the hypotheses that the surface and interior of the bone would respond
differently to a model of metabolic acidosis, with depletion of
HCO
During metabolic acidosis, a probable mechanism for proton buffering by
bone involves dissolution of bone carbonate. As the hydrogen
concentration is increased, carbonate would combine with a hydrogen ion
to form HCO
Bone contains 80% of the total CO2 (including
carbonate, HCO
position), and the third is an unstable location that
occurs principally in rapidly forming precipitates where carbonate can substitute for phosphate (44). The total carbonate content
in bone mineral increases with age, whereas the carbonate in the unstable location decreases with age (45). Studies with
biological and synthetic apatites have shown that carbonate markedly
disturbs the crystal structure and increases the reactivity of the
mineral to acid (28, 32, 36, 38-40). The present
study demonstrates a marked reduction of surface, but not
cross-sectional, HCO
The loss of surface, but not cross-sectional,
HCO
Bone primarily consists of the calcium phosphate complex apatite (2, 4, 27, 42). The extracellular fluid and our culture medium are both highly supersaturated with respect to apatite, making it unlikely that this phase of bone mineral is affected by an acidic medium. Indeed, we have previously shown that apatite is not in equilibrium with our culture medium (19). Extracellular fluid is slightly undersaturated with respect to brushite, which has been found in newly mineralized bone (47). While there has been a report that phosphate does not appear to be released from bone during acid infusion in dogs (5), our present in vitro data clearly indicate a loss of mineral phosphate during incubation in acidic medium. This is consistent with our prior in vivo data, which demonstrated a loss of mineral phosphate after chronic metabolic acidosis (11). The type of calcium phosphate complex reduced by incubation in acidic medium is not clear from this study. The cross-sectional location of the acidic medium-induced reduction in mineral phosphate is consistent with an older mineral phase. Further studies with sophisticated analysis comparing the mineral phases of bone incubated in neutral and acidic media will be necessary to resolve this important issue.
Previously, we studied changes in midcortical ion concentrations after
7 days of in vivo metabolic acidosis induced by oral ammonium chloride
(11). We found that compared with mice drinking only
distilled water, the ammonium chloride induced a loss of bone sodium,
potassium, and, as also shown in this study, a depletion of mineral
HCO
In this, as in previous studies (6, 7, 17), we demonstrate that proton buffering by bone cannot simply involve dissolution of the principle mineral phases. As observed in Fig. 1, for each millimole of calcium released by the bone, there were ~12 mmol of protons neutralized by the bone. If proton buffering by bone involved dissolution of apatite [Ca10(PO4)6.inclusion], the ratio should be ~5:3, varying slightly with the inclusion, and, with brushite (CaHPO4), the ratio should be 1:1. Were proton buffering simply dissolution of calcium carbonate (CaCO3), the ratio should be 1:1. Thus other buffering mechanisms, in addition to mineral dissolution, must be involved. We have previously shown that there is sodium and potassium efflux from bone in association with proton influx, which serves to further reduce the proton concentration and buffers the acid load (13, 14, 20, 25, 26).
In this study, we found that an acute model of metabolic acidosis
induces a depletion of surface, but not cross-sectional, HCO
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants AR-46289 and DK-56788.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. A. Bushinsky, Univ. of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 675, Rochester, New York 14642 (E-mail: David_Bushinsky{at}URMC.Rochester.edu).
The 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.
August 21, 2002;10.1152/ajprenal.00155.2002
Received 22 April 2002; accepted in final form 10 July 2002.
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