Acute acidosis-induced alteration in bone bicarbonate and phosphate

David A. Bushinsky1, Susan B. Smith1, Konstantin L. Gavrilov2, Leonid F. Gavrilov2, Jianwei Li2, and Riccardo Levi-Setti2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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During an acute fall in systemic pH due to a decrease in the concentration of serum bicarbonate ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]), metabolic acidosis, there is an influx of hydrogen ions into the mineral phase of bone, buffering the decrement in pH. When bone is cultured in medium modeling acute metabolic acidosis, the influx of hydrogen ions is coupled to an efflux of sodium and potassium and a depletion of mineral carbonate. These ionic fluxes would be expected to neutralize some of the excess hydrogen ions and restore the pH toward normal. Approximately one-third of bone carbonate is located on the hydration shell of apatite, where it is readily accessible to the systemic circulation, whereas the remainder is located in less accessible areas. We hypothesize that the surface of bone would respond to acidosis in a different manner than the interior of bone, with depletion of carbonate preferentially occurring on the bone surface. We utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to localize the changes in bone carbonate, as measured by HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and phosphate and determine their relative contribution to the buffering of hydrogen ions during acute metabolic acidosis. Neonatal mouse calvariae were incubated in control medium (pH ~7.44, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] ~27 mM) or in medium acidified by a reduction in [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] (pH ~7.14, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] ~13). Compared with control, after a 3-h incubation in acidic medium there is a fivefold decrease in surface HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with respect to the carbon-carbon bond (C2) and a threefold decrease in surface HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with respect to the carbon-nitrogen bond (CN) with no change in cross-sectional HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Compared with control, after a 3-h incubation in acidic medium there is a 10-fold decrease in cross-sectional phosphate with respect to C2 and a 10-fold decrease in cross-sectional phosphate with respect to CN, with no change in surface phosphate. On the bone surface, there is a fourfold depletion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in relation to phosphate, and, in cross section, a sevenfold depletion of phosphate in relation to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Thus acute hydrogen ion buffering by bone involves preferential dissolution of surface HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and of cross-sectional phosphate.

ion microprobe; calcium; proton


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>], induces a marked efflux of calcium from cultured neonatal mouse calvariae (14, 16, 21, 25, 35), whereas metabolic alkalosis induces an influx of calcium into bone (10). During short-term (3-h) cultures, this acid-induced calcium efflux appears due to physiochemical bone mineral dissolution (16, 25). However, over longer time periods (>24 h), the acid-induced calcium efflux from bone appears due to cell-mediated bone resorption (9, 14, 21, 35). We have shown that metabolic acidosis leads to an increase in osteoclastic beta -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<UP><SUB>3</SUB><SUP>−</SUP></UP>), but not acute respiratory (increased PCO2), acidosis (26). In vivo, we have shown that chronic metabolic acidosis causes a depletion of mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and phosphate (11).

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<UP><SUB>3</SUB><SUP>−</SUP></UP>, and phosphate during acute acidosis and determine the relative contribution of dissolution of mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and dissolution of mineral phosphate to the buffering of the additional hydrogen ions. We found that a model of acute metabolic acidosis induces a depletion of surface, but not cross-sectional, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and cross-sectional, but not surface, phosphate. There is depletion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in preference to phosphate on the surface and depletion of phosphate in preference to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the interior of the bone. Thus acute hydrogen ion buffering by bone involves predominantly the dissolution of surface HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and cross-sectional phosphate.


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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 [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]. At the conclusion of this single incubation, the medium was removed and analyzed for pH, PCO2, and calcium. Calvariae were then removed from the culture dishes, washed with ice-cold PBS, rapidly frozen (-80°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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Table 1.   Medium ion concentrations and fluxes

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, phosphate, the carbon-nitrogen bond (CN), and carbon on the surface and on the cross section. The analyses were repeated on six representative areas of each calvaria. Data were recorded after erosion of ~5 nm of material; this procedure ensured the removal of any contamination and permitted the acquisition of highly reproducible measurements and is consistent with our previously published studies (11, 13-15, 20, 25, 26). Given the beam current of 30 pA and an image-acquisition time not exceeding 524 s (maximum time in this study), the depth of erosion for these elemental measurements, over areas 40 × 40 to 160 × 160 µm2, never exceeded ~5 nm, which did not result in significant sample depletion. Given the extremely small area being examined, 40 × 40 to 160 ×160 µm2, all cross-sectional measurements were far from the surface of the calvariae, which is ~1 mm thick.

We compared the ratios of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and total phosphate to the carbon-carbon (C2) and CN bonds. Carbon appears to be a suitable denominator, as it would not be expected to be affected by acid in these relatively short-term experiments. We use C2 rather than C simply because it gives a stronger signal; the ratio of C2 to C is constant (Levi-Setti R and Bushinsky DA, unpublished observations). CN is present in areas of organic material. PO4 gives a very weak signal, presumably due to breakup of this large molecule into PO2 and PO3 by the gallium beam and there is little P, which is not associated with O. The [(PO2 + PO3)/C2] or [(PO2 + PO3)/CN] ratio is not significantly influenced by inclusion of small amounts of PO4 or P in the numerator (Levi-Setti R and Bushinsky DA, unpublished observations). Carbonate has an atomic mass of 60. Given that mass 60 is also C5, we would not expect a detectable decrease in mass 60 during metabolic acidosis given the large mass of organic carbon in bone (50), which would not be expected to be affected by acidosis. In contrast, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (mass 61) readily accepts hydrogen ions and is a known buffer in the extracellular fluid (8). There are no other common compounds at mass 61, making this an unambiguous marker for bone total CO2 (carbonate + HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>). In this study, we do not report positive ions, such as calcium, sodium, and potassium, because we do not know of a standard positive ion or ion cluster that would not be expected to be influenced by acidosis. We have previously demonstrated that acute acidosis induces a marked loss of bone sodium and potassium in relation to calcium (13). Without a standard for the positive ions, we cannot determine the localization of the loss of sodium and potassium.

Correction methods similar to those that we have previously reported (11, 13-15, 20, 25, 26) were applied to the observed mass-resolved counting rates to obtain secondary ion yields proportional to the elemental concentration in the sample. Corrections are necessary because of the species-dependent sputtering and ionization probabilities of the emitted atoms. The total ion counts in a micrograph are a function not only of the emission properties of ions from a sample but also of the fraction of the field of view occupied by the sample, which in the case of the calvariae may have physical holes. In addition, the detected ion yields are dependent on the degree of sample surface roughness. Because of these considerations, we express our results in terms of the ratios of counts obtained for the same area of a sample. Such ratios are independent of the fraction of the field of view occupied by the sample and of the surface topography.

Conventional 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<UP><SUB>3</SUB><SUP>−</SUP></UP>] was calculated from medium pH and PCO2 as described previously (7, 17). Net calcium flux (JCa) was calculated as Vm([Ca]f - [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.


    RESULTS
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METHODS
RESULTS
DISCUSSION
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Medium ion concentrations and fluxes. Compared with Ctl medium, the initial and final medium pH and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] were significantly reduced in Acid medium (Table 1). There were no differences in PCO2 between Ctl and Acid media. Compared with calvariae incubated in Ctl medium, during the 3-h incubation there was a significant increase in JCa from bones and a significant proton influx (JH) into bones incubated in Acid medium. The initial medium pH was correlated inversely with JCa (r = -0.840, 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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Fig. 1.   Correlation of proton and calcium fluxes. Calvariae were incubated for 3 h in either control () or acidic (black-triangle) medium. In the control group, the calvariae were cultured in basal medium, and, in the acidic group, the calvariae were cultured in medium in which the pH was lowered by the addition of 10 µl of 2.4 mM HCl/ml of medium to lower HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]). Relationship is best described by a first-order polynomial: net calcium flux (nmol · bone-1 · 3 h-1) = -0.0417 × net proton flux (nmol · bone-1 · 3 h-1) + 63.6. A positive flux indicates net ion movement from the bone into the medium, and a negative flux indicates net ion movement from the medium into the bone.

HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP>. The surface of calvariae incubated in Ctl medium contains significantly more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to C2 and significantly more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to CN than the cross section of calvariae incubated in Ctl (Fig. 2A, representative spectra of Ctl surface; Fig. 3A, representative spectra of Ctl cross section; Fig. 4A, compiled data). Compared with incubation in Ctl medium, incubation in Acid medium led to a marked fall in both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to C2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to CN on the surface of calvariae [Figs. 2 (Control vs. Acid in figure) and 4A]. Compared with incubation in Ctl medium, incubation in Acid medium did not alter either HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to C2 or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to CN in the cross section of the calvariae. Thus Acid medium induces a significant fall in mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> on the surface, but not in the cross section, of calvariae.


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Fig. 2.   Mass spectra of the negative secondary ions on the surface of neonatal mouse calvariae incubated for 3 h in either control (Control; A) or acidic (Acid; B) medium. Counts per channel are counts per second of detected secondary ions uncorrected for species-dependent ionization probabilities. Observed spectra were measured in 1,000 channels equally divided among mass 10-90 atomic mass units. CN, carbon-nitrogen bond; C2, carbon-carbon bond.



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Fig. 3.   Mass spectra of negative secondary ions in the cross section of neonatal mouse calvariae incubated for 3 h in either Control or Acid medium. Counts per channel are counts per second of detected secondary ions uncorrected for species-dependent ionization probabilities. Observed spectra were measured in 1,000 channels equally divided among mass 10-90 atomic mass units.



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Fig. 4.   A: ratio of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to C2 and ratio of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to CN on the surface and in the cross section of neonatal mouse calvariae incubated for 3 h in either control (Ctl) or acidic (Acid) medium. B: ratio of total phosphate (PO2 + PO3) to C2 and (PO2 + PO3) to CN on the surface and in the cross section of neonatal mouse calvariae incubated for 3 h in either Ctl or Acid medium. Values are mean plus SE. *P < 0.05 vs. surface Ctl. +P < 0.05 vs. surface Acid. oP < 0.05 vs. cross-sectional Ctl.

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<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP> in relation to phosphorus. The surface of calvariae incubated in Ctl medium contains significantly more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> relative to (PO2 + PO3) than the cross section of calvariae incubated in Ctl medium (Fig. 5). Compared with incubation in Ctl, incubation in Acid medium led to a significant reduction in the ratio of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to (PO2 + PO3) on the surface of the calvariae. However, compared with incubation in Ctl, incubation in Acid led to a significant increase in the ratio of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to (PO2 + PO3) in the cross section of the calvariae, indicating a depletion of phosphorus in relation to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Thus metabolic acidosis induces a fall in mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in relation to mineral PO2 + PO3 on the surface and an increase in the ratio of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to PO2 + PO3 in the cross section of calvariae.


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Fig. 5.   Ratio of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to (PO2 + PO3) on the surface and in the cross section of neonatal mouse calvariae incubated for 3 h in either control (Ctl) or acidic (Acid) medium. Values are expressed as mean plus SE. *P < 0.05 vs. surface Ctl. +P < 0.05 vs. surface Acid. oP < 0.05 vs. cross-sectional Ctl.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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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<UP><SUB>3</SUB><SUP>−</SUP></UP> preferentially occurring on the surface of the bone. We found that a model of acute metabolic acidosis induces depletion of surface, but not cross-sectional, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and cross-sectional, but not surface, phosphate. There is depletion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in preference to phosphate on the surface and depletion of phosphate in preference to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the interior of the bone. This indicates that acute proton buffering by bone involves predominantly a dissolution of surface HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and cross-sectional phosphate.

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<UP><SUB>3</SUB><SUP>−</SUP></UP>, which would then combine with an additional hydrogen ion to form CO2 and water (8). We and others have shown a loss of bone carbonate during metabolic acidosis (3, 18), and we have shown that in vivo metabolic acidosis causes a reduction in mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and phosphate (11). In vivo, Irving and Chute (33) demonstrated that several days of metabolic acidosis led to a loss of bone carbonate. Burnell (5) also demonstrated a loss of bone carbonate after metabolic acidosis, and Bettice (3) showed that the metabolic acidosis-induced loss of bone carbonate correlated with the fall in extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Using cultured neonatal mouse calvariae, we demonstrated that mineral calcium and carbonate, in the form of carbonated apatite, are in equilibrium with the culture medium (19). We have shown that acidosis induces the release of calcium and carbonate from bone (19), leading to a progressive loss of bone carbonate during metabolic, but not respiratory, acidosis (18).

Bone contains approx 80% of the total CO2 (including carbonate, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and CO2) in the body (41). Approximately two-thirds of this is in the form of carbonate complexed with a hydrogen ion (as HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>), calcium, potassium, and sodium and other cations, and is located in the lattice of the bone crystals, where it is relatively inaccessible to the systemic circulation. The other third is located in the hydration shell of hydroxyapatite, where it is readily available to the systemic circulation or the medium. Bone consists of 4-6% carbonate (1, 2, 4, 27, 38, 42), and the carbonate can occupy three sites in the apatite crystal lattice (45). The primary site is as a substitution for phosphate and approximately one in four or five phosphates appears to be substituted by carbonate (1, 2, 4, 27, 38, 42). The second is in the monovalent anionic site (OH- 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<UP><SUB>3</SUB><SUP>−</SUP></UP> with respect to two ion complexes that should not be altered by acidosis, CN and C2.

The loss of surface, but not cross-sectional, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> suggests mobilization of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> located in the hydration shell, perhaps in the unstable location (44). There appears to be an equal amount of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and phosphorus on the surface of the bone (Fig. 5), yet only the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is depleted by the acidic medium, suggesting at this location this ionic species is more susceptible to the acid challenge. There is ample carbonate present in the calvariae to account for a substantial amount of proton buffering by bone. Each calvariae weighs ~3-4 mg, so ~0.18-0.24 mg or 300-400 µmol of carbonate are present in the entire bone and ~100-133 µmol of carbonate are present on the surface. This carbonate is more than sufficient to buffer the influx of ~50 nmol of protons into each bone that was observed in this study. However, this observation would suggest that a longer duration of acidosis might deplete mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and decrease the buffering capacity of the calvariae. In this study, we utilized 4- to 6-day-old neonatal calvariae. Further studies, using older calvariae, will be necessary to determine whether bone age will alter the effect of hydrogen ions on mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and phosphate.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> and phosphate. The previous study questioned whether there were regional differences in the response of mineral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and phosphate to acid. The present study clearly demonstrates these regional differences and suggests that they arise from the differences in the location of the bone HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> and cross-sectional, but not surface, phosphate. There is depletion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in preference to phosphate on the surface and depletion of phosphate in preference to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the interior of the bone. Thus acute proton buffering by bone involves predominantly dissolution of surface HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and cross-sectional phosphate.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants AR-46289 and DK-56788.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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