Effects of in vivo metabolic acidosis on midcortical bone ion composition

David A. Bushinsky1, Jan M. Chabala2, Konstantin L. Gavrilov2, 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
REFERENCES

Chronic metabolic acidosis increases urine calcium excretion without altering intestinal calcium absorption, suggesting that bone mineral is the source of the additional urinary calcium. During metabolic acidosis there appears to be an influx of protons into bone mineral, lessening the magnitude of the decrement in pH. Although in vitro studies strongly support a marked effect of metabolic acidosis on the ion composition of bone, there are few in vivo observations. We utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to determine whether in vivo metabolic acidosis would alter bone mineral in a manner consistent with its purported role in buffering the increased proton concentration. Postweanling mice were provided distilled drinking water with or without 1.5% NH4Cl for 7 days; arterial blood gas was then determined. The addition of NH4Cl led to a fall in blood pH and HCO-3 concentration. The animals were killed on day 7, and the femurs were dissected and split longitudinally. The bulk cortical ratios Na/Ca, K/Ca, total phosphate/carbon-nitrogen bonds [(PO2 + PO3)/CN], and HCO-3/CN each fell after 1 wk of metabolic acidosis. Because metabolic acidosis induces bone Ca loss, the fall in Na/Ca and K/Ca indicates a greater efflux of bone Na and K than Ca, suggesting H substitution for Na and K on the mineral. The fall in (PO2 + PO3)/CN indicates release of mineral phosphates, and the fall in HCO-3/CN indicates release of mineral HCO-3. Each of these mechanisms would result in buffering of the excess protons and returning the systemic pH toward normal.

ion microprobe; calcium; mouse femurs; proton


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN HUMANS AND OTHER MAMMALS, chronic metabolic acidosis increases urine calcium excretion (19, 46), secondary to a direct reduction of renal tubular calcium reabsorption (57), without increasing intestinal calcium absorption (38), resulting in a net negative calcium balance (2, 44, 45). Because the vast majority of body calcium is located within the mineral stores of bone, the negative calcium balance implies depletion of bone mineral (60). In vivo studies have shown that metabolic acidosis, induced by ammonium chloride (NH4Cl), leads to a loss of bone mineral (3).

An in vitro model of metabolic acidosis, produced by a decrement in medium bicarbonate concentration [HCO-3], induces a marked efflux of calcium from cultured neonatal mouse calvariae (11, 21, 22, 27, 33, 42), whereas metabolic alkalosis induces an influx of calcium into bone (14). During short-term (3 h) cultures the acid-induced calcium efflux appears to be caused by physiochemical bone mineral dissolution (22, 33). However, over longer time periods (>24 h), the calcium efflux from bone appears to be caused by cell-mediated bone resorption (11, 21, 27, 42). We have shown that metabolic acidosis leads to an increase in osteoclastic beta -glucuronidase activity and a decrease in osteoblastic collagen synthesis (11, 37, 42). In addition, acidosis inhibits the stimulation of some, but not all, early-immediate response genes (37) and reversibly inhibits expression of certain extracellular matrix genes (36).

During in vitro (8, 9, 23, 26) and in vivo (45, 55, 58) metabolic acidosis, bone appears to buffer some of the additional protons, resulting in an increase in medium or systemic pH, respectively. In a classic study, Swann and Pitts (58) infused fixed amounts of acid into dogs and demonstrated that ~60% of the additional protons appear to be buffered outside of the extracellular fluid, presumably by soft tissues (1, 52) and/or bone (6, 45). The negative calcium balance may reflect proton buffering by bone. During the chronic metabolic acidosis of chronic renal failure, blood pH can remain stable, although substantially reduced, in spite of progressive proton retention, suggesting the availability of large stores of proton buffers (55). Given its large mass of potential proton buffers, bone is an obvious site for proton buffering during metabolic acidosis (12). Using in vitro models, we showed (8, 9, 23, 26) that in response to metabolic, but not respiratory, acidosis there is an influx of protons into bone mineral, buffering the medium proton concentration. In vitro metabolic acidosis causes the release of mineral potassium (20) and a depletion of mineral carbonate (CO2-3) (24).

Examining changes in bone ion composition during metabolic acidosis is an important part of understanding how bone may mitigate increased systemic acidity. In this study, we tested the hypothesis that mild, physiologically relevant in vivo metabolic acidosis would alter bone mineral in a manner consistent with its purported role in buffering the increased proton concentration.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Groups

Mice were studied in two groups. NH4Cl (1.5%) was added to the deionized, distilled drinking water of five mice immediately after they were weaned; five other mice from the same litter drank only distilled water. All mice were fed unlimited amounts of standard mouse chow (1.2% calcium). The average daily fluid consumption by the mice drinking 1.5% NH4Cl was 8.3 ml, indicating that each mouse received, on average, 2.3 mmol of NH4Cl per day. The average fluid consumption by the mice drinking distilled water was 10.3 ml. We previously showed (19, 28) in rats that 1.5% NH4Cl leads to a moderate, physiologically relevant metabolic acidosis. After 7 days systemic arterial blood gas was determined by cardiac puncture, and the animals were killed. The femurs were immediately dissected, washed with ice-cold PBS, rapidly frozen in an acetone-dry ice bath (-77°C) for 5 min and then lyophilized while frozen until dry (at least 16 h) (15-17, 20, 21, 26, 32-34). The femurs were then split longitudinally, 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. We examined an area in the midcortex (midway between the marrow space and the superficial cortex of the longitudinally split femur) midway down the bone shaft.

Scanning Ion Microprobe

The scanning ion microprobe utilized for these studies was conceptualized and built at the University of Chicago and uses a 40-keV gallium beam focused to a spot 40 nm in diameter (15-17, 20, 21, 26, 32-34, 48, 50). 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 secondary ion mass spectroscopy (SIMS), a technique that separates the sputtered ions according to their mass-to-charge ratio.

Microanalysis of bone by SIMS can be performed in four distinct modes. In the imaging SIMS mode, the mass spectrometer is tuned to transmit a single ion species while the probe is scanned over a square region of the sample surface. The result is a monoisotopic elemental distribution image, called a SIMS map. Because the mass-resolved counting rates during the image acquisition are recorded, these SIMS maps provide both a local quantitative record and a two-dimensional representation of the distribution of a particular element on the surface being scanned. In the nonimaging SIMS mode, the spectrometer is rapidly and sequentially retuned to filter several chosen ion species. At the same time, the probe is 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. In the mass analysis mode, the spectrometer mass tuning can be 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. In the fourth microanalysis mode, the gallium beam is used to erode the surface, continually exposing deeper layers for analysis. As in the nonimaging SIMS mode, the peak-switching procedure is used to simultaneously analyze several ion species. As deeper layers are exposed, a depth profile can be created that characterizes composition as a function of depth. Erosion depth is estimated from the primary ion current, the average density of bone, the duration and area of the scan, and a secondary particle sputter yield of 10, which is typical of these materials (15-17, 20, 21, 26, 32-34, 43, 47, 49).

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 (AMU) measured at 40 AMU]. 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 that 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 counts · s-1 · pA-1 of primary current were observed. 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, three right femurs from each group of five mice. For each femur we measured the concentration of sodium, potassium, calcium, HCO-3, total phosphate, carbon-nitrogen bond (CN), and carbon (9-16 measurements of each ion or ion complex). The analyses were repeated on representative areas of the femurs. Data were recorded after erosion of ~5 nm of material; this procedure ensured the removal of any contamination, permitted the acquisition of highly reproducible measurements, and is consistent with our previously published studies (15-17, 20, 21, 26, 32-34). Given the beam current of 30 pA and image acquisition time not exceeding 524 s (maximum time in this study), the depth of erosion for these elemental measurements, over areas of 160 × 160 µm2, never exceeded ~5 nm, which did not result in significant sample depletion.

Correction methods similar to those we previously reported (15-17, 20, 21, 26, 32-34) 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. Relative to calcium taken as standard, the corrected yields for sodium and potassium are obtained by dividing the observed counting rates by the sensitivity factor 1.9 for apatites, assumed to apply to other bone crystals as well.

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 femur 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

Blood pH and PCO2 were measured with a blood gas analyzer (Radiometer BMS 2 Mk III, Copenhagen, Denmark). Blood [HCO-3] was calculated from the pH and PCO2 using the Henderson-Hasselbalch equation (11, 33).

Mean Ion Ratio Calculations

Mean ion ratios were calculated as the difference between the log of the counts in the numerator and the log of the counts in the denominator. The mean of the differences in each group was then obtained, and analysis of variance was calculated. Values expressed are the antilog of the mean of the differences in each group plus the upper 95% confidence limit. The confidence limit was obtained by taking the mean of the differences in each group and adding the product of 2.24 times the standard error of this mean and then calculating the antilog of the resulting value. The number 2.24 is the T score for a one-tailed P < 0.025, so 2.5% of the data lies above the upper confidence limit (15-17, 20, 21, 26, 32-34).

Statistics

All tests of significance were calculated using T-tests (BMDP, University of California at Los Angeles, CA) on a digital computer. Mean ion ratios are expressed as means plus the upper 95% confidence limit for ion ratios; P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Blood pH, PCO2, and [HCO-3]

The addition of 1.5% NH4Cl to the drinking water of the mice led to a decrease in the systemic pH (7.36 to 7.31, control vs. NH4Cl), [HCO-3] (22 to 19 meq/l), and PCO2 (40 to 38 mmHg) from the pooled blood of several mice in each group. These metabolic changes are consistent with a mild, physiologically relevant metabolic acidosis (13).

Bone Ion Composition

Positive ions. The midcortex of a femur from a mouse drinking distilled water has abundant sodium and potassium relative to calcium (Fig. 1). Mass spectra indicate that the peak height of 23Na is greater than that of 40Ca and those of 39K and 41K are each greater than that of 40Ca.


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Fig. 1.   Mass spectra of positive secondary ions located in midcortex of a representative neonatal mouse femur after drinking only distilled water for 7 days. Mass spectra indicate that abundance of calcium is less than that of either sodium or potassium. Observed spectra were measured in 1,000 channels equally divided among mass 20-50 atomic mass units. Counts per channel are counts per second of detected secondary ions uncorrected for species-dependent ionization probabilities. Relative to calcium as a standard, corrected yields for sodium and potassium are obtained by dividing their observed counting rates by a sensitivity factor of 1.9. Note that 2 stable isotopes of potassium are indicated.

The midcortex of a femur from a mouse drinking 1.5% NH4Cl for 7 days demonstrates a marked change in the mass spectra of the positive ions. After exposure to systemic acidosis, the peak height of 40Ca is greater than that of 23Na and that of 39K and 41K (Fig. 2). Compared with mice drinking distilled water, consumption of NH4Cl leads to a marked fall in the ratios 23Na/40Ca and 39K/40Ca (Fig. 3).


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Fig. 2.   Mass spectra of positive secondary ions located in midcortex of a representative neonatal mouse femur after drinking distilled water with 1.5% NH4Cl for 7 days. Mass spectra indicate that abundance of calcium is greater than that of either sodium or potassium. Observed spectra were measured in 1,000 channels equally divided among mass 20-50 atomic mass units. Counts per channel are counts per second of detected secondary ions uncorrected for species-dependent ionization probabilities. Relative to calcium as a standard, corrected yields for sodium and potassium are obtained by dividing their observed counting rates by a sensitivity factor of 1.9. Note that 2 stable isotopes of potassium are indicated.



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Fig. 3.   Ratio of sodium to calcium (Na/Ca) and potassium to calcium (K/Ca) in midcortex of neonatal mouse femurs after drinking only distilled water (Ctl) or water with 1.5% NH4Cl (Acid) for 7 days measured in 3 right femurs in each group, with 9-16 measurements of each ion or ion complex in each femur. Values are expressed as means + upper 95% confidence limit. Compared with Ctl, there was a significant fall in Na/Ca and K/Ca after acid treatment. * P < 0.05.

Negative ions. We also examined certain of the negative ionic complexes that are relevant to proton buffering in the midcortex of a femur from a mouse drinking distilled water. Phosphate is a principal component of the mineral, and the release of phosphate would result in proton buffering. The secondary ions P and PO4 are difficult to detect with SIMS. Therefore, the ions PO2 and PO3, which are detected more easily, were recorded. The total signal PO2 + PO3 is taken as a marker for total phosphate, that is, first-order changes in total phosphate concentration are reflected by linear changes in the sum of PO2 + PO3. We also examined the abundance of bone HCO-3. We could not study bone CO2-3, because this complex is at mass 60 which is also the mass of C5, a common organic material.

We compared the abundance of detectable phosphates to carbon and to CN. Carbon is a suitable denominator because it would not be expected to be affected by acid in these relatively short-term experiments. We used C2 rather than C because it gives a stronger signal. CN is present in areas of organic material and also would not be expected to change over these relatively short-term experiments.

We found that there was as much PO2 + PO3 as C2 and almost as much PO2 + PO3 as CN in the midcortex of a femur from a mouse drinking distilled water (Fig. 4). There was far less HCO-3 than either C2 or CN; however, there was almost as much HCO-3 as mass 60, which consists predominantly of C5 and CO2-3.


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Fig. 4.   Mass spectra of negative secondary ions located in midcortex of a representative neonatal mouse femur after drinking only distilled water for 7 days. Mass spectra indicate that abundance of total phosphates (PO2 + PO3) is slightly less than of carbon-nitrogen bond (CN) and of C2 and that there is far less HCO-3 than CN or C2. Observed spectra were measured in 1,000 channels equally divided among mass 10-90 atomic mass units. Counts per channel are counts per second of detected secondary ions uncorrected for species-dependent ionization probabilities.

The midcortex of a femur from a mouse drinking 1.5% NH4Cl for 7 days demonstrates a marked change in the mass spectra of these negative ions. There was a fall in the peak height of PO2 + PO3 and of HCO-3, each relative to C2 and to CN (Fig. 5). Compared with mice drinking distilled water, consumption of NH4Cl leads to a marked fall in the ratio of PO2 + PO3 to C2 and CN (Fig. 6) and of HCO-3 relative to C2 and to CN (Fig. 7).


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Fig. 5.   Mass spectra of negative secondary ions located in midcortex of a representative neonatal mouse femur after drinking distilled water with 1.5% NH4Cl for 7 days. Mass spectra indicate that abundance of PO2 + PO3 is far less than of CN and of C2 and that there is far less HCO-3 than CN or C2. Observed spectra were measured in 1,000 channels equally divided among mass 10-90 atomic mass units. Counts per channel are counts per second of detected secondary ions uncorrected for species-dependent ionization probabilities.



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Fig. 6.   Ratio of PO2 + PO3 to CN and PO2 + PO3 to C2 in midcortex of neonatal mouse femurs after drinking only distilled water (Ctl) or water with 1.5% NH4Cl (Acid) for 7 days measured in 3 right femurs in each group, with 9-16 measurements of each ion or ion complex in each femur. Values are expressed as means + upper 95% confidence limit. Compared with Ctl, there was a significant fall in (PO2 + PO3)/CN and (PO2 + PO3)/C2 after acid treatment. * P < 0.05.



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Fig. 7.   Ratio of HCO-3 to CN and HCO-3 to C2 in midcortex of neonatal mouse femurs after drinking only distilled water (Ctl) or water with 1.5% NH4Cl (Acid) for 7 days measured in 3 right femurs in each group, with 9-16 measurements of each ion or ion complex in each femur. Values are expressed as means + upper 95% confidence limit. Compared with Ctl, there was a significant fall in HCO-3/CN and HCO-3/C2 after acid treatment. * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Metabolic acidosis has multiple effects on bone mineral (20, 24, 30, 33, 56) and on bone cell function (11, 27, 36, 37). In vitro, the excess protons present during metabolic acidosis are buffered by bone (8, 9, 23, 26). In vivo, bone has been proposed as a proton buffer during acidosis (45, 55); however, there is little firm experimental confirmation of this hypothesis. The current in vivo study is the first to demonstrate that after a period of metabolic acidosis there are multiple, concurrent changes in the bone mineral, each of which is consistent with bone acting as a proton buffer.

In acute in vitro studies we previously demonstrated that isolated neonatal mouse calvariae buffer a physiological reduction in medium pH when the reduction is produced by a decrement in [HCO-3], a model of metabolic acidosis (8, 23), but not when produced by an increment in PCO2, a model of respiratory acidosis (9). During metabolic acidosis, there was a strong inverse correlation between the initial medium proton concentration and the net proton influx into bone (23). It is clear that acute in vitro buffering is not caused by simple dissolution of the bone mineral (23). For every 16-21 neq of proton influx into bone there was 1 neq of calcium efflux. This stoichiometry is not consistent with simple dissolution of apatite [Ca10(PO4)6(OH)2], where the ratio would be 5:3 (39), brushite (CaHPO4), where the ratio would be 1:1 (39), or calcium carbonate (CaCO3), where the ratio would be 1:1 (51). The results of the current study, in which we found changes in bone sodium, potassium, phosphate, and carbonate are consistent with the hypothesis that bone buffers protons concurrently through a number of mechanisms.

A possible mechanism for proton buffering by bone is the exchange of protons for sodium or potassium. Bone is rich in sodium and potassium (15, 16, 20, 21, 26, 32-34), and sodium has been shown to exchange freely with the surrounding fluid (59). Bone contains numerous organic ions that have fixed negative sites that are normally bonded with sodium and potassium, which may exchange for protons during acidosis. There is experimental evidence for sodium-for-proton exchange during acidosis (4, 6). The sodium content of dried rat bone falls by 28%, without a change in mineral calcium, 4 h after intraperitoneal injection of NH4Cl (4). Bones prelabeled with 22Na lost 7 meq/kg of bone sodium after 5 h of metabolic acidosis (6). We previously (20, 21, 26, 34) used cultured calvariae to demonstrate that acidosis causes a loss of mineral sodium and potassium in relation to calcium. In the current in vivo study, we demonstrate a loss of mineral sodium and potassium relative to calcium that is consistent with the hypothesis that acidosis induces sodium-for-proton and potassium-for-proton exchange in the mineral. This uptake of protons by bone during metabolic acidosis would lessen the degree of systemic acidity. As metabolic acidosis has repeatedly been shown to result in a loss of mineral calcium (9-11, 23, 27, 29, 31, 42), the acidosis-induced fall in the ratios sodium/calcium and potassium/calcium suggests that acidosis causes a greater release of sodium and potassium than calcium from the mineral.

Another possible mechanism for proton buffering by bone during metabolic acidosis is the consumption of bone CO2-3. Bone contains ~4-6% CO2-3, indicating that approximately one in four or five phosphates is substituted by CO2-3 (35, 54). CO2-3 cannot only substitute for phosphate but can bind at a monovalent anionic site and at an unstable location that occurs principally in rapidly formed precipitates (53). Previous studies, in a variety of systems, have shown a loss of bone CO2-3 during in vivo metabolic acidosis (5, 7, 41). An early study by Irving and Chute (41) demonstrated a loss of bone CO2-3 after several days of metabolic acidosis. Burnell (7) demonstrated a loss of bone CO2-3 5-10 days after metabolic acidosis, and Bettice (5) demonstrated that a decrease in bone CO2-3 after metabolic acidosis was correlated with the fall in extracellular HCO-3. Using cultured neonatal mouse calvariae, we showed (25) that the mineral calcium and CO2-3, in the form of carbonated apatite, are in equilibrium with the culture medium. Additional experiments with cultured calvariae demonstrate a loss of mineral CO2-3 during metabolic, but not respiratory, acidosis (24). The current in vivo study demonstrates a loss of mineral HCO-3, with respect to two ion complexes that should not be affected by acidosis, CN, and C2. HCO-3 readily accepts protons and is a known buffer in the extracellular fluid (13). CO2-3 itself has an atomic mass of 60. Because mass 60 is also C5 we should not have expected a detectable decrease in mass 60 during metabolic acidosis, given the large mass of organic carbon in bone (60) that would not be expected to be affected by acidosis.

Yet another possible mechanism for proton buffering by bone during metabolic acidosis is the protonation of bone phosphate. Phosphate is present in bone as apatite [Ca10(PO4)6(OH)2] and as brushite (CaHPO4), which would provide proton acceptors in the form of PO3-4 and HPO2-4, respectively (51). Chronic metabolic acidosis leads to an increase in urine phosphate excretion (40), suggesting consumption of bone phosphate and excretion of this ion complex as titratable acidity (12, 13). The lower the urine pH, the greater the protonation of phosphate and the greater the quantity of acid excreted per mole of phosphorus (13). In this study, the decline in bone phosphate relative to CN and to C2 during metabolic acidosis is consistent with a role for bone phosphates in buffering the additional protons during metabolic acidosis.

The data from this in vivo study indicating that there is a loss of bone sodium and potassium in excess of a loss of calcium during metabolic acidosis are consistent with our previous in vitro microprobe studies (20, 26, 33, 34). We used the high-resolution scanning ion microprobe to determine the changes in cultured neonatal mouse calvariae after incubation in medium simulating metabolic acidosis. We found that after in vitro culture the surface of calvariae was rich in sodium and potassium relative to calcium and that acidosis resulted in a marked decline in these ratios. We also found that acidosis caused an efflux of calcium from bone, suggesting that the release of sodium and potassium was far greater than that of calcium. In these previous in vitro studies we were not able to measure bone phosphates or carbonates in relation to the carbon-nitrogen bond or to carbon itself.

In this study we examined midcortical bone. It is possible that there will be differences in the osseous response to metabolic acidosis depending on the age, depth, or type of bone examined. Although the total carbonate content in bone mineral increases with age, the carbonate in the unstable location decreases with age (54). Further studies, in which we examine the effect of animal age on proton buffering by bone and whether there is a difference in the buffering properties of superficial cortical bone versus trabecular bone, will be required to understand the integrated in vivo bone response to metabolic acidosis. Similarly, the duration of acidosis may have a substantial effect on the changes in bone mineral. In vitro studies have demonstrated that the osseous response to short-term acidosis (~3 h) is physicochemical whereas the longer-term response is, in addition, cell mediated (11, 21, 22, 27, 33, 42). Further studies, in which we use the ion microprobe to examine the osseous response to in vivo acidosis as a function of time, will be necessary to determine whether there is a difference in the ion composition of bone in response to acute compared with chronic metabolic acidosis.

Clinically, the initial human response to metabolic acidosis involves titration of extracellular fluid HCO-3 (13). Although the apparent "space of distribution" of HCO-3 is ~0.6% of total body weight, this apparent "space" increases as the systemic HCO-3 concentration declines (13, 18), indicating the recruitment of additional HCO-3 and nonbicarbonate buffers. The current study suggests that the multiple potential buffers in bone may be involved not only in the initial proton buffering but in the increase in this apparent space of distribution.

Thus metabolic acidosis appears to induce changes in the midcortical bone mineral that are consistent with its purported role as a proton buffer. The decreases in mineral sodium, potassium, carbonate, and phosphate will each buffer protons and lead to an increase in systemic pH toward the physiological normal. This apparent protective function of bone will come, in part, at the expense of its mineral stores. Future studies will be necessary to determine whether the proton buffering properties of bone can be described by a dynamic equilibrium: protonation of phosphate and carbonate and release of sodium and potassium during acidosis coupled to deprotonation and uptake of sodium and potassium during alkalosis. This attractive hypothetical mechanism has a clear survival advantage for mammals.


    ACKNOWLEDGEMENTS

We thank Daniel R. Riordon for expert technical assistance.


    FOOTNOTES

This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-39906 (D. A. Bushinsky) and by the Materials Research Science and Engineering Center at the University of Chicago of the National Science Foundation Grant DMR-9400379 (J. M. Chabala, K.L. Gavrilov, and R. Levi-Setti).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. A. Bushinsky, Univ. of Rochester School of Medicine, 601 Elmwood Ave., Box 675, Rochester, NY 14642 (E-mail: David_Bushinsky{at}URMC.Rochester.edu).

Received 23 December 1998; accepted in final form 1 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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12.   Bushinsky, D. A. The contribution of acidosis to renal osteodystrophy. Kidney Int. 47: 1816-1832, 1995[Medline].

13.   Bushinsky, D. A. Metabolic Acidosis. In: The Principles and Practice of Nephrology, edited by H. R. Jacobson, G. E. Striker, and S. Klahr. St. Louis, MO: Mosby, 1995, p. 924-932.

14.   Bushinsky, D. A. Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F216-F222, 1996[Abstract/Free Full Text].

15.   Bushinsky, D. A., J. M. Chabala, and R. Levi-Setti. Ion microprobe analysis of bone surface elements: effects of 1,25(OH)2D3. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E815-E822, 1989[Abstract/Free Full Text].

16.   Bushinsky, D. A., J. M. Chabala, and R. Levi-Setti. Ion microprobe analysis of mouse calvariae in vitro: evidence for a "bone membrane." Am. J. Physiol. 256 (Endocrinol. Metab. 19): E152-E158, 1989[Abstract/Free Full Text].

17.   Bushinsky, D. A., J. M. Chabala, and R. Levi-Setti. Comparison of in vitro and in vivo 44Ca labeling of bone by scanning ion microprobe. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E586-E592, 1990[Abstract/Free Full Text].

18.   Bushinsky, D. A., F. L. Coe, C. Katzenberg, J. P. Szidon, and J. H. Parks. Arterial PCO2 in chronic metabolic acidosis. Kidney Int. 22: 311-314, 1982[Medline].

19.   Bushinsky, D. A., M. J. Favus, A. B. Schneider, P. K. Sen, L. M. Sherwood, and F. L. Coe. Effects of metabolic acidosis on PTH and 1,25(OH)2D3 response to low calcium diet. Am. J. Physiol. 243 (Renal Fluid Electrolyte Physiol. 12): F570-F575, 1982[Medline].

20.   Bushinsky, D. A., K. Gavrilov, J. M. Chabala, J. D. B. Featherstone, and R. Levi-Setti. Effect of metabolic acidosis on the potassium content of bone. J. Bone Miner. Res. 12: 1664-1671, 1997[Medline].

21.   Bushinsky, D. A., K. Gavrilov, V. M. Stathopoulos, N. S. Krieger, J. M. Chabala, and R. Levi-Setti. Effects of osteoclastic resorption on bone surface ion composition. Am. J. Physiol. 271 (Cell Physiol. 40): C1025-C1031, 1996[Abstract/Free Full Text].

22.   Bushinsky, D. A., J. M. Goldring, and F. L. Coe. Cellular contribution to pH-mediated calcium flux in neonatal mouse calvariae. Am. J. Physiol. 248 (Renal Fluid Electrolyte Physiol. 17): F785-F789, 1985[Abstract/Free Full Text].

23.   Bushinsky, D. A., N. S. Krieger, D. I. Geisser, E. B. Grossman, and F. L. Coe. Effects of pH on bone calcium and proton fluxes in vitro. Am. J. Physiol. 245 (Renal Fluid Electrolyte Physiol. 14): F204-F209, 1983[Abstract/Free Full Text].

24.   Bushinsky, D. A., B. C. Lam, R. Nespeca, N. E. Sessler, and M. D. Grynpas. Decreased bone carbonate content in response to metabolic, but not respiratory, acidosis. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F530-F536, 1993[Abstract/Free Full Text].

25.   Bushinsky, D. A., and R. J. Lechleider. Mechanism of proton-induced bone calcium release: calcium carbonate dissolution. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F998-F1005, 1987[Abstract/Free Full Text].

26.   Bushinsky, D. A., R. Levi-Setti, and F. L. Coe. Ion microprobe determination of bone surface elements: effects of reduced medium pH. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F1090-F1097, 1986[Abstract/Free Full Text].

27.   Bushinsky, D. A., and E. L. Nilsson. Additive effects of acidosis and parathyroid hormone on mouse osteoblastic and osteoclastic function. Am. J. Physiol. 269 (Cell Physiol. 38): C1364-C1370, 1995[Abstract/Free Full Text].

28.   Bushinsky, D. A., G. S. Riera, M. J. Favus, and F. L. Coe. Response of serum 1,25(OH)2D3 to variation of ionized calcium during chronic acidosis. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F361-F365, 1985[Medline].

29.   Bushinsky, D. A., and N. E. Sessler. Critical role of bicarbonate in calcium release from bone. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F510-F515, 1992[Abstract/Free Full Text].

30.   Bushinsky, D. A., N. E. Sessler, R. E. Glena, and J. D. B. Featherstone. Proton-induced physicochemical calcium release from ceramic apatite disks. J. Bone Miner. Res. 9: 213-220, 1994[Medline].

31.   Bushinsky, D. A., N. E. Sessler, and N. S. Krieger. Greater unidirectional calcium efflux from bone during metabolic, compared with respiratory, acidosis. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F425-F431, 1992[Abstract/Free Full Text].

32.   Bushinsky, D. A., S. Sprague, P. Hallegot, C. Girod, J. M. Chabala, and R. Levi-Setti. Effects of aluminum on bone surface ion composition. J. Bone Miner. Res. 10: 1988-1997, 1995[Medline].

33.   Bushinsky, D. A., W. Wolbach, N. E. Sessler, R. Mogilevsky, and R. Levi-Setti. Physicochemical effects of acidosis on bone calcium flux and surface ion composition. J. Bone Miner. Res. 8: 93-102, 1993[Medline].

34.   Chabala, J. M., R. Levi-Setti, and D. A. Bushinsky. Alteration in surface ion composition of cultured bone during metabolic, but not respiratory, acidosis. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F76-F84, 1991[Abstract].

35.   Driessens, F. M. C., J. W. E. van Dijk, and J. M. P. M. Borggreven. Biological calcium phosphates and their role in the physiology of bone and dental tissues. I. Composition and solubility of calcium phosphates. Calcif. Tissue Res. 26: 127-137, 1978[Medline].

36.   Frick, K. K., and D. A. Bushinsky. Chronic metabolic acidosis reversibly inhibits extracellular matrix gene expression in mouse osteoblasts. Am. J. Physiol. 275 (Renal Physiol. 44): F840-F847, 1998[Abstract/Free Full Text].

37.   Frick, K. K., L. Jiang, and D. A. Bushinsky. Acute metabolic acidosis inhibits the induction of osteoblastic egr-1 and type 1 collagen. Am. J. Physiol. 272 (Cell Physiol. 41): C1450-C1456, 1997[Abstract/Free Full Text].

38.   Gafter, U., J. A. Kraut, D. B. N. Lee, V. Silis, M. W. Walling, K. Kurokawa, M. R. Haussler, and J. W. Coburn. Effect of metabolic acidosis in intestinal absorption of calcium and phosphorus. Am. J. Physiol. 239 (Gastrointest. Liver Physiol. 2): G480-G484, 1980[Abstract/Free Full Text].

39.   Glimcher, M. J. Composition, structure, and organization of bone and other mineralized tissues and the mechanism of calcification. In: Handbook of Physiology. Endocrinology. Parathyroid Gland. Washington, DC: Am. Physiol. Soc., 1976, sect. 7, vol. VII, chapt. 2, p. 25-116.

40.   Guntupalli, J., B. Eby, and K. Lau. Mechanism for the phosphaturia of NH4Cl: dependence on acidemia but not on diet PO3-4 or PTH. Am. J. Physiol. 242 (Renal Fluid Electrolyte Physiol. 11): F552-F560, 1982[Medline].

41.   Irving, L., and A. L. Chute. The participation of carbonates of bone in the neutralization of ingested acid. J. Cell. Comp. Physiol. 2: 157-176, 1932.

42.   Krieger, N. S., N. E. Sessler, and D. A. Bushinsky. Acidosis inhibits osteoblastic and stimulates osteoclastic activity in vitro. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F442-F448, 1992[Abstract/Free Full Text].

43.   Legeais, J. M., P. Hallegot, J. Chabala, G. Renard, R. Levi-Setti, and P. Galle. Trifluorothymidine localization in the rabbit cornea by secondary ion mass spectrometry imaging microanalysis. Curr. Eye Res. 8: 971-973, 1989[Medline].

44.   Lemann, J., Jr., N. D. Adams, and R. W. Gray. Urinary calcium excretion in human beings. N. Engl. J. Med. 301: 535-541, 1979[Medline].

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47.   Levi-Setti, R. Structural and microanalytical imaging of biological materials by scanning microscopy with heavy-ion probes. Annu. Rev. Biophys. Biophys. Chem. 17: 325-347, 1988[Medline].

48.   Levi-Setti, R., and M. Le Beau. Cytogenetic applications of high resolution secondary ion imaging microanalysis: detection and mapping of tracer isotopes in human chromosomes. Biol. Cell 74: 51-58, 1992[Medline].

49.   Levi-Setti, R., Y. L. Wang, and G. Crow. Scanning ion microscopy: elemental maps at high lateral resolution. Appl. Surface Sci. 26: 249-264, 1986.

50.   Lundgren, T., E. U. Engstrom, R. Levi-Setti, A. Linde, and J. G. Noren. The use of the stable isotope 44Ca in studies of calcium incorporation into dentin. J. Microsc. 173: 149-154, 1994[Medline].

51.   Neuman, W. F., and M. W. Neuman. The Chemical Dynamics of Bone Mineral. Chicago: Univ. of Chicago Press, 1958.

52.   Poole-Wilson, P. A., and I. R. Cameron. Intracellular pH and K+ of cardiac and skeletal muscle in acidosis and alkalosis. Am. J. Physiol. 229: 1305-1310, 1975[Medline].

53.   Rey, C., B. Collins, T. Goehl, I. R. Dickson, and M. J. Glimcher. The carbonate environment in bone mineral: a resolution-enhanced Fourier transform infrared spectroscopy study. Calcif. Tissue Int. 45: 157-164, 1989[Medline].

54.   Rey, C., V. Renugopalakrishnan, B. Collins, and J. Glimcher. Fourier transform infrared spectroscopic study of the carbonate ions in bone mineral during aging. Calcif. Tissue Int. 49: 251-258, 1991[Medline].

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56.   Sprague, S. M., N. S. Krieger, and D. A. Bushinsky. Greater inhibition of in vitro bone mineralization with metabolic than respiratory acidosis. Kidney Int. 46: 1199-1206, 1994[Medline].

57.   Sutton, R. A. L., N. L. M. Wong, and J. H. Dirks. Effects of metabolic acidosis and alkalosis on sodium and calcium transport in the dog kidney. Kidney Int. 15: 520-533, 1979[Medline].

58.   Swan, R. C., and R. F. Pitts. Neutralization of infused acid by nephrectomized dogs. J. Clin. Invest. 34: 205-212, 1955.

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