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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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 HCO3 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
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INTRODUCTION |
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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
[HCO3], 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
-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
(CO23) (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.
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METHODS |
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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 (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 · s1 · 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, HCO3, 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 [HCOMean 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 |
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Blood pH, PCO2, and
[HCO3]
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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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 HCO3. 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.
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DISCUSSION |
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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 [HCO3], 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
CO23. 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
PO34 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 HCO3 (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.
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ACKNOWLEDGEMENTS |
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We thank Daniel R. Riordon for expert technical assistance.
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FOOTNOTES |
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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.
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