1Nephrology Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York 14642; and 2Department of Physics, Enrico Fermi Institute, University of Chicago, Chicago, Illinois 60637
Submitted 1 April 2003 ; accepted in final form 11 May 2003
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ABSTRACT |
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ion microprobe; calcium; proton; metabolic acidosis
An in vitro model of metabolic acidosis, produced by a decrement in medium
HCO3- concentration ([HCO3-]),
induces a marked efflux of calcium from cultured neonatal mouse calvariae
(11,
22,
24,
30,
36,
48), whereas metabolic
alkalosis induces an influx of calcium into bone
(13). During short-term (3 h)
cultures, this acid-induced calcium efflux appears due to physicochemical bone
mineral dissolution (24,
36). However, over longer time
periods (>24 h), the calcium efflux from bone appears, in addition, due to
cell-mediated bone resorption
(11,
22,
30,
48). We have shown that
metabolic acidosis leads to an increase in osteoclastic -glucuronidase
activity and a decrease in osteoblastic collagen synthesis
(11,
42,
48). In addition, acidosis
inhibits the stimulation of some, but not all, immediate early response genes
(42) and reversibly inhibits
expression of certain extracellular matrix genes
(40). This cell-mediated
resorption is a result of increased prostaglandin E2 synthesis,
which stimulates osteoclastic resorption and suppresses osteoblastic function
(31,
44,
47,
58). In vitro metabolic
acidosis causes the release of mineral potassium and sodium
(21,
28,
36,
37) and a depletion of mineral
carbonate (26,
27),
HCO3-, and phosphate
(34).
Previously, we studied changes in midcortical ion concentrations after 7
days of in vivo metabolic acidosis induced by oral ammonium chloride
(15). We found that, compared
with mice drinking only distilled water, the ammonium chloride induced a loss
of bone sodium and potassium and a depletion of mineral
HCO3- and phosphate. In the previous study, we
questioned whether there were regional differences in the response of mineral
HCO3- and phosphate to acute and chronic metabolic
acidosis. We have shown that acute metabolic acidosis induces a depletion of
surface, but not cross-sectional, HCO3-, and
cross-sectional, but not surface, phosphate
(34). There was depletion of
HCO3- in preference to phosphate on the bone surface and
depletion of phosphate in preference to HCO3- in the
interior of bone. The effects of acid medium on bone during acute metabolic
acidosis are due to physicochemical dissolution of the mineral
(24,
36), while during more chronic
acidosis the effects are, in addition, due to cell-mediated resorption
(9,
11,
30,
31,
40,
48). Given that
physicochemical bone dissolution has very different effects on bone sodium and
calcium release than cell-mediated resorption
(22), we suspected that a
model of chronic acidosis would alter the proton buffers in the mineral
differently than for acute acidosis. As the ratio of carbonate to phosphate in
mouse calvariae 3 and 7 days postnatal is 0.12
(63), indicating that there is
far more phosphate than HCO3- available to buffer the
additional protons during metabolic acidosis, we would also suspect that an
acidic medium would decrease bulk (cross-sectional) bone phosphate to a
greater extent than bone carbonate.
Thus in the present study we utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy (SIMS) to test the hypothesis that chronic acidosis would decrease bulk (cross-sectional) bone phosphate to a greater extent than bone carbonate by localizing and comparing the changes in bone HCO3- and phosphate after chronic incubation of neonatal mouse calvariae in an acidic medium. We found that chronic acidosis induced a fall in both cross-sectional HCO3- and phosphate with no change in surface HCO3- and phosphate and the fall in phosphate predominated over the fall in HCO3-. Depletion of these proton buffers, HCO3- and phosphate, would help to mitigate the reduction in pH during chronic acidosis.
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METHODS |
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Experimental groups. Calvariae were incubated for 51 h in either
control (Ctl) or acidic (Acid) medium. In the Ctl group, the calvariae were
cultured in a neutral-pH medium (pH 7.45, [HCO3-]
26 meq/l). In the Acid group, the calvariae were cultured in medium in
which the pH was lowered (pH
7.14, [HCO3-]
13
meq/l) by the addition of 10 µl of 2.4 M HCl/ml medium to lower
[HCO3-] (Table
1). We have previously shown that calvariae are viable and exhibit
similar [3H] proline incorporation whether they are cultured for up
to 120 h under conditions of physiological pH or of metabolic acidosis
(9). Calvariae were incubated
for a total of 51 h. Bones were transferred to fresh preincubated medium at 24
and 48 h. Before and after each incubation, the medium was immediately
analyzed for pH, PCO2, and calcium. Fifty-one hours of
incubation were chosen to represent chronic acidosis as this is the time
period during which acidosis induces predominantly cell-mediated bone
resorption (9,
11,
30,
31,
40,
48). During more acute
acidosis, fewer then 24 h in culture, there is predominantly physicochemical
mineral dissolution (24,
36). At the conclusion of the
experiments, calvariae were removed from the culture dishes, 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)
(15-18,
21-23,
28,
34-37).
The frontal and parietal bones of some calvariae were split in half to reveal
the interior of the bone for cross-sectional analysis. All bones were then
mounted on aluminum supports with conductive glue and coated with a thin layer
(
5 nm) of gold. This layer, which is rapidly sputtered away by the ion
probe from the area being scanned, prevents artifact-inducing electrical
charging.
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Scanning ion microprobe. The scanning ion microprobe utilized for these studies was conceptualized and built at the University of Chicago and employs a 40-keV gallium beam focused to a spot 40 nm in diameter (15-18, 21-23, 28, 34-37, 53, 54). 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 microanalysis of bone by SIMS was performed in two nonimaging SIMS modes. In the first, 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 second, a 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 (15-18, 21-23, 28, 34-37, 53, 54).
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 unit 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 x 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 (sodium,
potassium, and calcium), counting rates as high as 4.0 x 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, four calvariae from each group. For each calvaria,
we measured the concentration of HCO3-, phosphate,
carbon-nitrogen bond, 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
(15-18,
21-23,
28,
34-37,
53,
54). 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
ranging from 40 x 40 to 160 x 160 µm2, never
exceeded
5 nm, which did not result in significant sample depletion.
Given the extremely small area being examined, 40 x 40 to 160 x
160 µm2, all cross-sectional measurements were far from the
calvarial surface, which is
1 mm thick.
We compared the ratios of HCO3- and total phosphate to the carbon-carbon bond (C2) and carbon-nitrogen bond (CN). 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). The CN is present in areas of organic material. PO4 gives a very weak signal, presumably due to the 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 ratio [(PO2 + PO3)/C2] or [(PO2 + PO3)/CN] 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 (64), which would not be expected to be affected by acidosis. In contrast HCO3- (mass 61), readily accepts hydrogen ions and is a known buffer in the extracellular fluid (10). There are no other common compounds at mass 61, making this an unambiguous marker for bone total CO2 (carbonate + HCO3-). As in previous studies, we have used C2, CN, total phosphate, and HCO3- to study the effects of acid on bone (15, 34). 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, which 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 (28). 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 (15-18, 21-23, 28, 34-37, 53, 54) 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 electrode (Nova Biomedical, Waltham, MA). The medium [HCO3-] was calculated from medium pH and PCO2 as described previously (8, 11, 25, 36). Net calcium flux 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 concentration, respectively. 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 tests of significance were calculated using analysis of variance with a Bonferroni correction for multiple comparisons (BMDP, University of California, Los Angeles, CA) on a digital computer. All values are expressed as means ± SE; P < 0.05 was considered significant.
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RESULTS |
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HCO3-. After 51 h of incubation in Ctl medium, the ratio of HCO3- relative to the C2 bond was greater in the cross section than on the surface of calvariae incubated in Ctl medium; however, the ratio of HCO3- relative to the CN bond on the surface was not different from that found on the cross section of calvariae incubated in Ctl medium [Fig. 2, top, representative spectra from surface of calvariae in Ctl medium (Control in figure); Fig. 3, top, representative spectra from cross section of calvariae in Ctl medium; Fig. 4, top, compiled data]. Compared with incubation in Ctl medium, incubation in Acid medium did not alter the ratio of HCO3- to C2 or the ratio of HCO3- to CN on the surface of calvariae (Fig. 2, cf. Ctl and Acid and Fig. 4, top).
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However, compared with incubation in Ctl medium, incubation in Acid medium led to a decrease in the ratio of HCO3- to C2 and a decrease in the ratio of HCO3- to CN in the cross section of the calvariae (Fig. 3, cf. Ctl and Acid and Fig. 4, top). Thus incubation in Acid medium induces a significant fall in mineral HCO3- in the cross section, but not on the surface, of calvariae.
Phosphate. After 51 h of incubation in Ctl medium, the ratio of total phosphate (PO2 + PO3) relative to C2 and the ratio of (PO2 + PO3) relative to CN on the surface were not different from that found on the cross section of calvariae incubated in Ctl medium (Fig. 2, top, representative spectra of Ctl surface; Fig. 3, top, representative spectra of Ctl cross section; Fig. 4, bottom, compiled data). Compared with incubation in Ctl medium, incubation in Acid medium did not alter the ratio of (PO2 + PO3) to C2 or the ratio of (PO2 + PO3) to CN on the surface of calvariae (Fig. 2, cf. Ctl and Acid and Fig. 3, bottom).
However, compared with incubation in Ctl medium, incubation in Acid medium led to a decrease in the ratio of (PO2 + PO3) to C2 and a decrease in the ratio of (PO2 + PO3) to CN in the cross section of the calvariae (Fig. 3, cf. Ctl and Acid and Fig. 4, bottom). Thus incubation in Acid medium induces a significant fall in mineral phosphate in the cross section, but not on the surface, of calvariae.
HCO3- in relation to phosphate. The amount of HCO3- relative to (PO2 + PO3) on the surface of calvariae does not differ significantly from that on the cross section of calvariae incubated in Ctl medium (Fig. 5). Compared with incubation in Ctl medium, incubation in Acid medium did not alter the ratio of HCO3- relative to (PO2 + PO3) on the surface of the calvariae. However, compared with incubation in Ctl medium, incubation in Acid medium led to a significant increase in the ratio of HCO3- to (PO2 + PO3) in the cross section of the calvariae. Because incubation in Acid medium led to a reduction of both (PO2 + PO3) and HCO3- in the cross section of calvariae (Figs. 3 and 4), an increase in the ratio of HCO3- to (PO2 + PO3) on the cross section of calvariae must indicate depletion of (PO2 + PO3) in relation to HCO3-. Thus metabolic acidosis induces a fall in mineral (PO2 + PO3) in relation to mineral HCO3- in the cross section of calvariae.
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DISCUSSION |
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We previously studied changes in bulk midcortical ion concentrations after 7 days of in vivo metabolic acidosis induced by oral ammonium chloride (15). We found that compared with mice drinking only distilled water, the ammonium chloride-induced acidosis led to a loss of bone sodium and potassium, and, as also shown in this study, a depletion of mineral HCO3- and phosphate. In the previous study, we questioned whether there were regional differences in the response of mineral HCO3- and phosphate to chronic metabolic acidosis. The present study clearly demonstrates regional differences in the response of bone to a model of chronic acidosis; the additional protons deplete cross-sectional, but not surface, bone phosphate and carbonate.
We have previously studied the effects of acute acidosis on surface and cross-sectional HCO3- and phosphate (34). We found that compared with control, after a 3-h incubation in acidic medium there was a marked decrease in surface HCO3- with respect to C2 and CN with no change in cross-sectional HCO3-. Compared with control, after a 3-h incubation in acidic medium, there was also a marked decrease in cross-sectional phosphate with respect to C2 and also to CN with no change in surface phosphate. On the bone surface, there is a fourfold depletion of HCO3- in relation to phosphate and in cross section a sevenfold depletion of phosphate in relation to HCO3-. These results indicate that acute H+ buffering by bone involves preferential dissolution of surface HCO3- and of cross-sectional phosphate.
The present study extends the acute observations on bone surface and cross-sectional HCO3- and phosphate (34) to a model of chronic acidosis. In the acute study, the surface HCO3- fell, whereas in the chronic study there was no change in surface HCO3-. However, in the acute study the control surface HCO3- was higher than the control surface HCO3- in this chronic study, suggesting that 51 h of culture in control medium may have led to a depletion in surface HCO3-. Perhaps the surface HCO3- is buffering the acids generated through normal cellular metabolism. Indeed, the medium HCO3- fell by the end of the two 48-h incubations of the control bones (Table 1). In the acute study, there was no change in surface phosphate, similar to the results of this study, and the levels of phosphate in the two studies were comparable. In the acute study, there was no change in cross-sectional HCO3-, whereas in this chronic study the cross-sectional HCO3- fell. This suggests that with time the cross-sectional HCO3- is depleted by acidosis in the process of buffering the additional hydrogen ions. In the acute study, the acidic medium resulted in a marked fall in cross-sectional phosphate, as also occurred in this chronic study. Taken together, the results of the two studies suggest that the surface of the control bone cultured for 3 h is rich in HCO3- and the cross section is rich in phosphate. An acidic medium rapidly depletes the surface HCO3- and cross-sectional phosphate. With more prolonged incubation in acidic medium, the cross-sectional, but not the surface, phosphate and HCO3- are further depleted. The consumption of these proton buffers during incubation in acidic medium helps to mitigate the fall in pH.
That the acid-induced depletion of phosphate would predominate over the
depletion of HCO3- in a chronic study of neonatal mouse
bone is not unexpected. Using Raman vibrational microspectroscopy, Tarnowski
et al. (63) found that the
ratio of carbonate to phosphate in mouse calvariae 3 and 7 days postnatal is
0.12, indicating that there is far more phosphate than
HCO3- available as a potential proton buffer. Each
released PO4 would accept a proton in the ratio of four
HPO42- to one
H2PO4- at pH 7.4. The lower the pH, the
greater the ratio of H2PO4- in relation to
HPO42-. Bone
CO32- would combine with H+ to
form HCO3- and then with an additional H+ to
form H2CO3, which rapidly dissociates to H2O
and CO2.
Other studies have shown that during in vitro (7, 8, 25, 28) and in vivo (51, 59, 62) metabolic acidosis, the mineral phases of bone appear to buffer some of the additional protons, resulting in an increase in medium or systemic pH, respectively (10, 14, 20). In cultured bone, we have previously shown that an acute reduction of medium pH is associated with an influx of H+ into the bone (7, 8, 25), an efflux of sodium and potassium from bone (22, 22, 28, 36, 37), and a loss of mineral carbonate (26, 27). The sodium and potassium exchange for H+ decreases the ambient H+ concentration. Because the majority of bone consists of calcium phosphate complexes, the acid-induced, cell-mediated bone resorption that occurs during chronic metabolic acidosis (11, 22, 30, 48) would result in the release of mineral phosphate. We and others have shown a loss of bone carbonate during metabolic acidosis (4, 30), and we have shown that in vivo metabolic acidosis causes a reduction in mineral HCO3- and phosphate (15).
Clinical observations in patients with renal tubular acidosis support the hypothesis that acidosis has deleterious effects on bone mineral. Metabolic acidosis has been shown to have a significant effect on bone density, formation, and growth (38, 39, 55, 56). Domrongkitchaiporn and co-workers (38) compared 14 adult patients with distal renal tubular acidosis who had never received HCO3- therapy with 28 well-matched controls. They measured bone mineral density and also performed bone biopsies with histomorphometric analysis (38). They found that patients with distal renal tubular acidosis had a lower bone mineral density in most areas compared with normal controls. The patients also had a decreased bone formation rate. After treating 12 renal tubular acidosis patients with KHCO3 for 1 yr, they found that bone mineral density significantly improved in the trochanter of the femur and the total femur (39). The bone formation rate normalized with treatment. Initially, the levels of serum parathyroid hormone were suppressed; they too improved with KHCO3 therapy (38, 39). McSherry and Morris (55, 56) studied the effect of renal tubular acidosis on growth in 10 children. Six were found to be stunted (height <2.5 SD), two were too young to determine whether they were short (<2 wk old), and two were previously not acidemic. With sustained alkali therapy, each patient attained and maintained normal stature, mean height increased from the 1.4 to the 37th percentile, and the rate of growth increased two- to threefold.
The results of the present study are consistent with those of previous
investigators. In a classic study, Swann and Pitts
(62) 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,
57) and/or bone
(5,
51). In vivo, Irving and Chute
(45) demonstrated that several
days of metabolic acidosis led to a loss of bone carbonate. Burnell
(6) also demonstrated a loss of
bone carbonate after metabolic acidosis, and Bettice
(4,
5) showed that the metabolic
acidosis-induced loss of bone carbonate correlated with the fall in
extracellular HCO3-. 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
(27). We have shown that
acidosis induces the release of calcium and carbonate from bone
(27), leading to a progressive
loss of bone carbonate during metabolic, but not respiratory, acidosis
(26).
In the present study, we used a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to localize the changes in bone HCO3- and phosphate in response to a model of chronic metabolic acidosis. We found that chronic acidosis induced a fall in both cross-sectional HCO3- and phosphate with no change in surface HCO3- and phosphate and that the fall in phosphate predominated over the fall in HCO3-. Consumption of these proton buffers, HCO3- and phosphate, would help to lessen the fall in pH during chronic acidosis at the expense of the bone mineral content.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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