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作者：David A. Bushinsky, Susan B. Smith, Konstantin L. Gavrilov, Leonid F. Gavrilov, Jianwei Li, Riccardo Levi-Setti作者单位：1 Nephrology Unit, Department of Medicine,University of Rochester School of Medicine, Rochester, New York 14642; and Department of Physics, Enrico Fermi Institute,University of Chicago, Chicago, Illinois 60637
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【摘要】
+ j' S6 R# t& `3 y$ e# y' n Chronic metabolic acidosis increases urinary calcium excretion withoutaltering intestinal calcium absorption, suggesting that bone mineral is thesource of the additional urinary calcium. In vivo and in vitro studies haveshown that metabolic acidosis causes a loss of mineral calcium while bufferingthe additional hydrogen ions. Previously, we studied changes in femoral,midcortical ion concentrations after 7 days of in vivo metabolic acidosis induced by oral ammonium chloride. We found that, compared with mice drinkingonly distilled water, ammonium chloride induced a loss of bone sodium andpotassium and a depletion of mineral HCO 3 - andphosphate. There is more phosphate than carbonate in neonatal mouse bone. Inthe present in vitro study, we utilized a high-resolution scanning ionmicroprobe with secondary ion mass spectroscopy to test the hypothesis thatchronic acidosis would decrease bulk (cross-sectional) bone phosphate to agreater extent than HCO 3 - by localizing andcomparing changes in bone HCO 3 - and phosphateafter chronic incubation of neonatal mouse calvariae in acidic medium.Calvariae were cultured for a total of 51 h in medium acidified by a reductionin HCO 3 - concentration ([HCO 3 - ]; pH 7.14,[HCO 3 - ] 13) or in control medium (pH 7.45, HCO 3 - 26). Compared withincubation in control medium, incubation in acidic medium caused no change insurface total phosphate but a significant fall in cross-sectional phosphate,with respect to the carbon-carbon bond (C 2 ) and the carbon-nitrogenbond (CN). Compared with incubation in control medium, incubation in acidicmedium caused no change in surface HCO 3 - but asignificant fall in cross-sectional HCO 3 - withrespect to C 2 and CN. The fall in cross-sectional phosphate wassignificantly greater than the fall in cross-sectionalHCO 3 -. The fall in phosphate indicates release ofmineral phosphates, and the fall in HCO 3 - indicates release of mineral HCO 3 -, both of whichwould be expected to buffer the additional protons and help restore the pHtoward normal. Thus a model of chronic acidosis depletes bulk bone protonbuffers, with phosphate depletion exceeding that ofHCO 3 -. * I+ M4 Z. U$ G: b3 Z2 T; I
【关键词】 ion microprobe calcium proton metabolic acidosis
+ v; y5 _% }7 E6 N$ ]* N3 n IN HUMANS AND OTHER MAMMALS, chronic metabolic acidosisincreases urinary calcium excretion( 19, 52 ), secondary to a direct reduction of renal tubular calcium reabsorption (67), without increasingintestinal calcium absorption( 43 ), resulting in a netnegative calcium balance ( 2, 49, 51 ). Because the vast majorityof body calcium is located within the mineral stores of bone( 29 ), the negative calciumbalance implies loss of bone mineral( 64 ). In vivo studies haveshown that metabolic acidosis, induced by ammonium chloride, leads to a lossof bone mineral ( 3 ) and thatpatients with proximal renal tubular acidosis are shorter in height and havedecreased radial bone densities and thinner iliac cortices than unaffectedrelatives ( 50 ). Patients withdistal renal tubular acidosis also have decreased bone density and boneformation rate ( 38 ); both parameters improve after a year of HCO 3 - treatment( 39 ). During the ongoingmetabolic acidosis of chronic renal failure, blood pH can remain stable,although substantially reduced, despite progressive hydrogen ion (proton)retention, suggesting the availability of large stores of proton buffers( 59 ). Given its large mass ofpotential proton buffers, bone is an obvious site for proton buffering duringmetabolic acidosis ( 12 ).
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% q5 y* p1 z" Y0 rAn in vitro model of metabolic acidosis, produced by a decrement in mediumHCO 3 - concentration ([HCO 3 - ]),induces a marked efflux of calcium from cultured neonatal mouse calvariae( 11, 22, 24, 30, 36, 48 ), whereas metabolicalkalosis induces an influx of calcium into bone( 13 ). During short-term (3 h) cultures, this acid-induced calcium efflux appears due to physicochemical bonemineral dissolution ( 24, 36 ). However, over longer time 24 h), the calcium efflux from bone appears, in addition, due tocell-mediated bone resorption( 11, 22, 30, 48 ). We have shown thatmetabolic acidosis leads to an increase in osteoclastic -glucuronidaseactivity and a decrease in osteoblastic collagen synthesis( 11, 42, 48 ). In addition, acidosisinhibits the stimulation of some, but not all, immediate early response genes( 42 ) and reversibly inhibitsexpression of certain extracellular matrix genes( 40 ). This cell-mediated resorption is a result of increased prostaglandin E 2 synthesis, which stimulates osteoclastic resorption and suppresses osteoblastic function( 31, 44, 47, 58 ). In vitro metabolicacidosis causes the release of mineral potassium and sodium( 21, 28, 36, 37 ) and a depletion of mineralcarbonate ( 26, 27 ),HCO 3 -, and phosphate( 34 ).
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2 d: C7 w, M* }; A, `! nPreviously, we studied changes in midcortical ion concentrations after 7days of in vivo metabolic acidosis induced by oral ammonium chloride( 15 ). We found that, comparedwith mice drinking only distilled water, the ammonium chloride induced a lossof bone sodium and potassium and a depletion of mineral HCO 3 - and phosphate. In the previous study, wequestioned whether there were regional differences in the response of mineral HCO 3 - and phosphate to acute and chronic metabolicacidosis. We have shown that acute metabolic acidosis induces a depletion ofsurface, but not cross-sectional, HCO 3 -, andcross-sectional, but not surface, phosphate( 34 ). There was depletion ofHCO 3 - in preference to phosphate on the bone surface anddepletion of phosphate in preference to HCO 3 - in theinterior of bone. The effects of acid medium on bone during acute metabolicacidosis are due to physicochemical dissolution of the mineral( 24, 36 ), while during more chronicacidosis the effects are, in addition, due to cell-mediated resorption( 9, 11, 30, 31, 40, 48 ). Given thatphysicochemical bone dissolution has very different effects on bone sodium andcalcium release than cell-mediated resorption( 22 ), we suspected that amodel of chronic acidosis would alter the proton buffers in the mineraldifferently 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 isfar more phosphate than HCO 3 - available to buffer theadditional protons during metabolic acidosis, we would also suspect that anacidic medium would decrease bulk (cross-sectional) bone phosphate to agreater extent than bone carbonate.
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/ T8 j! F4 n" o( g/ z% U8 I5 FThus in the present study we utilized a high-resolution scanning ionmicroprobe with secondary ion mass spectroscopy (SIMS) to test the hypothesisthat chronic acidosis would decrease bulk (cross-sectional) bone phosphate toa greater extent than bone carbonate by localizing and comparing the changesin bone HCO 3 - and phosphate after chronic incubation ofneonatal mouse calvariae in an acidic medium. We found that chronic acidosis induced a fall in both cross-sectional HCO 3 - andphosphate with no change in surface HCO 3 - and phosphateand the fall in phosphate predominated over the fall inHCO 3 -. Depletion of these proton buffers,HCO 3 - and phosphate, would help to mitigate thereduction in pH during chronic acidosis.
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METHODS( s5 d* L! s& o6 o1 c! S
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Organ culture of bone. Neonatal (4- to 6-day-old) CD-1 mice (Charles River, Wilmington, MA) were killed, their calvariae were removed bydissection, the adherent cartilaginous material was trimmed, and theperiosteum was left intact ( 8, 9, 11, 13, 21 - 28, 30 - 34, 36, 37, 40 - 42, 46 - 48, 60 ). Exactly 2.8 ml ofDulbecco's modified Eagle's medium (M. A. Bioproducts, Walkersville, MD)containing heat-inactivated horse serum (15%), heparin sodium (10 U/ml), andpenicillin potassium (100 U/ml) were preincubated at aP CO 2 of 40 Torr at 37°C for 3 h in 35-mm dishes( 8, 9, 11, 13, 21 - 28, 30 - 34, 36, 37, 40 - 42, 46 - 48, 60 ). We found that 3 h aresufficient for P CO 2 equilibration between the incubatorand the medium ( 25 ). After preincubation, 1 ml of medium was removed to determine initial medium pH andP CO 2 and total calcium concentration and two calvariaewere placed in each dish on a stainless steel wire grid. Total bone content ineach culture was controlled by using pups that were the same age and size, byusing a standardized dissection procedure, and by placing two bones in eachdish. Experimental and control cultures were performed in parallel and inrandom order.. I9 D2 s5 W% k* E9 X" v( {
) h" x J7 k. s/ YExperimental groups. Calvariae were incubated for 51 h in either control (Ctl) or acidic (Acid) medium. In the Ctl group, the calvariae werecultured in a neutral-pH medium (pH 7.45, [HCO 3 - ] 26 meq/l). In the Acid group, the calvariae were cultured in medium inwhich the pH was lowered (pH 7.14, [HCO 3 - ] 13meq/l) by the addition of 10 µl of 2.4 M HCl/ml medium to lower [HCO 3 - ] ( Table1 ). We have previously shown that calvariae are viable and exhibitsimilar [ 3 H] proline incorporation whether they are cultured for upto 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 24and 48 h. Before and after each incubation, the medium was immediatelyanalyzed for pH, P CO 2, and calcium. Fifty-one hours ofincubation were chosen to represent chronic acidosis as this is the timeperiod during which acidosis induces predominantly cell-mediated boneresorption ( 9, 11, 30, 31, 40, 48 ). During more acuteacidosis, fewer then 24 h in culture, there is predominantly physicochemicalmineral dissolution ( 24, 36 ). At the conclusion of theexperiments, 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 thenmounted 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 ionprobe from the area being scanned, prevents artifact-inducing electricalcharging.
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; h+ d/ L2 V2 KTable 1. Medium ion concentrations and fluxes; P" z) ~2 X& l, d! B6 a( G D
$ _3 ^, m1 p/ i. F9 X- B/ ]& VScanning ion microprobe. The scanning ion microprobe utilized forthese studies was conceptualized and built at the University of Chicago andemploys 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 scannedacross a sample surface in a controlled sequence, resulting in the emission ofsecondary electrons, ions, and neutral atoms. These secondary particlesoriginate within, and consequently carry information about, the mostsuperficial 1-2 nm of the sample. The charged secondary particles can be collected to generate images of the surface topography of a sample similar tothose obtained using a scanning electron microscope. The particles can also becollected and analyzed by SIMS, a technique that separates the sputtered ionsaccording to their mass-to-charge ratio., C8 Z4 B6 |4 W: d1 j5 z
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In this study, the microanalysis of bone by SIMS was performed in twononimaging SIMS modes. In the first, the spectrometer is rapidly andsequentially retuned to filter several chosen ion species. At the same time,the probe is quickly scanned over a square area, so that the measured signalsare secondary ion intensities averaged over the entire field of view. Using this "peak-switching" technique, the relative concentrations ofseveral elements can be acquired simultaneously from one area and at onesample depth. In the second, a mass analysis mode, the spectrometer masstuning can be systematically varied (as in a conventional mass spectrometer)to yield mass spectra. If the spectra data are corrected by element-dependentsensitivity factors, they provide quantitative relative abundance measurements for a given area of sample( 15 - 18, 21 - 23, 28, 34 - 37, 53, 54 ).8 C: H h3 F5 A0 i3 g* ]. v
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For SIMS analysis, the secondary ions emerging from the sample aretransported through a high-transmission optical system containing anelectrostatic energy analyzer and a magnetic sector mass spectrometer (massresolution 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 (eachcollected ion yields 1 digital pulse). Mass spectra are accumulated with amultichannel scaler, which counts each detected ion by ramping the magneticfield of the mass spectrometer to scan a preselected mass region of thespectrum while the probe is scanning an area of arbitrary dimensions. Thechoice of the scanned area determines the depth over which the target composition is sampled for a given probe current and time. Elemental maps areconstructed by recording the individual, detected pulses in a 512 x 512array of computer memory, each element of the array corresponding to aposition of the probe on the sample. For the most abundant elements (sodium,potassium, and calcium), counting rates as high as 4.0 x 10 5 counts·s - 1 ·pA - 1 ofprimary current were observed. In such cases, statistically significantelemental images could be obtained in scan times of
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We selected, at random, four calvariae from each group. For each calvaria,we measured the concentration of HCO 3 -, phosphate, carbon-nitrogen bond, and carbon on the surface and on the cross section. Theanalyses were repeated on six representative areas of each calvaria. Data wererecorded after erosion of 5 nm of material; this procedure ensured theremoval of any contamination and permitted the acquisition of highlyreproducible measurements and is consistent with our previously publishedstudies( 15 - 18, 21 - 23, 28, 34 - 37, 53, 54 ). Given the beam current of30 pA and image acquisition time not exceeding 524 s (maximum time in thisstudy), the depth of erosion for these elemental measurements, over areasranging from 40 x 40 to 160 x 160 µm 2, 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 µm 2, all cross-sectional measurements were far from thecalvarial surface, which is 1 mm thick./ M; O6 K& ]$ D2 B$ t
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We compared the ratios of HCO 3 - and total phosphateto the carbon-carbon bond (C 2 ) and carbon-nitrogen bond (CN).Carbon appears to be a suitable denominator, as it would not be expected to beaffected by acid in these relatively short-term experiments. We useC 2 rather than C simply because it gives a stronger signal; theratio of C 2 to C is constant (Levi-Setti R and Bushinsky DA,unpublished observations). The CN is present in areas of organic material.PO 4 gives a very weak signal, presumably due to the breakup of thislarge molecule into PO 2 and PO 3 by the gallium beam, andthere is little P which is not associated with O. The ratio [(PO 2 PO 3 )/C 2 ] or [(PO 2 PO 3 )/CN] isnot significantly influenced by inclusion of small amounts of PO 4 or P in the numerator (Levi-Setti R and Bushinsky DA, unpublishedobservations). Carbonate has an atomic mass of 60. Given that mass 60 is alsoC 5, we would not expect a detectable decrease in mass 60 duringmetabolic acidosis given the large mass of organic carbon in bone( 64 ), which would not beexpected to be affected by acidosis. In contrast HCO 3 - (mass 61), readily accepts hydrogen ions and is a known buffer in theextracellular fluid ( 10 ).There are no other common compounds at mass 61, making this an unambiguousmarker for bone total CO 2 (carbonate HCO 3 - ). As in previous studies, we have usedC 2, CN, total phosphate, and HCO 3 - to studythe effects of acid on bone( 15, 34 ). In this study, we do not report positive ions, such as calcium, sodium, and potassium, because we donot know of a standard positive ion or ion cluster, which would not beexpected to be influenced by acidosis. We have previously demonstrated thatacute acidosis induces a marked loss of bone sodium and potassium in relationto calcium ( 28 ). Without astandard for the positive ions, we cannot determine the localization of theloss of sodium and potassium.
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# w. u% |- e1 t: P. Z' z4 UCorrection methods similar to those that we have previously reported( 15 - 18, 21 - 23, 28, 34 - 37, 53, 54 ) were applied to theobserved mass-resolved counting rates to obtain secondary ion yieldsproportional to the elemental concentration in the sample. Corrections arenecessary because of the species-dependent sputtering and ionizationprobabilities of the emitted atoms. The total ion counts in a micrograph are afunction 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 ofthe calvariae may have physical holes. In addition, the detected ion yieldsare dependent on the degree of sample surface roughness. Because of theseconsiderations, we express our results in terms of the ratios of countsobtained for the same area of a sample. Such ratios are independent of thefraction of the field of view occupied by the sample and of the surface topography. D( ]$ Z5 X; j$ e6 ]
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Conventional measurements. Medium pH andP CO 2 were determined with a blood-gas analyzer(Radiometer model ABL 30) and calcium by electrode (Nova Biomedical, Waltham,MA). The medium [HCO 3 - ] was calculated from medium pHand P CO 2 as described previously ( 8, 11, 25, 36 ). Net calcium flux wascalculated as V m ( f - i ), whereV m is the medium volume (1.8 ml), and f and i are the final and initial medium calcium concentration, respectively. A positive flux value indicates movement of the ion from thebone into the medium, and a negative value indicates movement from the mediuminto the bone.
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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. Allvalues are expressed as means ± SE; P
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RESULTS
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Medium pH, P CO 2, and calcium. Compared withCtl medium, the initial and final pH and [HCO 3 - ] weresignificantly reduced in the Acid medium during the 0- to 24-h, 24- to 48-h,and 48- to 51-h incubation ( Table1 ). There were no differences in P CO 2 between Ctl and Acid media during any time period. Compared with calvariaeincubated in Ctl medium, during each of the three individual incubations andover the entire time period studied, there was a significant increase in netcalcium flux from bones incubated in Acid medium( Fig. 1 ).
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9 |9 Z" |/ Z* {2 E! Y4 a/ L! MFig. 1. Net calcium flux from neonatal mouse calvariae incubated in either control(Ctl; initial medium pH 7.45, HCO 3 - 26) oracidic (Acid; initial medium pH 7.14, HCO 3 - 13) medium for a total of 51 h. Calvariae were transferred to similarfresh preincubated medium at 24 and 48 h. Before and after each incubation,the medium was analyzed for calcium. Total represents the sum of the netcalcium flux from the 3 separate incubations in each group. A positive fluxrepresents movement from the bone into the medium. * P
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, |- z' s6 B$ Q" O- sHCO 3 -. After 51 h of incubation inCtl medium, the ratio of HCO 3 - relative to theC 2 bond was greater in the cross section than on the surface ofcalvariae incubated in Ctl medium; however, the ratio ofHCO 3 - relative to the CN bond on the surface was notdifferent from that found on the cross section of calvariae incubated in Ctlmedium [ Fig. 2, top,representative spectra from surface of calvariae in Ctl medium (Control infigure); Fig. 3, top,representative spectra from cross section of calvariae in Ctl medium; Fig. 4, top, compileddata]. Compared with incubation in Ctl medium, incubation in Acid medium didnot alter the ratio of HCO 3 - to C 2 or theratio of HCO 3 - to CN on the surface of calvariae( Fig. 2, cf. Ctl and Acid and Fig. 4, top ).. a( h: g3 L/ a( v' a" ~
$ Z9 s5 w6 {' a. c, }9 {Fig. 2. Mass spectra of the negative secondary ions on the surface of neonatalmouse calvariae incubated for 51 h in either Ctl (Control in figure) or Acidmedium. Calvariae were transferred to similar fresh preincubated medium at 24and 48 h. Counts/channel are counts/s of detected secondary ions uncorrectedfor species-dependent ionization probabilities. Observed spectra were measuredin 1,000 channels equally divided among mass 10-90 atomic mass units. CN,carbon-nitrogen bond; C 2, carbon-carbon bond./ ~* i: e! i& ~1 a; w/ c
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Fig. 3. Mass spectra of the negative secondary ions on a cross section of neonatalmouse calvariae incubated for 51 h in either Ctl or Acid medium. Calvariaewere transferred to similar fresh preincubated medium at 24 and 48 h.Counts/channel are counts/s of detected secondary ions uncorrected forspecies-dependent ionization probabilities. Observed spectra were measured in1,000 channels equally divided among mass 10-90 atomic mass units., u# D5 k" t- I* X B7 V& Z9 r
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Fig. 4. Ratio of HCO 3 - to the C 2 and ratio ofHCO 3 - to the CN on the surface and in the cross sectionof neonatal mouse calvariae incubated for 51 h in either Ctl or Acid medium( top ). Calvariae were transferred to similar, fresh preincubatedmedium at 24 and 48 h. Bottom : ratio of total phosphate(PO 2 PO 3 ) to C 2 and (PO 2 PO 3 ) to CN on the surface and in the cross section of neonatalmouse calvariae incubated for 51 h in either Ctl or Acid medium. Calvariaewere transferred to similar fresh preincubated medium at 24 and 48 h. Valuesare means ± SE. * P P P" A' g" k. L$ q2 ?$ _7 _8 S
, y: y6 u8 @0 U. E: {) I* [However, compared with incubation in Ctl medium, incubation in Acid mediumled to a decrease in the ratio of HCO 3 - to C 2 and a decrease in the ratio of HCO 3 - to CN in the crosssection of the calvariae ( Fig.3, cf. Ctl and Acid and Fig.4, top ). Thus incubation in Acid medium induces asignificant fall in mineral HCO 3 - in the cross section,but not on the surface, of calvariae.
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# g/ l2 x8 s3 e6 Q& |4 D- c: }7 vPhosphate. After 51 h of incubation in Ctl medium, the ratio oftotal phosphate (PO 2 PO 3 ) relative to C 2 and the ratio of (PO 2 PO 3 ) relative to CN on thesurface were not different from that found on the cross section of calvariaeincubated 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 Acidmedium did not alter the ratio of (PO 2 PO 3 ) toC 2 or the ratio of (PO 2 PO 3 ) to CN on the surface of calvariae ( Fig. 2,cf. Ctl and Acid and Fig. 3, bottom ).
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However, compared with incubation in Ctl medium, incubation in Acid mediumled to a decrease in the ratio of (PO 2 PO 3 ) toC 2 and a decrease in the ratio of (PO 2 PO 3 )to CN in the cross section of the calvariae( Fig. 3, cf. Ctl and Acid and Fig. 4, bottom ). Thusincubation in Acid medium induces a significant fall in mineral phosphate inthe cross section, but not on the surface, of calvariae.4 g+ \$ |1 s. f' B7 }
! ]2 O( u/ m7 }9 e0 v" BHCO 3 - in relation tophosphate. The amount of HCO 3 - relative to(PO 2 PO 3 ) on the surface of calvariae does not differ significantly from that on the cross section of calvariae incubated in Ctlmedium ( Fig. 5 ). Compared withincubation in Ctl medium, incubation in Acid medium did not alter the ratio ofHCO 3 - relative to (PO 2 PO 3 ) onthe surface of the calvariae. However, compared with incubation in Ctl medium,incubation in Acid medium led to a significant increase in the ratio ofHCO 3 - to (PO 2 PO 3 ) in the crosssection of the calvariae. Because incubation in Acid medium led to a reductionof both (PO 2 PO 3 ) and HCO 3 - inthe cross section of calvariae (Figs. 3 and 4 ), an increase in the ratio ofHCO 3 - to (PO 2 PO 3 ) on the crosssection of calvariae must indicate depletion of (PO 2 PO 3 ) in relation to HCO 3 -. Thus metabolicacidosis induces a fall in mineral (PO 2 PO 3 ) inrelation to mineral HCO 3 - in the cross section ofcalvariae.
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' B9 u( y% W7 hFig. 5. Ratio of HCO 3 - to (PO 2 PO 3 )on the surface and in the cross section of neonatal mouse calvariae incubatedfor 51 h in either Ctl or Acid medium. Calvariae were transferred to similarfresh preincubated medium at 24 and 48 h. Values are means ± SE. * P P P! i% x! _9 {: ]8 x
% P* t& l( y& |) \5 uDISCUSSION
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The purpose of the present study was to test the hypothesis that chronicmetabolic acidosis would decrease bulk (cross-sectional) bone phosphate to agreater extent than bone carbonate. Using a high-resolution scanning ionmicroprobe with secondary ion mass spectroscopy, we localized and compared thechanges in bone HCO 3 - and phosphate after chronicincubation of neonatal mouse calvariae in acidic medium. We found thatcompared with bones incubated in a neutral-pH medium, chronic acidosis induced a fall in both cross-sectional HCO 3 - and phosphate withno change in surface HCO 3 - and phosphate and that thefall in phosphate predominated over the fall in HCO 3 -.Consumption of these proton buffers, HCO 3 - andphosphate, would help to lessen the fall in pH during chronic acidosis.
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* F# m- z# U0 }# W! I7 }6 n1 mWe previously studied changes in bulk midcortical ion concentrations after7 days of in vivo metabolic acidosis induced by oral ammonium chloride( 15 ). We found that comparedwith mice drinking only distilled water, the ammonium chloride-inducedacidosis led to a loss of bone sodium and potassium, and, as also shown inthis study, a depletion of mineral HCO 3 - and phosphate. In the previous study, we questioned whether there were regional differencesin the response of mineral HCO 3 - and phosphate tochronic metabolic acidosis. The present study clearly demonstrates regionaldifferences in the response of bone to a model of chronic acidosis; theadditional protons deplete cross-sectional, but not surface, bone phosphateand carbonate.3 u3 D7 j+ b- P
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We have previously studied the effects of acute acidosis on surface andcross-sectional HCO 3 - and phosphate( 34 ). We found that comparedwith control, after a 3-h incubation in acidic medium there was a markeddecrease in surface HCO 3 - with respect to C 2 and CN with no change in cross-sectional HCO 3 -. Compared with control, after a 3-h incubation in acidic medium, there was also a markeddecrease in cross-sectional phosphate with respect to C 2 and alsoto CN with no change in surface phosphate. On the bone surface, there is afourfold depletion of HCO 3 - in relation to phosphate andin cross section a sevenfold depletion of phosphate in relation toHCO 3 -. These results indicate that acute H buffering by bone involves preferential dissolution of surfaceHCO 3 - and of cross-sectional phosphate.
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The present study extends the acute observations on bone surface andcross-sectional HCO 3 - and phosphate( 34 ) to a model of chronicacidosis. In the acute study, the surface HCO 3 - fell, whereas in the chronic study there was no change in surface HCO 3 -. However, in the acute study the control surfaceHCO 3 - was higher than the control surfaceHCO 3 - in this chronic study, suggesting that 51 h ofculture in control medium may have led to a depletion in surfaceHCO 3 -. Perhaps the surface HCO 3 - is buffering the acids generated through normal cellular metabolism. Indeed,the medium HCO 3 - fell by the end of the two 48-hincubations of the control bones ( Table1 ). 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 twostudies were comparable. In the acute study, there was no change incross-sectional HCO 3 -, whereas in this chronic study thecross-sectional HCO 3 - fell. This suggests that with timethe cross-sectional HCO 3 - is depleted by acidosis in theprocess of buffering the additional hydrogen ions. In the acute study, theacidic medium resulted in a marked fall in cross-sectional phosphate, as alsooccurred in this chronic study. Taken together, the results of the two studiessuggest that the surface of the control bone cultured for 3 h is rich inHCO 3 - and the cross section is rich in phosphate. Anacidic medium rapidly depletes the surface HCO 3 - andcross-sectional phosphate. With more prolonged incubation in acidic medium, the cross-sectional, but not the surface, phosphate andHCO 3 - are further depleted. The consumption of theseproton buffers during incubation in acidic medium helps to mitigate the fall in pH.
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That the acid-induced depletion of phosphate would predominate over thedepletion of HCO 3 - in a chronic study of neonatal mousebone is not unexpected. Using Raman vibrational microspectroscopy, Tarnowskiet al. ( 63 ) found that theratio of carbonate to phosphate in mouse calvariae 3 and 7 days postnatal is 0.12, indicating that there is far more phosphate thanHCO 3 - available as a potential proton buffer. Eachreleased PO 4 would accept a proton in the ratio of fourHPO 4 2 - to oneH 2 PO 4 - at pH 7.4. The lower the pH, thegreater the ratio of H 2 PO 4 - in relation toHPO 4 2 -. BoneCO 3 2 - would combine with H toform HCO 3 - and then with an additional H toform H 2 CO 3, which rapidly dissociates to H 2 Oand CO 2.5 L. h, t/ `* L- I2 f, D
0 P7 ^6 H; v& p7 GOther studies have shown that during in vitro( 7, 8, 25, 28 ) and in vivo( 51, 59, 62 ) metabolic acidosis, themineral 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 havepreviously 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 andpotassium from bone ( 22, 22, 28, 36, 37 ), and a loss of mineralcarbonate ( 26, 27 ). The sodium and potassiumexchange 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 chronicmetabolic acidosis ( 11, 22, 30, 48 ) would result in therelease of mineral phosphate. We and others have shown a loss of bonecarbonate during metabolic acidosis( 4, 30 ), and we have shown that invivo metabolic acidosis causes a reduction in mineralHCO 3 - and phosphate( 15 ).
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+ s: a. e7 }0 {3 x2 b& }Clinical observations in patients with renal tubular acidosis support thehypothesis that acidosis has deleterious effects on bone mineral. Metabolicacidosis has been shown to have a significant effect on bone density,formation, and growth ( 38, 39, 55, 56 ). Domrongkitchaiporn andco-workers ( 38 ) compared 14adult patients with distal renal tubular acidosis who had never receivedHCO 3 - therapy with 28 well-matched controls. Theymeasured bone mineral density and also performed bone biopsies withhistomorphometric analysis( 38 ). They found that patientswith distal renal tubular acidosis had a lower bone mineral density in mostareas compared with normal controls. The patients also had a decreased boneformation rate. After treating 12 renal tubular acidosis patients withKHCO 3 for 1 yr, they found that bone mineral density significantlyimproved in the trochanter of the femur and the total femur( 39 ). The bone formation ratenormalized with treatment. Initially, the levels of serum parathyroid hormonewere suppressed; they too improved with KHCO 3 therapy( 38, 39 ). McSherry and Morris ( 55, 56 ) studied the effect ofrenal tubular acidosis on growth in 10 children. Six were found to be stunted(height SD), two were too young to determine whether they were short ( alkalitherapy, each patient attained and maintained normal stature, mean heightincreased from the 1.4 to the 37th percentile, and the rate of growthincreased two- to threefold.2 F( n: K/ o) q/ H1 y
# y: e. i1 v. m" i- U& [: A+ GThe results of the present study are consistent with those of previousinvestigators. In a classic study, Swann and Pitts ( 62 ) infused fixed amounts ofacid into dogs and demonstrated that 60% of the additional protons appearto 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 severaldays of metabolic acidosis led to a loss of bone carbonate. Burnell( 6 ) also demonstrated a loss ofbone carbonate after metabolic acidosis, and Bettice( 4, 5 ) showed that the metabolicacidosis-induced loss of bone carbonate correlated with the fall inextracellular HCO 3 -. Using cultured neonatal mousecalvariae, we demonstrated that mineral calcium and carbonate, in the form ofcarbonated apatite, are in equilibrium with the culture medium( 27 ). We have shown thatacidosis induces the release of calcium and carbonate from bone( 27 ), leading to a progressiveloss of bone carbonate during metabolic, but not respiratory, acidosis( 26 ).
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In the present study, we used a high-resolution scanning ion microprobewith secondary ion mass spectroscopy to localize the changes in boneHCO 3 - and phosphate in response to a model of chronicmetabolic acidosis. We found that chronic acidosis induced a fall in bothcross-sectional HCO 3 - and phosphate with no change insurface HCO 3 - and phosphate and that the fall inphosphate predominated over the fall in HCO 3 -.Consumption of these proton buffers, HCO 3 - andphosphate, would help to lessen the fall in pH during chronic acidosis at theexpense of the bone mineral content.
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7 v. y2 v; f% `6 E7 `DISCLOSURES
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/ a2 L4 m) K1 `3 CThis work was supported in part by National Institutes of Health GrantsAR-46289 and DK-56788.3 i' Z- t, v0 `3 s( z$ Q
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发表于 2009-4-21 13:42
