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The physical state of bone carbonate. A comparative infra-red study in several mineralized tissues.

The physical state of bone carbonate. A comparative infra-red study in several mineralized tissues. JOHN D. BAXTER* Department of Medicine, ROBERT M. BILTZ* * University of Kentucky, Lexington, Kentucky EDMUND D. PELLEGRINOt THE PHYSICAL STATE OF BONE CARBONATE: A COMPARATIVE INFRA-RED STUDY IN SEVERAL MINERALIZED TISSUES A precise understanding of the crystal properties of bone and other calci- and physiological mineraliza- fications is essential in defining pathological tion. Bone mineral is predominately calcium and orthophosphate, but contains about 6 per cent carbonate. The physical state of carbonate in but has attracted considerable interest since bone has been controversial carbonate probably influences bone solubility and crystallinity and provides a reservoir of in metabolic and respiratory diseases. CO2 In 1953, Neuman and Neuman1 proposed a unifying concept of the salt for which the chemical and physical properties of nature of the bone "impure," microcrystalline-OH-apatite provided the working model. In contradistinction to those who suggested that CO2 was an essential part of the bone crystal complex" Neuman regarded CO2 as an impurity. magnesium, sodium, citrate, and other The view that CO2 together with in small quantities are contaminants absorbed or trapped elements present in the OH-apatite crystal surfaces persists as the predominant working hypothesis. Recent investigations in our laboratory indicate that bone carbonate that are physicochemically and physiologically may exist in two forms in uremia and distinct. One form may be lost completely long-standing acidosis. The other form is intimately related to the crystal, possibly as and lost even in the most severe uremia. These a double salt, is not that is somewhat different from the con- observations provide a concept with to the ventional OH-apatite model, and its implications respect state of CO3.' Direct evidence for the physical state of C03 in bone and some other The of carbonate mineralized tissues is wanting. poor crystallinity many * Cancer Teaching Grant T2- Student summer fellow, N.I.H. Undergraduate CA5096. **Research Assistant. of Medicine. Professor and t Chairman, Department Received for publication 2 September 1965. 456 Carbonate in bone BAXTER, BILTZ, PELLEGRINO causes difficulty in interpreting X-ray diffraction and other crystal- salts7'8 lographic data. Consequently, other methods, particularly infra-red spectro- scopy have been used to evaluate the location and orientation of CO3 in mineralized tissues."0 Infra-red spectroscopy has a distinct advantage in the study of these crystalline materials since absorption is dependent upon the symmetry of individual groups, rather than on a number of repeating lattice units."0 infra-red data have been conflicting. Pobeguin' " concluded However, the that carbonate in bone and other mineralized tissues was in an amorphous state by comparing infra-red data with X-ray diffraction and polarizing data which showed no crystalline calcium carbonate. Posner microscopic that the presence of a carbonate peak of absorp- and Duyckaerts concluded tion (mode) near 876 cm-' in bone indicated that the carbonate in the latter was indistinguishable from that in calcite.* Elliotte0 could find no evidence amount of calcite in frankolite, a naturally occurring for an appreciable carbonate containing apatite, or enamel because they showed double carbon- cm-' whereas a single peak is seen in oxygen stretching modes near 1,430 basis of polarized infra-red spectroscopy, he further con- calcite. On the no evidence of an amorphous calcium-carbonate cluded'0 that there was frankolite. Emerson and Fisher'0 concluded in elephant enamel or phase that the carbonate ion in the calcified tissues is present in two non-equivalent basis of double v2 carbon-oxygen out-of-plane deformations sites on the in dentin, and enamel. We have shown that the two near 876 cm-' bone, sites Emerson and Fisher"0 suggested are not the same carbonate that sites that can be demonstrated by elution methods.6 two carbonate vibration which is present in some mineral- The V4 (712 cm-') ized tissues and not in others has received little attention or analysis as an indicator of the carbonate state. Herman and Dallemagne'7 stated was Emerson and Fisher' reported that they that its absence unexplained. mineralized this band in the tissues had never seen the spectra of a number of mineralized The present study compares and that vary widely in their carbonate composition tissues compounds on the and absence of the V4 carbonate with emphasis presence special It is as shown here in the spectra of clam shell absorption peak. present some of the lobster shell, but is absent in other calcified tissues, and parts and salts containing ap- C03-apatite, synthetic CO3 naturally occurring of preciable amounts phosphate. * of CaC03 and arag- Calcite refers to the hexagonal-R-scalenohedral configuration refers to the orthorhombic of CaCO3.2' onite dipyramidal configuration 457 YALE JOURNAL OF BIOLOGY AND MEDICINE Volume 1966 38, April, MATERIALS AND METHODS The diversified collection of specimens used in this study were selected as repre- sentative of the wide range of carbonate-containing minerals CaCOs and/or carbonate- apatites found in biological calcifications. These included fresh water clam containing salt clam shell (Arca), crab shell (Callinectes sapidus), and shell (Anodonta), water from the principal layer of the tail and lobster shell (Homarus americanus) samples human material of whole bone, a renal calculus "matrix carapace. The consisted stone" and a fragment of tissue from metastatic calcification of muscle. Fossil bone (mammoth) from Irvington, California was also included. 10.0 11.0 WAVELENGTH ;MICRONS) between 625 and cm' of clam shell and FIG. 1. Infra-red absorption spectra 2,000 lobster shell (tail). for sodium dahllite The infra-red absorption spectra calcite, aragonite, bicarbonate, from and two (a naturally occurring carbonate-containing apatite thermopolis shale"), were studied for synthetic calcium-phosphate-carbonate compounds comparative pur- poses. content and The were made such that the carbonate Ca/P synthetic preparations ratios in the of that of human bone (see Table 4). Precipi- molar were general range 1 100 of acacia calcium salt of an acidic tate No. was prepared by adding mg. (a which used as calcium source and as a gum from Acacia senegal was a nucleating to 10 ml. solution 25 millimoles of as agent) a containing carbonate (added KHCO3) 1 2 and millimole of phosphate (added as HPO,P). Precipitate No. was prepared to Trautz and Zapanta' by adding 75 ml. Ca acetate (0.1 M) to a mixture according of 100 ml. H20 250 ml. (0.1 M) + 50 ml. Na2PO4 (0.1 M). + NaHCO3 KBr tech- Samples were studied by infra-red spectroscopy employing the pellet nique." TI The biological materials and dahllite were untreated prior to grinding and 458 Carbonate in bone | BAXTER, BILTZ, PELLEGRINO the pellet preparation. They were ground with a dental grinder when necessary and then in a mortar and pestle. Perkin-Elmer model 237 and model 21 infra-red spectrophotometers were used with a of 25 microns in the model 237 and with a variable slit width with slit width 15 microns was usually examined. the model 21. The spectrum from 2.5 to RESULTS The infra-red (IR) absorption frequencies for the carbonate group' and each of the type of carbonate vibration causing peak absorption' (mode) 900 CM., 11.0 12.0 13.0 14.0 15.0 10.0 (MIC*ONS) WAVELENGTH FIG. 2. cm-' of crab shell Infra-red absorption spectra between 625 and 2,000 (carapace) and lobster shell (carapace). are given for calcite,= aragonite,' NaHCO8, and fresh and salt water clam shell in Table 1. The IR spectrum of clam shell, which is relatively is identical to that of aragonite (Table 1). Small amounts pure CaCO3, of calcite can be identified by the presence of calcite vibrations in parts of the lobster shell (tail) (Fig. 1) but neither calcite nor aragonite is identi- in other of lobster shell or in crab fiable parts the (carapace), carapace of the of 712 cm-' and cm-' by virtue absence peaks at 1,795 (Fig. 2). summarized in Table 2. These absorption peaks are calcium- in the of whole bone and the Similarly, spectra synthetic 3), fossil bone and dahllite (Fig. 4), phosphate-carbonate compound (Fig. "matrix stone" modes and the metastatic calcification and (Fig. 5), -459 YrALE JOURNAL OF Volume 38, BIOLOGY AND 1966 April, MEDICINE I.4 %O %O C14 C4 cn cn C%4 P-4 P-4 114 C4 cn CA cn r. cis0 z 0 CI C) C> cis :D I: 5. E ..q> .." CD 0 N N C) C114 N V-4 " <D C> I", .o 00 Ln in NO 6n 00 00 cl \0 O., \0 0\ r Cd t,-. in -O- \0 z 00 00 00 cn bO 4i v) > 0. cu Cd C) 00 tl% \0 m Cq M CI Co (=> 8\\ cn 0 0 0 co bo Ul r. Q cn r tN, tz \6 (U Ei cn V" W-4 O "14 V" 0 P4 44 W-4 "-O ot v) CIS cn m 0 fT-4z cn V) Ca Ei v CN ai 1.0 cn 4-&W 10 0 8 bO rn cis > cis 0 (L) Cd co cd 0 O C.) (u .- ci 4-& Cd (u 4a bo ...4 W (A . 14-4 4i cn Cd W cd Cd 0 = Cd -" CIS > %" u U 40 Carbonate in bone BAXTER, BILTZ, PELLEGRINO 3. Infra-red absorption spectra between 625 and 2,000 cm1 of whole bone FIG. and the synthetic calcium-carbonate-phosphate precipitates. CM1 700 2000 1600 1400 1200 900 800 .. .. , ,- u.v 0.0 ~~~~~~~~~~~~~FOSSIL B3ONE (Irvinq10n) >i. u,.20 cc.30 co <.40 i I ' I"" .70 ', 1 ''r , .~~~~~~~~~~~~~~~~~~~~~~~~'. 1.0 1.0 | dn--I . -iC- 14.0 15.0 16.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 WAVfIENGTH IMICEONSI FIG. 4. Infra-red absorption spectra between 625 and 2,000 cm14 of fossil bone (Irvington) and dahllite. 41 YALE JOURNAL OF BIOLOGY AND MEDICINE Volutize 1966 38, April, attributable to carbonate are seen only near 1,430 and 875 cm-' and no near 710 or 1,790 cm-- are seen (see also Table 3). In these speci- modes strong absorption from ortho- mens and in the crab and lobster shell, phosphate is seen from 900 to 1,200 cm-" with the largest peak of absorp- near tion 1,031 cm-1. near 1,400-1,450 cm-' appear as two distinguishable The absorptions v3 differences in the resolution, peaks in all samples in Figures 2-5. However, broadening and peak intensities of these and the peaks near 870 cm-' (v2) are seen. 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 WAVELENGTH ;MICRONS) between 625 and 2,000 cm1 of the metastatic FIG. 5. Infra-red absorption spectra calcification and the renal calculus studied. of the carbonate content and of the relative con- Chemical analyses centrations of calcium and are shown in Table 4. phosphate small amounts of Since only those salts containing relatively phosphate, and lobster shell i.e. clam shell which is relatively pure CaCO3 (tail) exhibit and which has molar ratio of as high as 23.40, the V4 a Ca/P the correlation between increasing phosphate concentra- the + V4 modes, vj of these latter carbonate modes can be seen. tion and the disappearance V4 vi modes occurs in the we have Absence of the and the + V4 specimens as with molar ratios as 11.20 (lobster carapace) examined Ca/P high listed in Table 4 with ratios lower than and in all the specimens Ca/P that. 42 Carbonate in bone BAXTER, BILTZ, I PELLEGRINO TABLE 2. SUMMARY OF CARBONATE ABSORPTION PEAKS IN CRAB SHELL AND LOBSTER SHELL Assignment of + 1e2 4 VI the mode Wavelength cm1 (microns) cm-"(microns) cm-'(microns) cm:`(microns) Crab (carapace) Absent 1475(6.78) 870(11.50) Absent 1419(7.05) 862(11.60) Lobster shell (tail) 1795(5.57) 1400-1450 870(11.50) .712(14.05) Poorly (6.9-7.15) (Much weaker resolved Poorly than or '2 Ps) resolved Lobster (carapace) Absent 1456(6.87) 862(11.60) Absent 1400(7.15) TABLE 3. SUMMARY OF ABSORPTION PEAKS ATTRIuTED TO CARBONATE BETWEEN 625 AND 4,000 CM'1 IN HUMAN BONE, FossIL BONE, DAHLLITE, RENAL CALCULUS, METASTATIC CALCIFICATION, AND SYNTHETIC CALCIUM- PHOSPHATE-CARBONATE COMPOUNDS STUDIED Assignment of the mode Ps cm1 cm'1 (microns) Frequency (microns) (wavelength) 1450 865-877( (11.4-11.6) Bone (6.9) 1409 (7.1) Poorly resolved Fossil bone 1459 (6.85) 872 (11.45) 1429 (7.05) 866 (11.55) (mammoth) Dahllite 1449 862 (11.60) (6.9) 1409 (7.1) Synthetic compound No. 1471 (6.8) 877 (11.4) (7.05) 869 (11.5) Poorly resolved No. 2 1492 (6.70) 866-877 (11.40-11.55) 1470 Poorly resolved (6.80) (6.87) (7.00) (7.05) Renal calculus 1466 (6.82) (11.4) "matrix stone" 1412 (7.08) 870 (11.5) Poorly resolved Metastatic 1445 (6.92) 876 (11.42) calcification 1412 (7.08) 870 (11.50) Poorly resolved 463 Volunie YALE JOURNAL OF BIOLOGY AND MEDICINE 1966 38, April, near Additional vibrations 1,540 cm-' and 1,660 cm-" are seen in most of the tissue specimens and these are attributed to matrix usually organic as can be diminished when the matrix is removed vibrationsl' they by or extraction confirmed ignition at 600° C. by by ethylenediaminei° (also in our laboratory). Vibrations due to water can also be seen near 1,660 cm.-"2' Some observers have noted carbonate near absorption 1,538 cm-110, THE AND THE TABLE 4. CHEMICAL ANALYSIS OF CARBONATE CONITENT OF RELATIVE MOLAR CONCENTRATIONS OF CALCIUM AND PHOSPHATE IN THE SPECIMENS STUDIED Specimen (mg/100 mg) Ca/P molar ratio Cog Clam shell 58.2 > 100.00 Lobster shell (tail) 40.0 23.40 Lobster 31.8 (carapace) 11.20 34.5 Crab (carapace) 9.50 Whole bone (human) 5.8 1.76 Fossil bone (mammoth) 11.5 2.32 Dahllite 7.2 1.76 Synthetic compound 1 9.0 1.83 No. 2 3.1 No. 1.67 Metastatic calcification 5.7 1.67 Renal calculus 2.0 0.53 "Matrix stone" DISCUSSION AND CONCLUSIONS The carbonate group has four normal modes of vibration's (see Table 1). The vibration is ordinarily infra-red but inactive, appears when carbonate v, is lowered as in symmetry aragonite' (Fig. 1). This mode is difficult to in the salts due evaluate phosphate-containing to phosphate absorption from 1,100 900 to cm-'.'2 The v2 absorption is due to carbon-oxygen out-of-plane bending vibra- tions' and is seen in all the specimens. The and vibrations are due to v3 V4 carbon-oxygen asymmetrical stretch- vibrations ing and in-plane bending respectively and are both doubly Both when degenerate.' are seen as single carbonate exists in peaks higher environments as in the free ion in solution or symmetry as in (D3h) These modes become into two calcite (D3).'° split peaks when carbonate is reduced as in or when symmetry aragonite carbonate oxvgens (CQ)' 464 Carbonate in bone BAXTER, PELLEGRINO BILTIZ, are covalently or hydrogen bonded and C2) ."' a The splitting of V3 (C. in aragonite is slight and is not clearly demonstrated without better resolu- tion. The location of the peaks may also vary with the cation precipitated with carbonate.' The V4 carbonate mode, however, is not seen in many carbonate com- pounds, i.e., in whole bone, fossil bone, dahllite, crab shell, some parts of the lobster shell, the synthetic compounds described, in metastatic calci- fication and renal calculus. All of these compounds contain, in addition to of phosphate. In addition v3 absorption carbonate, a significant amount in appears as two peaks these tissues. The splitting of V3 and the disappearance of v4 and v4 + vi appears to be related to the co-precipitation of carbonate with phosphate (or vice whether it occurs in the exoskeleton of the lobster and crab, in the versa) in The data seem to indicate that where apatites or synthetic compounds. as in the lobster shell shown in Figure 1, the v4 less phosphate is present the and v4 + peaks are seen which can be interpreted as indicating vi presence of a separate CaCO3 phase, most likely calcite. These data are that most of the carbonate in the specimens, compatible with the postulation in 2-5, is present in a state stabilized whose spectra are shown Figures unlike that in calcite or by the presence of phosphate, aragonite. is absent of absorption It is unlikely that V4 absorption because strong in 700 cm-' region from other species, since the peak distinctly appears the of calcite are added to the specimensl4.. '7 (confirmed when small amounts with than 1 cent calcite). Additional confirma- in our laboratory less per the combination band + V4) appears tion from the present data is that (vl not in the other when V4 is seen (Fig. 1) but is seen samples. might be concluded that V4 was IR inactive. This might occur if It had than and that the planar con- carbonate a higher symmetry D3h implies was with formation for a figuration of carbonate upset of, example, or an octahedron with coordinated This terahedron (Oh) oxygens. (Td) since the and near Furthermore, V2 V3 absorptions appear seems unlikely. of carbonate 1), it is more likely that the fundamental frequencies (Table as As even when one or the carbonate is present planar C03. mentioned, are bound in covalent and bonds as in more carbonate hydrogen oxygens remains active rise to two unidentate and bidentate ligands, v4 IR giving is seen near cm-'.'- We therefore feel that least one of which 700 peaks, at even distinct are not seen. V4 iS IR active at some frequency though peaks that carbonate are in these materials It seems possible the groups present is or of the in- in a way that there broadening decreasing V4 peak such the chemical nature of the carbonate tensities without changing seriously is seen near 700 cm-' in lobster A broadened area of group. absorption 46S YALE JOURNAL OF Volume 1966 BIOLOGY AND MEDICINE April, and crab shell (Fig. 2), but this may represent water of hydration.' Although v4 strong in calcite, the absorption becomes much weaker when iS there is splitting as in aragonite (Fig. 1). If there was significant broaden- ing or weakening of the peak intensities in bone, for example, the area under the too be absorption peak might be small to visualized. However, for there is no proof this and alternate possibilities have not been excluded, e.g., that the peak has shifted to a frequency where it is not seen. Nor is it known that the same explanation holds in all the tissues. Broadening of the carbonate absorption peaks has been noted in poorly crystalline synthetic calcium-carbonate-phosphate precipitates (Racquel Zapanta-LeGeros, personal communication) and seems to be more prominent in in the more amorphous materials. It is present the crab and lobster shell specimens shown in Figure 2. Even though the carbonate absorption peaks are in some cases broad- v3 ened, two peaks can be distinguished (Figs. 2-5) in the phosphate-contain- ing mineralized tissues reported here. These are more poorly resolved in whole human bone, lobster and crab, but are seen distinctly in some of the other samples, particularly in fossil bone (Fig. 4). This has been interpreted as indicative of of the is of splitting doubly degenerate absorptions."' It vs greater magnitude than seen in aragonite and therefore may indicate a reduced carbonate symmetry from D3h of the free ion or from D3 of calcite to as low as but the extent to which this splitting represents a C., perturbation is unknown. Herman and have attributed one of the carbonate Dallemagne67 absorp- v3 to tion peaks in bone calcite and other carbonate peaks to carbonate bound for to the phosphate. These data question that the same interpretation the splitting of the v3 carbonate absorption applies to all the specimens or that the double do of the peaks represent splitting doubly degenerate absorption. are must mean that there is some However, that two peaks distinguished the orientation of the force field on carbonate and that physical state of This carbonate in bone and these tissues is not completely amorphous. conclusion can be made whether two distinct the splitting represents phases of or two carbonate sites. There- carbonate, split doubly degenerate modes, of lines in the diffraction of these materials fore, lack CaCO3 X-ray patterns should not be taken as evidence for a separate completely amorphous in the shown the all exhibit CaCO3 phase. Although spectra present study splitting this is not always observed. In these cases it is possible that the v3 on carbonate is less oriented. force field Fisher'0 raised the of two Emerson and and Elliott'8 question carbonate of near 875 cm-" and on the sites in enamel on the basis two basis peaks and Elliott of at cm-1. also noted three peaks 1,542 cm-', 1,454 cm-', 1,410 4" Carbonate in bone BAXTER, BILTZ, PELLEGRINO that the absorption bands differed in some of the specimens he studied and concluded that the carbonate ions must enter the structure in several ways. Examination of Tables 2 and 3 and Figures 2-5 shows that although all these samples exhibit splitting of the V3 and absence of the V4 peaks some differences are present. The resolution, broadening, and the fre- quencies of the V3 peaks differ slightly. The V2 peaks are seen as either distinct doublets (fossil bone), poorly resolved doublets as in the renal single peaks as in dahllite, or as more poorly calculus or crab shell, peaks. These data suggest some differences in the physical state resolved of carbonate in these materials. The differences are seen not only when the exoskeleton of lobster or crab is compared to bone, but slight differences bone, whole human bone, the human pathological calcification and in fossil which the the synthetic compounds are also seen. The extent to degree carbonate groups, the of hydration or crystallinity, the location of the presence of impurities or other factors are responsible for these differences these data. It should be emphasized, however, cannot be determined from in carbonate or as are seen in that large differences bonding symmetry in other metallic carbonates' hydrogen bonded and chelated carbonates9" much greater and in bicarbonate (see Table 1), are often accompanied by differences than are seen above. The discussion has not con- absorption some have noted near 1,538 cm-'."0"7"1 sidered the carbonate absorption contaminated with atmospheric CO2 shows V2, V3, Reagent Ca (OH)2 carbonate These in and sometimes V4, and + V4 absorption peaks.' peaks vj attributed reagent Ca (OH)2 and in ashed bone were previously incorrectly to Ca(OH) 2. The better resolution of the V3 peaks near 1,400-1,450 cm-' bone indicates a more oriented force field on the in heated probably in the carbonate latter samples. SUMMARY A of the state of carbonate in bone and precise understanding physical in the influence of carbonate other mineralizations is essential understanding of the mineralized tissues as well as the on the solubility and crystallinity a carbon dioxide reservoir in metabolic disease. role of bone carbonte as state of carbonate in several mineralized tissues, minerals The physical and calcium-carbonate-phosphate compounds was studied by synthetic carbonate infra-red between 625 cm-' and examining their absorption cm-' 4,000 (2.5-15 microns). attributed to carbonate were distinguishable Double absorption peaks in from human cm-' some bone, dahllite, pathol- near 1,400-1,450 samples and of the lobster and crab shell and calcifications, parts suggest ogical 467 YALE JOURNAL OF BIOLOGY AND MEDICINE Volume 38, April, 1966 the presence of carbonate in these materials that is not in a completely state. amorphous Double absorption peaks near 1,400 cm-' may in some of the tissues vs indicate a splitting of this doubly degenerate mode and a reduced symmetry from D3h of the free ion or from of calcite for carbonate; however, D8 have not been clearly shown. causes for this splitting near in pure tissues and Absorption 712 cm-1 (V4) was seen CaCO3 in calcium-carbonate-phosphate compounds containing small amounts of phosphate but could not be seen in bone and in the mineralized tissues with These data are compatible and compounds a higher phosphate content. in tissues in a state with the view that the carbonate is present the latter stabilized by the phosphate unlike that in calcite or aragonite. It is concluded that V4 is infra-red active even though distinct peaks are not seen. Possible explanations are discussed. in broadening, and in the frequencies of Some differences the resolution, cm-' and near 870 cm-, were noted the absorption peaks near 1,400-1,450 was to bone and when when the exoskeleton of lobster and crab compared was compared to fossil bone, pathological calcifications, synthetic the latter dahllite which differences in the physical state of compounds and suggest the nature of which cannot be determined from these data. carbonate, ACKNOWLEDGMENTS Yale Dr. R. C. The authors wish to thank Dr. David Seligson, University, Gore, Perkin-Elmer Corp., Drs. R. Zapanta-LeGeros and John LeGeros, New York Univer- for valuable suggestions in the preparation of the manuscript. We are also sity, their of for the fossil bone indebted to Dr. H. C. Ezra, University California, providing of for her specimens and to Miss Peggy J. Rodgers, University Kentucky, very excellent technical assistance. Ferraro the National for Thanks are due to Dr. J. R. of Argonne Laboratory and for helpful suggestions and to Dr. R. C. Lord of the reading the manuscript Institute of for advice. Massachusetts Technology helpful REFERENCIES 1. Neuman, W. F. and Neuman, M. W.: The nature of the mineral phase of bone. Chem. Rev., 1953, 53, 1-45. 2. Roseberry, H. H., Hastings, A. B., and Morse, J. K.: X-ray analysis of bone and teeth. biol. Chem., 1931, 90, 395-407. J. 3. Bogert, L. J. and Hastings, A. B.: The calcium salts of bone. J. biol. Chem., 1931, 94, 473-481. 4. McConnell, Duncan: A structural investigation of isomorphism of the apatite group. Amer. Mineral., 1938, 23, 1-19. 5. Pellegrino, E. D. and Biltz, R. M.: The composition of human bone in uremia. Observations on the reservoir functions of bone and demonstration of a labile fraction of bone carbonate. Medicine 1965, 44, 397-418. 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B.: Spectra-structure correlations in the infra-red region. J. opt. Soc. Amer., 1950, 40, 397-400. 29. Barnes, R. B., Gore, R. C., Stafford, R. W., and Williams, V. Z.: Qualitative organic analysis and infrared spectrometry. Analyt. Chem., 1948, 20, 402-410. The infrared 30. Gatehouse, B. M., Livingston, S. E., and Nyholm, R. S.: spectra of some simple and complex carbonates. J. chem. Soc., 1958, V., 3137-3142. 31. Kazuo, Fujita, Junnosuke, Tanaka, Shizuo, and Kobayashi, Masahisa: Nakamoto, Infrared spectra of metallic complexes. IV. Comparison of the infrared spectra of unidentate and bidentate metallic complexes. J. Amer. chem. Soc., 1957, 79, 4904-4908. 32. Miller, F. A. and Wilkins, C. H.: Infrared spectra and characteristic frequencies of inorganic ions. Analyt. Chem., 1952, 24, 1253-1294. Articles are accepted for publication in The Yale Journal of Biology and Medicine on the basis of merit and suitability. The privilege of submitting articles is not limited to those associated with Yale University and contributions from others are cordially invited. The Board of Editors http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Yale Journal of Biology and Medicine Pubmed Central

The physical state of bone carbonate. A comparative infra-red study in several mineralized tissues.

Baxter, J. D.; Biltz, R. M.; Pellegrino, E. D.
The Yale Journal of Biology and Medicine , Volume 38 (5) – Apr 1, 1966
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Abstract

JOHN D. BAXTER* Department of Medicine, ROBERT M. BILTZ* * University of Kentucky, Lexington, Kentucky EDMUND D. PELLEGRINOt THE PHYSICAL STATE OF BONE CARBONATE: A COMPARATIVE INFRA-RED STUDY IN SEVERAL MINERALIZED TISSUES A precise understanding of the crystal properties of bone and other calci- and physiological mineraliza- fications is essential in defining pathological tion. Bone mineral is predominately calcium and orthophosphate, but contains about 6 per cent carbonate. The physical state of carbonate in but has attracted considerable interest since bone has been controversial carbonate probably influences bone solubility and crystallinity and provides a reservoir of in metabolic and respiratory diseases. CO2 In 1953, Neuman and Neuman1 proposed a unifying concept of the salt for which the chemical and physical properties of nature of the bone "impure," microcrystalline-OH-apatite provided the working model. In contradistinction to those who suggested that CO2 was an essential part of the bone crystal complex" Neuman regarded CO2 as an impurity. magnesium, sodium, citrate, and other The view that CO2 together with in small quantities are contaminants absorbed or trapped elements present in the OH-apatite crystal surfaces persists as the predominant working hypothesis. Recent investigations in our laboratory indicate that bone carbonate that are physicochemically and physiologically may exist in two forms in uremia and distinct. One form may be lost completely long-standing acidosis. The other form is intimately related to the crystal, possibly as and lost even in the most severe uremia. These a double salt, is not that is somewhat different from the con- observations provide a concept with to the ventional OH-apatite model, and its implications respect state of CO3.' Direct evidence for the physical state of C03 in bone and some other The of carbonate mineralized tissues is wanting. poor crystallinity many * Cancer Teaching Grant T2- Student summer fellow, N.I.H. Undergraduate CA5096. **Research Assistant. of Medicine. Professor and t Chairman, Department Received for publication 2 September 1965. 456 Carbonate in bone BAXTER, BILTZ, PELLEGRINO causes difficulty in interpreting X-ray diffraction and other crystal- salts7'8 lographic data. Consequently, other methods, particularly infra-red spectro- scopy have been used to evaluate the location and orientation of CO3 in mineralized tissues."0 Infra-red spectroscopy has a distinct advantage in the study of these crystalline materials since absorption is dependent upon the symmetry of individual groups, rather than on a number of repeating lattice units."0 infra-red data have been conflicting. Pobeguin' " concluded However, the that carbonate in bone and other mineralized tissues was in an amorphous state by comparing infra-red data with X-ray diffraction and polarizing data which showed no crystalline calcium carbonate. Posner microscopic that the presence of a carbonate peak of absorp- and Duyckaerts concluded tion (mode) near 876 cm-' in bone indicated that the carbonate in the latter was indistinguishable from that in calcite.* Elliotte0 could find no evidence amount of calcite in frankolite, a naturally occurring for an appreciable carbonate containing apatite, or enamel because they showed double carbon- cm-' whereas a single peak is seen in oxygen stretching modes near 1,430 basis of polarized infra-red spectroscopy, he further con- calcite. On the no evidence of an amorphous calcium-carbonate cluded'0 that there was frankolite. Emerson and Fisher'0 concluded in elephant enamel or phase that the carbonate ion in the calcified tissues is present in two non-equivalent basis of double v2 carbon-oxygen out-of-plane deformations sites on the in dentin, and enamel. We have shown that the two near 876 cm-' bone, sites Emerson and Fisher"0 suggested are not the same carbonate that sites that can be demonstrated by elution methods.6 two carbonate vibration which is present in some mineral- The V4 (712 cm-') ized tissues and not in others has received little attention or analysis as an indicator of the carbonate state. Herman and Dallemagne'7 stated was Emerson and Fisher' reported that they that its absence unexplained. mineralized this band in the tissues had never seen the spectra of a number of mineralized The present study compares and that vary widely in their carbonate composition tissues compounds on the and absence of the V4 carbonate with emphasis presence special It is as shown here in the spectra of clam shell absorption peak. present some of the lobster shell, but is absent in other calcified tissues, and parts and salts containing ap- C03-apatite, synthetic CO3 naturally occurring of preciable amounts phosphate. * of CaC03 and arag- Calcite refers to the hexagonal-R-scalenohedral configuration refers to the orthorhombic of CaCO3.2' onite dipyramidal configuration 457 YALE JOURNAL OF BIOLOGY AND MEDICINE Volume 1966 38, April, MATERIALS AND METHODS The diversified collection of specimens used in this study were selected as repre- sentative of the wide range of carbonate-containing minerals CaCOs and/or carbonate- apatites found in biological calcifications. These included fresh water clam containing salt clam shell (Arca), crab shell (Callinectes sapidus), and shell (Anodonta), water from the principal layer of the tail and lobster shell (Homarus americanus) samples human material of whole bone, a renal calculus "matrix carapace. The consisted stone" and a fragment of tissue from metastatic calcification of muscle. Fossil bone (mammoth) from Irvington, California was also included. 10.0 11.0 WAVELENGTH ;MICRONS) between 625 and cm' of clam shell and FIG. 1. Infra-red absorption spectra 2,000 lobster shell (tail). for sodium dahllite The infra-red absorption spectra calcite, aragonite, bicarbonate, from and two (a naturally occurring carbonate-containing apatite thermopolis shale"), were studied for synthetic calcium-phosphate-carbonate compounds comparative pur- poses. content and The were made such that the carbonate Ca/P synthetic preparations ratios in the of that of human bone (see Table 4). Precipi- molar were general range 1 100 of acacia calcium salt of an acidic tate No. was prepared by adding mg. (a which used as calcium source and as a gum from Acacia senegal was a nucleating to 10 ml. solution 25 millimoles of as agent) a containing carbonate (added KHCO3) 1 2 and millimole of phosphate (added as HPO,P). Precipitate No. was prepared to Trautz and Zapanta' by adding 75 ml. Ca acetate (0.1 M) to a mixture according of 100 ml. H20 250 ml. (0.1 M) + 50 ml. Na2PO4 (0.1 M). + NaHCO3 KBr tech- Samples were studied by infra-red spectroscopy employing the pellet nique." TI The biological materials and dahllite were untreated prior to grinding and 458 Carbonate in bone | BAXTER, BILTZ, PELLEGRINO the pellet preparation. They were ground with a dental grinder when necessary and then in a mortar and pestle. Perkin-Elmer model 237 and model 21 infra-red spectrophotometers were used with a of 25 microns in the model 237 and with a variable slit width with slit width 15 microns was usually examined. the model 21. The spectrum from 2.5 to RESULTS The infra-red (IR) absorption frequencies for the carbonate group' and each of the type of carbonate vibration causing peak absorption' (mode) 900 CM., 11.0 12.0 13.0 14.0 15.0 10.0 (MIC*ONS) WAVELENGTH FIG. 2. cm-' of crab shell Infra-red absorption spectra between 625 and 2,000 (carapace) and lobster shell (carapace). are given for calcite,= aragonite,' NaHCO8, and fresh and salt water clam shell in Table 1. The IR spectrum of clam shell, which is relatively is identical to that of aragonite (Table 1). Small amounts pure CaCO3, of calcite can be identified by the presence of calcite vibrations in parts of the lobster shell (tail) (Fig. 1) but neither calcite nor aragonite is identi- in other of lobster shell or in crab fiable parts the (carapace), carapace of the of 712 cm-' and cm-' by virtue absence peaks at 1,795 (Fig. 2). summarized in Table 2. These absorption peaks are calcium- in the of whole bone and the Similarly, spectra synthetic 3), fossil bone and dahllite (Fig. 4), phosphate-carbonate compound (Fig. "matrix stone" modes and the metastatic calcification and (Fig. 5), -459 YrALE JOURNAL OF Volume 38, BIOLOGY AND 1966 April, MEDICINE I.4 %O %O C14 C4 cn cn C%4 P-4 P-4 114 C4 cn CA cn r. cis0 z 0 CI C) C> cis :D I: 5. E ..q> .." CD 0 N N C) C114 N V-4 " <D C> I", .o 00 Ln in NO 6n 00 00 cl \0 O., \0 0\ r Cd t,-. in -O- \0 z 00 00 00 cn bO 4i v) > 0. cu Cd C) 00 tl% \0 m Cq M CI Co (=> 8\\ cn 0 0 0 co bo Ul r. Q cn r tN, tz \6 (U Ei cn V" W-4 O "14 V" 0 P4 44 W-4 "-O ot v) CIS cn m 0 fT-4z cn V) Ca Ei v CN ai 1.0 cn 4-&W 10 0 8 bO rn cis > cis 0 (L) Cd co cd 0 O C.) (u .- ci 4-& Cd (u 4a bo ...4 W (A . 14-4 4i cn Cd W cd Cd 0 = Cd -" CIS > %" u U 40 Carbonate in bone BAXTER, BILTZ, PELLEGRINO 3. Infra-red absorption spectra between 625 and 2,000 cm1 of whole bone FIG. and the synthetic calcium-carbonate-phosphate precipitates. CM1 700 2000 1600 1400 1200 900 800 .. .. , ,- u.v 0.0 ~~~~~~~~~~~~~FOSSIL B3ONE (Irvinq10n) >i. u,.20 cc.30 co <.40 i I ' I"" .70 ', 1 ''r , .~~~~~~~~~~~~~~~~~~~~~~~~'. 1.0 1.0 | dn--I . -iC- 14.0 15.0 16.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 WAVfIENGTH IMICEONSI FIG. 4. Infra-red absorption spectra between 625 and 2,000 cm14 of fossil bone (Irvington) and dahllite. 41 YALE JOURNAL OF BIOLOGY AND MEDICINE Volutize 1966 38, April, attributable to carbonate are seen only near 1,430 and 875 cm-' and no near 710 or 1,790 cm-- are seen (see also Table 3). In these speci- modes strong absorption from ortho- mens and in the crab and lobster shell, phosphate is seen from 900 to 1,200 cm-" with the largest peak of absorp- near tion 1,031 cm-1. near 1,400-1,450 cm-' appear as two distinguishable The absorptions v3 differences in the resolution, peaks in all samples in Figures 2-5. However, broadening and peak intensities of these and the peaks near 870 cm-' (v2) are seen. 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 WAVELENGTH ;MICRONS) between 625 and 2,000 cm1 of the metastatic FIG. 5. Infra-red absorption spectra calcification and the renal calculus studied. of the carbonate content and of the relative con- Chemical analyses centrations of calcium and are shown in Table 4. phosphate small amounts of Since only those salts containing relatively phosphate, and lobster shell i.e. clam shell which is relatively pure CaCO3 (tail) exhibit and which has molar ratio of as high as 23.40, the V4 a Ca/P the correlation between increasing phosphate concentra- the + V4 modes, vj of these latter carbonate modes can be seen. tion and the disappearance V4 vi modes occurs in the we have Absence of the and the + V4 specimens as with molar ratios as 11.20 (lobster carapace) examined Ca/P high listed in Table 4 with ratios lower than and in all the specimens Ca/P that. 42 Carbonate in bone BAXTER, BILTZ, I PELLEGRINO TABLE 2. SUMMARY OF CARBONATE ABSORPTION PEAKS IN CRAB SHELL AND LOBSTER SHELL Assignment of + 1e2 4 VI the mode Wavelength cm1 (microns) cm-"(microns) cm-'(microns) cm:`(microns) Crab (carapace) Absent 1475(6.78) 870(11.50) Absent 1419(7.05) 862(11.60) Lobster shell (tail) 1795(5.57) 1400-1450 870(11.50) .712(14.05) Poorly (6.9-7.15) (Much weaker resolved Poorly than or '2 Ps) resolved Lobster (carapace) Absent 1456(6.87) 862(11.60) Absent 1400(7.15) TABLE 3. SUMMARY OF ABSORPTION PEAKS ATTRIuTED TO CARBONATE BETWEEN 625 AND 4,000 CM'1 IN HUMAN BONE, FossIL BONE, DAHLLITE, RENAL CALCULUS, METASTATIC CALCIFICATION, AND SYNTHETIC CALCIUM- PHOSPHATE-CARBONATE COMPOUNDS STUDIED Assignment of the mode Ps cm1 cm'1 (microns) Frequency (microns) (wavelength) 1450 865-877( (11.4-11.6) Bone (6.9) 1409 (7.1) Poorly resolved Fossil bone 1459 (6.85) 872 (11.45) 1429 (7.05) 866 (11.55) (mammoth) Dahllite 1449 862 (11.60) (6.9) 1409 (7.1) Synthetic compound No. 1471 (6.8) 877 (11.4) (7.05) 869 (11.5) Poorly resolved No. 2 1492 (6.70) 866-877 (11.40-11.55) 1470 Poorly resolved (6.80) (6.87) (7.00) (7.05) Renal calculus 1466 (6.82) (11.4) "matrix stone" 1412 (7.08) 870 (11.5) Poorly resolved Metastatic 1445 (6.92) 876 (11.42) calcification 1412 (7.08) 870 (11.50) Poorly resolved 463 Volunie YALE JOURNAL OF BIOLOGY AND MEDICINE 1966 38, April, near Additional vibrations 1,540 cm-' and 1,660 cm-" are seen in most of the tissue specimens and these are attributed to matrix usually organic as can be diminished when the matrix is removed vibrationsl' they by or extraction confirmed ignition at 600° C. by by ethylenediaminei° (also in our laboratory). Vibrations due to water can also be seen near 1,660 cm.-"2' Some observers have noted carbonate near absorption 1,538 cm-110, THE AND THE TABLE 4. CHEMICAL ANALYSIS OF CARBONATE CONITENT OF RELATIVE MOLAR CONCENTRATIONS OF CALCIUM AND PHOSPHATE IN THE SPECIMENS STUDIED Specimen (mg/100 mg) Ca/P molar ratio Cog Clam shell 58.2 > 100.00 Lobster shell (tail) 40.0 23.40 Lobster 31.8 (carapace) 11.20 34.5 Crab (carapace) 9.50 Whole bone (human) 5.8 1.76 Fossil bone (mammoth) 11.5 2.32 Dahllite 7.2 1.76 Synthetic compound 1 9.0 1.83 No. 2 3.1 No. 1.67 Metastatic calcification 5.7 1.67 Renal calculus 2.0 0.53 "Matrix stone" DISCUSSION AND CONCLUSIONS The carbonate group has four normal modes of vibration's (see Table 1). The vibration is ordinarily infra-red but inactive, appears when carbonate v, is lowered as in symmetry aragonite' (Fig. 1). This mode is difficult to in the salts due evaluate phosphate-containing to phosphate absorption from 1,100 900 to cm-'.'2 The v2 absorption is due to carbon-oxygen out-of-plane bending vibra- tions' and is seen in all the specimens. The and vibrations are due to v3 V4 carbon-oxygen asymmetrical stretch- vibrations ing and in-plane bending respectively and are both doubly Both when degenerate.' are seen as single carbonate exists in peaks higher environments as in the free ion in solution or symmetry as in (D3h) These modes become into two calcite (D3).'° split peaks when carbonate is reduced as in or when symmetry aragonite carbonate oxvgens (CQ)' 464 Carbonate in bone BAXTER, PELLEGRINO BILTIZ, are covalently or hydrogen bonded and C2) ."' a The splitting of V3 (C. in aragonite is slight and is not clearly demonstrated without better resolu- tion. The location of the peaks may also vary with the cation precipitated with carbonate.' The V4 carbonate mode, however, is not seen in many carbonate com- pounds, i.e., in whole bone, fossil bone, dahllite, crab shell, some parts of the lobster shell, the synthetic compounds described, in metastatic calci- fication and renal calculus. All of these compounds contain, in addition to of phosphate. In addition v3 absorption carbonate, a significant amount in appears as two peaks these tissues. The splitting of V3 and the disappearance of v4 and v4 + vi appears to be related to the co-precipitation of carbonate with phosphate (or vice whether it occurs in the exoskeleton of the lobster and crab, in the versa) in The data seem to indicate that where apatites or synthetic compounds. as in the lobster shell shown in Figure 1, the v4 less phosphate is present the and v4 + peaks are seen which can be interpreted as indicating vi presence of a separate CaCO3 phase, most likely calcite. These data are that most of the carbonate in the specimens, compatible with the postulation in 2-5, is present in a state stabilized whose spectra are shown Figures unlike that in calcite or by the presence of phosphate, aragonite. is absent of absorption It is unlikely that V4 absorption because strong in 700 cm-' region from other species, since the peak distinctly appears the of calcite are added to the specimensl4.. '7 (confirmed when small amounts with than 1 cent calcite). Additional confirma- in our laboratory less per the combination band + V4) appears tion from the present data is that (vl not in the other when V4 is seen (Fig. 1) but is seen samples. might be concluded that V4 was IR inactive. This might occur if It had than and that the planar con- carbonate a higher symmetry D3h implies was with formation for a figuration of carbonate upset of, example, or an octahedron with coordinated This terahedron (Oh) oxygens. (Td) since the and near Furthermore, V2 V3 absorptions appear seems unlikely. of carbonate 1), it is more likely that the fundamental frequencies (Table as As even when one or the carbonate is present planar C03. mentioned, are bound in covalent and bonds as in more carbonate hydrogen oxygens remains active rise to two unidentate and bidentate ligands, v4 IR giving is seen near cm-'.'- We therefore feel that least one of which 700 peaks, at even distinct are not seen. V4 iS IR active at some frequency though peaks that carbonate are in these materials It seems possible the groups present is or of the in- in a way that there broadening decreasing V4 peak such the chemical nature of the carbonate tensities without changing seriously is seen near 700 cm-' in lobster A broadened area of group. absorption 46S YALE JOURNAL OF Volume 1966 BIOLOGY AND MEDICINE April, and crab shell (Fig. 2), but this may represent water of hydration.' Although v4 strong in calcite, the absorption becomes much weaker when iS there is splitting as in aragonite (Fig. 1). If there was significant broaden- ing or weakening of the peak intensities in bone, for example, the area under the too be absorption peak might be small to visualized. However, for there is no proof this and alternate possibilities have not been excluded, e.g., that the peak has shifted to a frequency where it is not seen. Nor is it known that the same explanation holds in all the tissues. Broadening of the carbonate absorption peaks has been noted in poorly crystalline synthetic calcium-carbonate-phosphate precipitates (Racquel Zapanta-LeGeros, personal communication) and seems to be more prominent in in the more amorphous materials. It is present the crab and lobster shell specimens shown in Figure 2. Even though the carbonate absorption peaks are in some cases broad- v3 ened, two peaks can be distinguished (Figs. 2-5) in the phosphate-contain- ing mineralized tissues reported here. These are more poorly resolved in whole human bone, lobster and crab, but are seen distinctly in some of the other samples, particularly in fossil bone (Fig. 4). This has been interpreted as indicative of of the is of splitting doubly degenerate absorptions."' It vs greater magnitude than seen in aragonite and therefore may indicate a reduced carbonate symmetry from D3h of the free ion or from D3 of calcite to as low as but the extent to which this splitting represents a C., perturbation is unknown. Herman and have attributed one of the carbonate Dallemagne67 absorp- v3 to tion peaks in bone calcite and other carbonate peaks to carbonate bound for to the phosphate. These data question that the same interpretation the splitting of the v3 carbonate absorption applies to all the specimens or that the double do of the peaks represent splitting doubly degenerate absorption. are must mean that there is some However, that two peaks distinguished the orientation of the force field on carbonate and that physical state of This carbonate in bone and these tissues is not completely amorphous. conclusion can be made whether two distinct the splitting represents phases of or two carbonate sites. There- carbonate, split doubly degenerate modes, of lines in the diffraction of these materials fore, lack CaCO3 X-ray patterns should not be taken as evidence for a separate completely amorphous in the shown the all exhibit CaCO3 phase. Although spectra present study splitting this is not always observed. In these cases it is possible that the v3 on carbonate is less oriented. force field Fisher'0 raised the of two Emerson and and Elliott'8 question carbonate of near 875 cm-" and on the sites in enamel on the basis two basis peaks and Elliott of at cm-1. also noted three peaks 1,542 cm-', 1,454 cm-', 1,410 4" Carbonate in bone BAXTER, BILTZ, PELLEGRINO that the absorption bands differed in some of the specimens he studied and concluded that the carbonate ions must enter the structure in several ways. Examination of Tables 2 and 3 and Figures 2-5 shows that although all these samples exhibit splitting of the V3 and absence of the V4 peaks some differences are present. The resolution, broadening, and the fre- quencies of the V3 peaks differ slightly. The V2 peaks are seen as either distinct doublets (fossil bone), poorly resolved doublets as in the renal single peaks as in dahllite, or as more poorly calculus or crab shell, peaks. These data suggest some differences in the physical state resolved of carbonate in these materials. The differences are seen not only when the exoskeleton of lobster or crab is compared to bone, but slight differences bone, whole human bone, the human pathological calcification and in fossil which the the synthetic compounds are also seen. The extent to degree carbonate groups, the of hydration or crystallinity, the location of the presence of impurities or other factors are responsible for these differences these data. It should be emphasized, however, cannot be determined from in carbonate or as are seen in that large differences bonding symmetry in other metallic carbonates' hydrogen bonded and chelated carbonates9" much greater and in bicarbonate (see Table 1), are often accompanied by differences than are seen above. The discussion has not con- absorption some have noted near 1,538 cm-'."0"7"1 sidered the carbonate absorption contaminated with atmospheric CO2 shows V2, V3, Reagent Ca (OH)2 carbonate These in and sometimes V4, and + V4 absorption peaks.' peaks vj attributed reagent Ca (OH)2 and in ashed bone were previously incorrectly to Ca(OH) 2. The better resolution of the V3 peaks near 1,400-1,450 cm-' bone indicates a more oriented force field on the in heated probably in the carbonate latter samples. SUMMARY A of the state of carbonate in bone and precise understanding physical in the influence of carbonate other mineralizations is essential understanding of the mineralized tissues as well as the on the solubility and crystallinity a carbon dioxide reservoir in metabolic disease. role of bone carbonte as state of carbonate in several mineralized tissues, minerals The physical and calcium-carbonate-phosphate compounds was studied by synthetic carbonate infra-red between 625 cm-' and examining their absorption cm-' 4,000 (2.5-15 microns). attributed to carbonate were distinguishable Double absorption peaks in from human cm-' some bone, dahllite, pathol- near 1,400-1,450 samples and of the lobster and crab shell and calcifications, parts suggest ogical 467 YALE JOURNAL OF BIOLOGY AND MEDICINE Volume 38, April, 1966 the presence of carbonate in these materials that is not in a completely state. amorphous Double absorption peaks near 1,400 cm-' may in some of the tissues vs indicate a splitting of this doubly degenerate mode and a reduced symmetry from D3h of the free ion or from of calcite for carbonate; however, D8 have not been clearly shown. causes for this splitting near in pure tissues and Absorption 712 cm-1 (V4) was seen CaCO3 in calcium-carbonate-phosphate compounds containing small amounts of phosphate but could not be seen in bone and in the mineralized tissues with These data are compatible and compounds a higher phosphate content. in tissues in a state with the view that the carbonate is present the latter stabilized by the phosphate unlike that in calcite or aragonite. It is concluded that V4 is infra-red active even though distinct peaks are not seen. Possible explanations are discussed. in broadening, and in the frequencies of Some differences the resolution, cm-' and near 870 cm-, were noted the absorption peaks near 1,400-1,450 was to bone and when when the exoskeleton of lobster and crab compared was compared to fossil bone, pathological calcifications, synthetic the latter dahllite which differences in the physical state of compounds and suggest the nature of which cannot be determined from these data. carbonate, ACKNOWLEDGMENTS Yale Dr. R. C. The authors wish to thank Dr. David Seligson, University, Gore, Perkin-Elmer Corp., Drs. R. Zapanta-LeGeros and John LeGeros, New York Univer- for valuable suggestions in the preparation of the manuscript. We are also sity, their of for the fossil bone indebted to Dr. H. C. Ezra, University California, providing of for her specimens and to Miss Peggy J. Rodgers, University Kentucky, very excellent technical assistance. Ferraro the National for Thanks are due to Dr. J. R. of Argonne Laboratory and for helpful suggestions and to Dr. R. C. Lord of the reading the manuscript Institute of for advice. Massachusetts Technology helpful REFERENCIES 1. Neuman, W. F. and Neuman, M. W.: The nature of the mineral phase of bone. Chem. Rev., 1953, 53, 1-45. 2. Roseberry, H. H., Hastings, A. B., and Morse, J. K.: X-ray analysis of bone and teeth. biol. Chem., 1931, 90, 395-407. J. 3. Bogert, L. J. and Hastings, A. B.: The calcium salts of bone. J. biol. Chem., 1931, 94, 473-481. 4. McConnell, Duncan: A structural investigation of isomorphism of the apatite group. Amer. Mineral., 1938, 23, 1-19. 5. Pellegrino, E. D. and Biltz, R. M.: The composition of human bone in uremia. Observations on the reservoir functions of bone and demonstration of a labile fraction of bone carbonate. Medicine 1965, 44, 397-418. 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B.: Spectra-structure correlations in the infra-red region. J. opt. Soc. Amer., 1950, 40, 397-400. 29. Barnes, R. B., Gore, R. C., Stafford, R. W., and Williams, V. Z.: Qualitative organic analysis and infrared spectrometry. Analyt. Chem., 1948, 20, 402-410. The infrared 30. Gatehouse, B. M., Livingston, S. E., and Nyholm, R. S.: spectra of some simple and complex carbonates. J. chem. Soc., 1958, V., 3137-3142. 31. Kazuo, Fujita, Junnosuke, Tanaka, Shizuo, and Kobayashi, Masahisa: Nakamoto, Infrared spectra of metallic complexes. IV. Comparison of the infrared spectra of unidentate and bidentate metallic complexes. J. Amer. chem. Soc., 1957, 79, 4904-4908. 32. Miller, F. A. and Wilkins, C. H.: Infrared spectra and characteristic frequencies of inorganic ions. Analyt. Chem., 1952, 24, 1253-1294. Articles are accepted for publication in The Yale Journal of Biology and Medicine on the basis of merit and suitability. The privilege of submitting articles is not limited to those associated with Yale University and contributions from others are cordially invited. The Board of Editors

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Published: Apr 1, 1966

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