The evolution of metazoan α-carbonic anhydrases and their roles in calcium carbonate biomineralization

The carbonic anhydrase (CA; EC 4.2.1.1) superfamily is a class of ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide. The α-CA family, present in all metazoan clades, is a key enzyme involved in a wide range of physiological functions including pH regulation, respiration, photosynthesis, and biocalcification. This paper reviews the evolution of the α-CA family, with an emphasis on metazoan α-CA members involved in biocalcification. Phylogenetic analyses reveal a complex evolutionary history of α-CAs, and suggest α-CA was independently co-opted into a variety of skeleton forming roles (e.g. as a provider of HCO₃^- ions, a structural protein, a nucleation activator, etc.) in multiple metazoan lineages. This evolutionary history is most likely the result of multiple gene duplications coupled with the insertion of repetitive or non-repetitive low-complexity domains (RLCDs/LCDs). These domains, of largely unknown function, appear to be lineage-specific, and provide further support for the hypothesis of independent recruitment of α-CAs to diverse metazoan biocalcification processes. An analysis of α-CA sequences associated with biocalcification processes indicates that the domains involved in the activity and conformation of the active site are extremely conserved among metazoans.     

Decrease in Hydraulic Conductivity of a Paddy Field using Biocalcification in situ

The hydraulic conductivity of a paddy field (Anthraquic Dystrustept), a silty clay soil containing more than 29% (w/w) of gravel, in Nagoya University Farm was reduced by in situ treatment of subsurface soil using bentonite and biocalcification (microbial calcium carbonate precipitation) through the addition of CaCl₂, urea, and corn steep liquor (CSL). The treatment decreased the hydraulic conductivity of the field from an average of 10^?3 cm/s to a range of 10^?5 to 10^?7 cm/s during 69 days, with reducing the proportion of pores of subsurface soil larger than 75 µm in diameter. The biocalcification effect was observed at 10-cm thickness from the treated subsurface. Laboratory soil core experiments demonstrated that the decrease in the hydraulic conductivity was not attributed to the effect of bentonite but mainly to the effect of biocalcification. The addition of CSL enhanced the urease activity of soil required for biocalcification, even at 4°C, as indicated by a decrease in urease activation energy temperature sensitivity. These experimental results agreed with the gradual decrease in hydraulic conductivity observed in the field when the average daily temperature was 7°C (days 24–69). It was suggested that the biocalcification is a potential technique to reduce the hydraulic conductivity of paddy field.     

Influence of Environmental Temperature on Biocalcification by Non-sporing Freshwater Bacteria

The process of biocalcification, recognised as precipitation of calcium carbonate, has been described as a widespread phenomenon associated with a wide range of different bacterial species. This biocalcifying activity, and factors that affect it, have been widely studied in moderately halophilic bacteria but there is a lack of information on factors that affect biocalcification by freshwater bacteria. In this paper, we study how temperature can affect biocalcification by freshwater bacteria that potentially could be used for the process of bioconsolidation during conservation. Ten isolates were characterised by standard biochemical and API 20NE tests. Their biocalcifying activity was studied at temperatures between 10 and 40°C in B4 liquid medium. Mineralogical and quantitative analyses of the crystals were carried out by XRD, and morphological studies by SEM. Biocalcification only occurred when bacteria were present and were able to grow. Carbonate precipitation by bacteria increased with time and temperature of incubation. Temperature affected not only the amount of precipitation but also crystal quality and morphology. As bioconsolidant agents, these organisms could be applied to stone when the temperature does not exceed 40°C depending on the type of isolate.     

Fertilization of the northwestern Tethys (Vocontian basin,SE France) during the Valanginian carbon isotope perturbation: Evidence from calcareous nannofossils and trace element data

A high resolution calcareous nannofossil study associated with a geochemical analysis (major, trace elements, and carbon and oxygen isotope stratigraphies) was carried out in the Angles section (hemipelagic setting of the Vocontian basin, SE France) during the Valanginian positive carbon isotope excursion. The behaviour of calcareous nannofossil taxa in relation to fertility conditions was studied to elaborate new nutrient indices in this environment: a high nutrient index based on Biscutum spp., Discorhabdus rotatorius, Zeugrhabdotus fissus, (high fertility indicators) and Watznaueria barnesae (low fertility indicator); and a medium nutrient index based on Lithraphidites carniolensis (medium fertility indicator) and W. barnesae (low fertility indicator). These two indices show a major fertilization from the Stephanophorus ammonite Zone to the Trinodosum ammonite Zone, with a maximum during the positive carbon isotope excursion.Since high values of the nutrient indices are in phase with high values of chemical elements related to terrigenous material and low values of the coccolith total abundance, it is proposed that pulses of detrital inputs into the basin triggered the nutrification which, in turn, caused a biocalcification crisis of the calcareous nannofossils. Nutrification is also responsible for the reef demise in the surrounded platforms, as indicated by the increased Sr/Ca seawater ratio at that time.The intensification of the Paranà–Etendeka volcanic activity, triggering CO₂ excess in the atmosphere, is probably responsible for an acceleration of the hydrological cycle, the increased weathering, and the subsequent higher terrigenous and nutrient transfer from continents to the Vocontian basin. In such a scenario, nutrification is a dominant factor controlling neritic and hemipelagic biocalcification. However, one cannot exclude that the global increase of atmospheric CO₂ could generate chemical changes of the sea-surface waters, acting with the nutrification, to modify the biocalcification of the carbonate producers.     

Dietary Silicon Intake of Korean Young Adult Males and Its Relation to their Bone Status

Accumulated data suggests a positive effect of silicon on bone health; however, limited research exists on the silicon content of foods. To further the understanding of the relationship between dietary silicon intake and bone health, a food composition database of commonly consumed foods in Korea is required. For quantitative data on the intake levels of silicon, we analyzed the silicon content of 365 food items commonly consumed in Korea using inductively coupled plasma—atomic emission spectrometry following microwave-assisted digestion. To investigate the dietary silicon intake status and to examine the potential role of dietary silicon intake in the bone status of men, a total of 400 healthy Korean adult males aged 19–25 were observed for their diet intake and calcaneus bone density using the 24-h recall method and quantitative ultrasound, respectively. Clinical markers reflecting bone metabolism such as serum total alkaline phosphatase, N-mid osteocalcin, and type 1 collagen C-terminal telopeptide concentrations were also analyzed. Silicon intake of the subjects was estimated as 37.5 ± 22.2 mg/day. Major food sources of dietary silicon in the Korean male were cereal and cereal products (25.6 % of total silicon intake), vegetables (22.7 %), beverages and liquors (21.2 %), and milk and milk products (7.0 %). Silicon intake correlated positively with age, weight, energy intake, protein intake, calcium intake, and alcohol intake. After adjusted for age, weight, energy intake, protein intake, calcium intake, alcohol intake, smoking cigarettes, and regular exercise status, daily total silicon intake had no correlation with calcaneus bone density and the bone metabolism markers, but silicon intake from vegetables had a positive correlation with serum total alkaline phosphatase activity, a bone formation maker. These findings show the possible positive relationship between dietary silicon intake from vegetables and the bone formation of young adult males. Further investigation in a larger (Korean) population and correcting for additional nutritional confounders is required to confirm these findings.     

Clarifying the relationship between body size and extinction risk in amphibians by complete mapping of model space

In vertebrates, large body size is often a key diagnostic feature of species threatened with extinction. However, in amphibians the link between body size and extinction risk is highly uncertain, with previous studies suggesting positive, negative, u-shaped, or no relationship. Part of the reason for this uncertainty is ‘researcher degrees of freedom’: the subjectivity and selectivity in choices associated with specifying and fitting models. Here, I clarify the size–threat association in amphibians using Specification Curve Analysis, an analytical approach from the social sciences that attempts to minimize this problem by complete mapping of model space. I find strong support for prevailing negative associations between body size and threat status, the opposite of patterns typical in other vertebrates. This pattern is largely explained by smaller species having smaller geographic ranges, but smaller amphibian species also appear to lack some of the life-history advantages (e.g. higher reproductive output) that are often assumed to ‘protect’ small species in other taxa. These results highlight the need for a renewed conservation focus on the smallest species of the world''s most threatened class of vertebrates, as aquatic habitats become increasingly degraded by human activity.     

Silicon: A neglected micronutrient essential for bone health

Bone matrix is predominantly made up of collagen, and in vitro and in animal models studies have shown that silicon is linked to glycosaminoglycans and plays an important role in the formation of cross-links between collagen and proteoglycans, determining the beneficial effects on strength, composition, and mechanical properties of bone. However, there are still no precise indications regarding a possible role of silicon on bone health in humans. Given this background, the aim of this narrative review was to consider the effectiveness of silicon dietary intake and silicon dietary supplementation (alone or with other micronutrients), in order to suggest a daily dosage of Si supplementation, on bone mineral density in humans. This review included eight eligible studies: four regarding dietary intake and four considering supplementation with silicon alone or with other nutrients. Despite the number of studies considered being low, the number of subjects studied is high (10012) and the results are interesting. Although to date the available scientific evidences are not considered valid enough to allow to establish an adequate level of Silicon intake, based on extrapolations from the data obtained with studies on animal and human models, it has been suggested that an adequate intake in order to promote beneficial effects for bone could be considered to be around 25 mg silicon/day. As for silicon dietary supplements, it has been shown that the combined treatment with orthosilicic acid (6 mg), calcium, and vitamin D has a potentially beneficial effect on femoral BMD compared to only use of calcium and vitamin D.     

The importance of mechanical loading in bone biology and medicine

This paper discusses the premise that the skeleton is primarily a mechanical organ, and reviews the reasons that mechanical factors play a major role in bone biology. It begins by considering three basic observations: (1) Galileo's observation that bone proportions become more robust as the species' overall size increases; (2) da Vinci's observation that larger structures are inherently weaker than smaller structures subjected to the same stress; and (3) the general observation that each unit of bone mass provides structural support for about 15 units of soft tissue organ mass. Together, these observations lead to the concept that it can be advantageous to minimize bone mass, consistent with constraints on other factors. This premise is discussed here in relation to the phenomenon of bone remodeling, which is seen to serve two purposes: the adjustment of bone mass and geometry to maintain peak bone strains at their maximum tolerable values, and the continual removal of fatigue damage produced at those strain levels. Finally, it is observed that bone remodeling apparently originated approximately 250 million years ago when the first vertebrates of substantial size became weight-bearing on land, suggesting that mechanical forces associated with weight-bearing were instrumental in the evolution of bone remodeling.     

Electrochemical study of a new methylene blue/silicon oxide nanocomposition mediator and its application for stable biosensor of hydrogen peroxide

A novel organic-inorganic nanocomposite of methylene blue (MB) and silicon oxide was synthesized and characterized by TEM, FTIR, and UV-vis. The as-prepared material was able to transfer the electron of the MB to electrode and was different from other SiO2 spheres structurally. It can be used as mediator to construct a biosensor with horseradish peroxidase (HRP) coimmobilized in the gelatine matrix and cross-linked with formaldehyde. The resulting biosensor exhibited fast amperometric response and good stability to hydrogen peroxide (H2O2). The linear range for H2O2 determination was from 1 x 10(-5) to 1.2 x 10(-3) M, with a detection limit of 4 x 10(-6) M based on S/N = 3. Moreover, the lifetime is more than 3 months under dry conditions at 4 degrees C.     

Vertebrate cranial evolution: Contributions and conflict from the fossil record

In modern vertebrates, the craniofacial skeleton is complex, comprising cartilage and bone of the neurocranium, dermatocranium and splanchnocranium (and their derivatives), housing a range of sensory structures such as eyes, nasal and vestibulo-acoustic capsules, with the splanchnocranium including branchial arches, used in respiration and feeding. It is well understood that the skeleton derives from neural crest and mesoderm, while the sensory elements derive from ectodermal thickenings known as placodes. Recent research demonstrates that neural crest and placodes have an evolutionary history outside of vertebrates, while the vertebrate fossil record allows the sequence of the evolution of these various features to be understood. Stem-group vertebrates such as Metaspriggina walcotti (Burgess Shale, Middle Cambrian) possess eyes, paired nasal capsules and well-developed branchial arches, the latter derived from cranial neural crest in extant vertebrates, indicating that placodes and neural crest evolved over 500 million years ago. Since that time the vertebrate craniofacial skeleton has evolved, including different types of bone, of potential neural crest or mesodermal origin. One problematic part of the craniofacial skeleton concerns the evolution of the nasal organs, with evidence for both paired and unpaired nasal sacs being the primitive state for vertebrates.     

Aspects of Parathyroid Physiology in Mammals

This report summarizes the parameters of parathyroid physiologyin the mammal. Emphasis is given to the part played by thishormone in the processes of growth and remodelling in bone aswell as to its function in the maintenance of a relatively constantlevel of ionic calcium in the extracellular fluid. The importanceof phosphate ions has been shown, particularly with respectto their role in the rate of Ca-transport through the extracellularfluid compartments, and their influence on the rate of parathyroidsecretion. In addition, the inter-relationship of the newlydiscovered hormone, thyrocalcitonin, is considered. The latterhormone, by suppressing resorption of bone, may have an influencein stabilizing plasma levels of calcium; it also has a moderate,but long range effect on the rate of bone remodelling. Finally,an attempt has been made to relate the vast amount of work donewith these two hormones in mammals to their phylogenetic developmentin vertebrates. It is suggested that the parathyroids developedat the time when the specialization of bone in vertebrates produceda solid structure which was incompatible with the fluid calciumconcentration needed for the maintenance of many physiologicalfunctions. The parathyroid hormone, by its action to increasethe transfer of calcium from bone against a concentration gradient,permitted these vertebrates to maintain the higher fluid calciumion concentration needed for the normal functioning of the animal.     

Analysis of the Proteinaceous Components of the Organic Matrix of Calcitic Sclerites from the Soft Coral Sinularia sp.

An organic matrix consisting of a protein-polysaccharide complex is generally accepted as an important medium for the calcification process. While the role this “calcified organic matrix” plays in the calcification process has long been appreciated, the complex mixture of proteins that is induced and assembled during the mineral phase of calcification remains uncharacterized in many organisms. Thus, we investigated organic matrices from the calcitic sclerites of a soft coral, Sinularia sp., and used a proteomic approach to identify the functional matrix proteins that might be involved in the biocalcification process. We purified eight organic matrix proteins and performed in-gel digestion using trypsin. The tryptic peptides were separated by nano-liquid chromatography (nano-LC) and analyzed by tandem mass spectrometry (MS/MS) using a matrix-assisted laser desorption/ionization (MALDI) – time-of-flight-time-of-flight (TOF-TOF) mass spectrometer. Periodic acid Schiff staining of an SDS-PAGE gel indicated that four proteins were glycosylated. We identified several proteins, including a form of actin, from which we identified a total of 183 potential peptides. Our findings suggest that many of those peptides may contribute to biocalcification in soft corals.     

Characterization of Urease and Carbonic Anhydrase Producing Bacteria and Their Role in Calcite Precipitation

Urease and carbonic anhydrase (CA) are key enzymes in the chemical reaction of living organisms and have been found to be associated with calcification in a number of microorganisms and invertebrates. Three bacterial strains designated as AP4, AP6, and AP9 were isolated from highly alkaline soil samples using the enrichment culture technique. On the basis of various physiological tests and 16S rRNA sequence analysis, these three bacteria were identified as Bacillus sp., B. megaterium, and B. simplex. Further, these Bacillus species have been characterized for the production of urease and CA in the process of biocalcification. One of the isolates, AP6 produced 553 U/ml of urease and 5.61 EU/ml CA. All the strains were able to produce significant amount of exopolymeric substances and biofilm. Further, efficacy of these strains was tested for calcite production ability and results were correlated with urease and CA. Isolate AP6 precipitated 2.26 mg calcite/cell dry mass (mg). Our observations strongly suggest that it is not only urease but CA also plays an important role in microbially induced calcium carbonate precipitation process. The current work demonstrates that urease and CA producing microbes can be utilized in biocalcification as a sealing agent for filling the gaps or cracks and fissures in constructed facilities and natural formations alike.     

有机无机肥分次配施对嘎啦苹果生长、15N-尿素吸收利用及损失的影响

以15年生嘎啦苹果/平邑甜茶为试材,采用¹⁵N同位素示踪技术,研究有机无机肥分次配施对嘎啦苹果/平邑甜茶生长、¹⁵N-尿素吸收利用及损失的影响.结果表明: 与不施有机肥相比,有机无机肥配施显著提高了植株根冠比、叶绿素含量、单株叶片总氮量和单果质量,且有机无机肥分次配施的效果要优于有机无机肥一次性配施.有机无机肥配施提高了各器官对氮的吸收征调能力(Ndff值),其中有机无机肥分次配施的效果更显著;而不同施肥处理各器官的Ndff值均表现为果实最高,其次是叶片、一年生枝、细根、粗根和多年生枝,中心干最低.有机无机肥分次配施植株总氮量和¹⁵N肥料利用率分别为395.39 g和28.4%,显著高于有机无机肥一次性配施处理(342.77 g和21.1%)和不施有机肥处理(296.41 g和14.6%),而¹⁵N损失率为51.3%,显著低于有机无机肥一次性配施处理(57.5%)和不施有机肥处理(60.6%).     

有机无机肥分次配施对嘎啦苹果生长、15N-尿素吸收利用及损失的影响

以15年生嘎啦苹果/平邑甜茶为试材,采用¹⁵N同位素示踪技术,研究有机无机肥分次配施对嘎啦苹果/平邑甜茶生长、¹⁵N-尿素吸收利用及损失的影响.结果表明: 与不施有机肥相比,有机无机肥配施显著提高了植株根冠比、叶绿素含量、单株叶片总氮量和单果质量,且有机无机肥分次配施的效果要优于有机无机肥一次性配施.有机无机肥配施提高了各器官对氮的吸收征调能力(Ndff值),其中有机无机肥分次配施的效果更显著;而不同施肥处理各器官的Ndff值均表现为果实最高,其次是叶片、一年生枝、细根、粗根和多年生枝,中心干最低.有机无机肥分次配施植株总氮量和¹⁵N肥料利用率分别为395.39 g和28.4%,显著高于有机无机肥一次性配施处理(342.77 g和21.1%)和不施有机肥处理(296.41 g和14.6%),而¹⁵N损失率为51.3%,显著低于有机无机肥一次性配施处理(57.5%)和不施有机肥处理(60.6%).     

Differentiation and growth of bone ornamentation in vertebrates: A comparative histological study among the Crocodylomorpha

Bone ornamentation, that is, hollow (pits and grooves) or protruding (ridges) repetitive reliefs on the surface of dermal bones, is a frequent, though poorly studied and understood, feature in vertebrates. One of the most typical examples of this characteristic is given by the Crurotarsi, a taxon formed by the crocodilians and their closest allies, which generally display deep ornamentation on skull roof and osteoderms. However, the ontogenetic process responsible for the differentiation and development of this character remains controversial. This study was conducted to settle the question on histological and microanatomical evidence in several crurotarsan taxa. Observational and experimental data in extant and extinct crocodyliforms show that bone ornamentation is initially created, and later maintained during somatic growth (that is indefinite in crocodilians), by a complex process of bone remodeling comprising local resorption of superficial bone cortices, followed by partial reconstruction. The superficial reliefs of crocodilian dermal bones are thus permanently modified through pit enlargement, drift, stretching, shrinking, or complete filling. Ridges are also remodeled in corresponding ways. These processes allow accommodation of unitary ornamental motifs to the overall dimensions of the bones during growth. A parsimony optimization based on the results of this study, but integrating also published data on bone histology in non‐crocodyliform crurotarsans and some non‐crurotarsan taxa, suggests that the peculiar mechanism described above for creating and maintaining bone ornamentation is a general feature of the Crurotarsi and is quite distinct from that attributed by previous authors to other vertebrates. J. Morphol. 276:425–445, 2015. © 2014 Wiley Periodicals, Inc.     

Combined Effects of Phytoestrogen Genistein and Silicon on Ovariectomy-Induced Bone Loss in Rat

This study was performed to evaluate the effect of concomitant supplementation of genistein and silicon on bone mineral density and bone metabolism-related markers in ovariectomized rat. Three-month-old Sprague Dawley female rats were subjected to bilateral ovariectomy (OVX) or sham surgery, and then the OVX rats were randomly divided into four groups: OVX-GEN, OVX-Si, OVX-GEN-Si, and OVX. Genistein and silicon supplementation was started immediately after OVX and continued for 10 weeks. In the OVX-GEN group, 5 mg genistein per gram body weight was injected subcutaneously. The OVX-Si group was given soluble silicon daily in demineralized water (Si 20 mg/kg body weight/day). The OVX-GEN-Si group was given subcutaneous injections of 5 mg genistein per gram body weight, at the same time, given soluble silicon daily (Si 20 mg/kg body weight/day). The results showed that the genistein supplementation in the OVX rats significantly prevented the loss of uterus weight; however, the silicon supplementation showed no effect on the uterus weight loss. The lumbar spine and femur bone mineral density was significantly decreased after OVX surgery; however, this decrease was inhibited by the genistein and/or silicon, and the BMD of the lumbar spine and femur was the highest in the OVX-GEN-Si-treated group. Histomorphometric analyses showed that the supplementation of genistein and/or silicon restored bone volume and trabecular thickness of femoral trabecular bone in the OVX group. Besides, the treatment with genistein and silicon for 10 weeks increased the serum levels of calcium and phosphorus in the OVX rats; serum calcium and serum phosphorus in the OVX-GEN-Si group were higher than those in the OVX-GEN and OVX-Si group (P < 0.05). At the same time, the treatment with genistein and/or silicon decreased serum alkaline phosphatase (ALP) and osteocalcin, which were increased by ovariectomy; serum ALP and osteocalcin in the OVX-GEN-Si group were lower than those in the OVX-GEN and OVX-Si groups (P < 0.05). The results above indicate that genistein and silicon have synergistic effects on bone formation in ovariectomized rats.     

Histology of the heterostracan dermal skeleton: Insight into the origin of the vertebrate mineralised skeleton

Living vertebrates are divided into those that possess a fully formed and fully mineralised skeleton (gnathostomes) versus those that possess only unmineralised cartilaginous rudiments (cyclostomes). As such, extinct phylogenetic intermediates of these living lineages afford unique insights into the evolutionary assembly of the vertebrate mineralised skeleton and its canonical tissue types. Extinct jawless and jawed fishes assigned to the gnathostome stem evidence the piecemeal assembly of skeletal systems, revealing that the dermal skeleton is the earliest manifestation of a homologous mineralised skeleton. Yet the nature of the primitive dermal skeleton, itself, is poorly understood. This is principally because previous histological studies of early vertebrates lacked a phylogenetic framework required to derive evolutionary hypotheses. Nowhere is this more apparent than within Heterostraci, a diverse clade of primitive jawless vertebrates. To this end, we surveyed the dermal skeletal histology of heterostracans, inferred the plesiomorphic heterostracan skeleton and, through histological comparison to other skeletonising vertebrate clades, deduced the ancestral nature of the vertebrate dermal skeleton. Heterostracans primitively possess a four‐layered skeleton, comprising a superficial layer of odontodes composed of dentine and enameloid; a compact layer of acellular parallel‐fibred bone containing a network of vascular canals that supply the pulp canals (L1); a trabecular layer consisting of intersecting radial walls composed of acellular parallel‐fibred bone, showing osteon‐like development (L2); and a basal layer of isopedin (L3). A three layered skeleton, equivalent to the superficial layer L2 and L3 and composed of enameloid, dentine and acellular bone, is possessed by the ancestor of heterostracans + jawed vertebrates. We conclude that an osteogenic component is plesiomorphic with respect to the vertebrate dermal skeleton. Consequently, we interpret the dermal skeleton of denticles in chondrichthyans and jawless thelodonts as independently and secondarily simplified. J. Morphol. 276:657–680, 2015. © 2015 The Authors Journal of Morphology Published by Wiley Periodicals, Inc.     

THE EVOLUTION OF BONE

Vertebrates are practically unique among the Metazoa in their possession of a skeleton made from calcium phosphate rather than calcium carbonate. Interpretation of the origin of a phosphatic skeleton in early vertebrates has previously centered primarily on systemic requirements for phosphate and/or calcium storage or excretion. These interpretations afford no anatomical or physiological advantage(s) that would not have been equally valuable to many invertebrates. We suggest the calcium phosphate skeleton is distinctly advantageous to vertebrates because of their relatively unusual and ancient pattern of activity metabolism: intense bursts of activity supported primarily by rapid intramuscular formation of lactic acid. Bursts of intense activity by vertebrates are followed by often protracted periods of marked systemic acidosis. This postactive acidosis apparently generates slight skeletal dissolution, associated with simultaneous vascular hypercalcemia. A variety of apparently unrelated histological features of the skeleton in a number of vertebrates may minimize this postactive hypercalcemia. We present new data that suggest that postactive skeletal dissolution would be significantly exacerbated if bone were composed of calcium carbonate rather than calcium phosphate. The former is far less stable both in vivo and in vitro than is calcium hydroxyapatite, under both resting and postactive physiological conditions.