The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 188-193

Calcium-Regulating Hormones, Bone Mineral Content, Breaking Load and Trabecular Remodeling Are Altered in Growing Pigs Fed Calcium-Deficient Diets^1,2,3

E. Eklou-Kalonji, E. Zerath^*, C. Colin, C. Lacroix, X. Holy^*, I. Denis, and A. Pointillart⁴

Laboratoire de Nutrition et Sécurité Alimentaire, Institut National de la Recherche Agronomique, 78 352 Jouy-en-Josas cedex, France and ^* Département de Physiologie Gravitationnelle, Institut de Médecine Aérospatiale du Service de Santé des Armées, 91 223 Brétigny-sur-Orge cedex, France

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Studies on calcium nutrition in appropriate large animal models can be directly relevant to humans. We have examined the effect of dietary Ca deficiency on various bone and bone-related variables, including plasma markers, histomorphometry, mineral content and breaking strength in pigs. Three groups of eight 38-d-old female pigs were fed adequate (0.9%; control), low (0.4%; LCa) or very low (0.1%; VLCa) Ca diets for 32 d. Plasma Ca significantly decreased over time only in the VLCa-deficient pigs. The concentrations of the parathyroid hormones (PTH) and calcitriol increased as Ca deficiency developed, and the plasma PTH and calcitriol levels varied inversely with dietary Ca. The total bone ash contents, bending moments, trabecular bone volume and the mineral apposition rate all decreased as the calcium intake decreased. The osteoclast surface areas were greater than those of controls in both Ca-deficient groups, whereas the osteoblast surface areas were greater only in the VLCa group. The plasma osteoblast-related markers (alkaline phosphatase, carboxy-terminal propeptide of type I procollagen and osteocalcin) were either greater or unaffected in the Ca-deficient pigs. The results indicate that deficient bone mineralization combined with an increased bone resorption led to bone loss and fragility. The differences in the changes in bone cells (number and activity) between LCa and VLCa groups might be due to differences (time and extent) of circulating PTH and calcitriol. The defective mineralization in both Ca-depleted groups resulted mainly from the lack of Ca because their osteoblast activity was either maintained or stimulated. The results also underline the progressive sensitivity of pigs to Ca supply and the usefulness of this model.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

An adequate calcium intake is critical for preserving the integrity of the skeleton in humans and animals, and a low Ca intake is one of the major nutritional factors involved in osteoporosis (Doige et al. 1975, Matkovic 1991, Peterson et al. 1995). Dietary Ca deficiency alters bone remodeling and causes significant osteopenia (Avioli 1984, Nordin and Morris 1989). This loss of bone is attributed to increased bone resorption mediated via the secondary hyperparathyroidism caused by hypocalcemia (Blum et al. 1974, Persson et al. 1993, Rader et al. 1979, Thomas et al. 1988). The effects of Ca deficiency on bone formation are less clear. Bone formation, evaluated by bone histomorphometry, may be augmented (Shen et al. 1995) or diminished (Drivdahl et al. 1984, Peterson et al. 1995, Stauffer et al. 1973) in growing rats receiving a low Ca diet.

The guidelines for using animal models to study osteoporosis recommend the use of two animal species, the rat and a second non-rodent (Thompson et al. 1995). Thus, there is a need for a large animal with a bone remodeling process and a nutritional behavior more similar to those of humans than those of rats, in which to evaluate the effects of nutritional factors on the skeleton. The growing pig is an interesting experimental model in which to investigate changes in bone and mineral metabolism caused by diets with an unbalanced mineral content (Mosekilde et al. 1993, Pointillart and Guéguen 1993, Pointillart et al. 1995, Schandler et al. 1991) or hormonal treatment (Denis et al. 1994, de Vernejoul et al. 1990). However, the effects of Ca deficiency on the relationship between Ca-regulating hormones, bone-related markers and bone histomorphometry have never been investigated in detail in pigs.

Therefore, we monitored the alterations in the plasma concentrations of Ca-regulating hormones and bone-related markers in growing pigs receiving Ca-deficient diets. Direct measurements of ash content, histomorphometry and breaking strength were performed on bones at slaughter.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and feeding.  Twenty-four 38-d-old female crossbred pigs (Cepra, Vermenton, France) weighing 9.5 ± 0.4 kg were randomly assigned to one of three groups each of which was fed a diet with a different Ca content as follows: control (0.9%), low calcium (LCa)⁵ (0.4%), and very low calcium (VLCa) (0.1%). The basal diet (Table 1) was formulated to meet the requirements for growing pigs (INRA 1989). All of the pigs were pair-fed the same basal diet, which consisted of cereals and soybean meal and provided 14.0 MJ digestible energy/kg diet, 18.6% protein (including 1.1% L-lysine), 0.53% phosphorus and 25 µg cholecalciferol/kg. The 0.9% Ca diet contained the recommended Ca level for growing pigs at this age (INRA 1989). Calcium carbonate (Prolabo, Paris, France) was used to vary the diet Ca content. The VLCa diet contained the Ca provided by the basal ingredients only. The pigs were housed individually (pens of 1 m × 2.5 m, concrete floor and walls); the growth rate was evaluated weekly for a total of 32 d. The animals were stunned by electronarcosis and killed by exsanguination. The study complied with the French regulations governing animal care.

Plasma parameters.  Venous blood was taken from the anterior vena cava to monitor plasma metabolites. The plasma concentrations of Ca, 1,25-dihydroxycholecalciferol (calcitriol) and immunoreactive parathyroid hormone (iPTH) were measured weekly (d 9, 16, 23 and 32). The plasma level of the carboxy-terminal propeptide of type I procollagen (PICP) was measured in samples taken on d 16, 23 and 32. Those of inorganic phosphorus (P), total alkaline phosphatase activity (ALP) (EC 3.1.3.1), osteocalcin, and hydroxyproline were determined at slaughter (d 32). The plasma concentration of PICP was considered to be a marker of the synthesis of bone collagen and that of hydroxyproline as a marker of its breakdown because no urine was collected. Under similar conditions, the plasma concentration of hydroxyproline is strongly correlated (r = 0.89) with its urinary excretion in pigs (Pointillart et al. 1997). Plasma total ALP is also closely correlated (r = 0.87) with the plasma bone isoenzyme (Pointillart et al. 1995). Therefore, only total ALP was determined. Plasma Ca, inorganic P, total ALP and hydroxyproline were measured as previously described (Pointillart et al. 1995 and 1997). PICP was determined by RIA (Orion Diagnostica, Espoo, Finland). The lowest detectable concentration was 1.2 µg/L and the intra- and interassay CV were 3 and 5%, respectively. Osteocalcin was determined by a modified RIA kit (Ostk-Pr, Oris, Gif sur Yvette, France) with purified bovine osteocalcin as standard and tracer and rabbit antiserum to bovine osteocalcin, adding an internal porcine standard (Pointillart et al. 1995). Calcitriol was measured by radioreceptor assay using a kit (Nichols Institute Diagnostics, San Juan Capistrano, CA) after extraction on modified C-18OH columns. Immunoreactive PTH was measured by a two-site immunoradiometric assay (Allegro intact PTH kit, Nichols Institute, Mallinckrodt Diagnostica, Evry-Lisses, France). The sensitivity of the method was 1 ng/L. The intra- and interassay CV were 3.4 and 5.6%, respectively.

Bone measurements.  Mechanical parameters. At slaughter, the internal (finger III) and external (finger IV) metatarsal bones were removed from the right hind-legs and dissected free of soft tissues. The "three-point bending test" at fracture was used to determine the mechanical parameters (Crenshaw et al. 1981). The failure load, i.e., the force (newtons) applied to the midpoint of the bone until breaking, was determined with a universal testing machine (AP 4000 Erichsen, Villetaneuse, France). The bending moment was calculated according to the following formula: bending moment = F × L/4, where F is the failure load, and L the length (m) between the two fulcrum points supporting the bone. It is expressed in N·m. This parameter was selected because of its great sensitivity to mineral nutrition in the pig (Pointillart and Guéguen 1993, Pointillart et al. 1995).

Ash contents. The volume, dry matter, ash, Ca and P contents of the right internal metatarsal bone were determined (Pointillart et al. 1995). Ash (g/100 g dry matter) was evaluated for the whole bone, the diaphyses (to represent cortical bone), and the proximal and distal epiphyses (to represent spongy bone). Values for ash, Ca and P relative to apparent whole-bone volume were calculated.

Histomorphometry. The pig bones were labeled by giving intramuscular injections of 10 mg calcein per kg body weight 8 and 2 d before slaughter. At slaughter, samples of trabecular bone from the distal metaphysis of the left external metacarpals were removed, fixed in formol/methanol, and embedded in methyl/methacrylate without decalcification. The histomorphometric procedure has been described previously (Denis et al. 1994, De Vernejoul et al. 1990). The following parameters were evaluated: bone volume (BV/TV, %), trabecular thickness (Tb Th, µm), osteoid thickness (O Th, µm), osteoblast (Ob S/BS, %) and osteoclast (Oc S/BS, %) surfaces, double-labeled trabecular surfaces (mineralizing surfaces, MS/BS, %) and mineral apposition rate (MAR, µm/d). The bone formation rate [BFR, µm³/(µm²·d)] was calculated by multiplying the MAR by the double-labeled trabecular surfaces. The formation period (FP, d) and mineralization lag time (Mlt, d) were calculated using the formulas wall thickness divided by mineral apposition rate (FP), and osteoid thickness divided by mineral apposition rate (Mlt).

Statistical methods.  The data were analyzed using Abacus statistical software (Abacus Concepts 1989). Growth parameters, plasma bone-related markers, bone mineral contents, and histomorphometric parameters were analyzed by one-way (diet) ANOVA and Newman-Keuls test (Snedecor and Cochran 1971) as post test. Failure load and the bending moment were analyzed by two-way (diet and bone) ANOVA. Plasma concentrations of Ca, PTH and calcitriol were analyzed by two-way (diet and time) ANOVA, followed by a Newman-Keuls test. Relationships between plasma and/or bone variables were assessed by linear correlation analyses with all individual data obtained at slaughter. The effect of the three dietary Ca levels on the MAR was analyzed by linear regression analysis.

	RESULTS

Abstract Introduction Methods Results Discussion References

Performance.  The performances of the three groups of pigs were similar. The body weights at slaughter did not differ: 22.6 ± 0.6 (controls), 22.9 ± 0.4 (LCa) and 22.4 ± 0.4 kg (VLCa), nor did the average daily weight gains, 0.42, 0.43 and 0.41 (± 0.01) kg/d or the feed-efficiency ratios (kg body weight gain/kg diet consumed), 0.57, 0.59 and 0.57 (± 0.02).

Plasma measurements.  Time course of variables related to calcium metabolism. The pigs fed the very low Ca diet (VLCa) showed a time-dependent decrease in plasma Ca concentration, but there was no significant change in the LCa pigs (Fig. 1). There were no significant differences among the three groups in the plasma concentrations of phosphorus at slaughter (data not shown). The plasma concentrations of iPTH and calcitriol were dramatically higher in both Ca-deficient groups (LCa and VLCa) than in controls. The differences in hormone concentrations were amplified over time and were proportional to the severity of the Ca deficiency. The plasma levels of iPTH of the three groups were already significantly different on d 9, i.e., the lower the dietary Ca content, the greater the plasma PTH concentration. Plasma iPTH of the Ca-deficient pigs was 70% (LCa) and 2.7-times (VLCa) higher than that of the control pigs at slaughter (d 32). The plasma calcitriol increased from d 16 in the VLCa pigs, but only at slaughter in the LCa pigs. The plasma concentrations of calcitriol in the Ca-deficient pigs were 1.7 (LCa ) and 3.9 times (VLCa) higher than those of the control pigs at slaughter. The plasma concentrations of iPTH and calcitriol were significantly correlated (r = 0.73, P < 0.001), and both were correlated with plasma Ca (r = 0.91 and r = 0.71, P < 0.001, respectively).

View larger version (15K): [in this window] [in a new window]   Fig 1. Plasma concentrations of calcium, immunoreactive parathyroid (iPTH) and 1,25-dihydroxycholecalciferol (calcitriol) in growing pigs fed diets containing 0.9% (control), 0.4% (low Ca, LCa) and 0.1% (very low Ca, VLCa) calcium for 32 d. Values are means ± sem, n = 8. Two-way (diet and time) ANOVA, P < 0.0001; overall diet effect, P < 0.003; overall time effect, P < 0.001; time × diet interactions, P < 0.001. Means at a time with no letters in common differ; lower case, P < 0.05; upper case, P < 0.01.

Plasma bone-related markers. The plasma total ALP in the LCa pigs did not differ, but it was significantly higher (+50%) in the VLCa pigs than in the control pigs (Table 2). The plasma level of PICP was significantly elevated in the VLCa pigs only. The plasma concentrations of osteocalcin were greater than in controls in both the Ca-deficient groups. The plasma concentration of hydroxyproline was higher in the VLCa pigs than in the other two groups. In addition, the plasma levels of ALP and PICP were correlated in the three groups (r = 0.55, P < 0.006).

Bone measurements.  Ash contents and mechanical parameters. The fresh weight of the right internal metatarsal of the VLCa pigs was lower (P < 0.05) than that of the two other groups (Table 3). Ash weights, mineral contents relative to dry matter (ash) or volume (ash, Ca and P) of the whole bone were significantly (P < 0.0001) affected by both calcium-deficient diets, i.e., the lower the Ca content of the diet, the lower the bone mineral content, with significant differences among all three groups (P < 0.05). The g ash/100 g bone dry matter in the spongy bone was reduced as dietary Ca was reduced (P < 0.0001), whereas it was reduced in the cortical bone only of the VLCa group (P < 0.01).

Failure loads and bending moments of the external and internal metatarsal bones were reduced (P < 0.0001) in the Ca-deficient pigs (Table 4), with significant differences among all three groups. The bending moments in the LCa were 30% lower than those of controls; in the VLCa pigs, they were 54% lower. Bone bending moments were correlated with bone mineral contents (total ash, ash relative to dry matter and ash, Ca and P relative to bone volume); the correlation coefficients between bending moments and ash or Ca contents of the whole bone were 0.89 and 0.78, respectively (P < 0.001). Bending moments and percentage dry matter of ash were inversely correlated with the plasma concentration of iPTH at the time of slaughter (r = 0.79 and r = 0.76, P < 0.001, respectively).

Histomorphometric parameters. The volume of trabecular bone was lower in the calcium-deficient pigs (21%, LCa and 29%, VLCa vs. controls) than in the control pigs, but there was no significant difference between the two calcium-deficient groups (Table 5). The osteoid thickness was 2.5 times greater in the VLCa pigs than in the control pigs. The trabecular thicknesses did not differ among groups. The osteoclast surface areas were greater in the calcium-deficient pigs (+23% in LCa and +37% in VLCa pigs vs. controls) than in the control group. The osteoblast surface areas were significantly greater in the VLCa pigs than in the two other groups (+40% vs. controls, P < 0.05). The mineral apposition rate decreased linearly (r = 0.85, P < 0.0001) as the dietary Ca decreased. The double-labeled areas tended (P = 0.054) to be greater in the VLCa group only. The bone formation rate was reduced (P < 0.001) in both LCa and VLCa pigs. The formation period and the mineralization lag time were longer (P < 0.001) in the VLCa pigs. The trabecular bone volume was significantly correlated with the ash content per unit bone volume (r = 0.75, P < 0.001). The osteoclast areas were correlated with the plasma hydroxyproline and the plasma iPTH concentrations at slaughter (r = 0.59 and r = 0.67, respectively, P < 0.003). The osteoblast areas were significantly (P < 0.009) correlated with the plasma levels of ALP (r = 0.56) or PICP (r = 0.54).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This comprehensive review of the effects of nutritional Ca deficiency in a large animal, the pig, is provided by the measurement and subsequent comparison of a wide spectrum of direct (mineral contents, breaking strength and remodeling) and indirect (bone-related plasma markers) bone assessment variables in conjunction with the changes in plasma calcium regulation. The bones of growing pigs fed low Ca diets were severely affected. Bone formation and mineralization rates, the trabecular volume, the mineral contents and strength were all reduced, and bone resorption was above normal. The plasma markers of osteoblast (PICP and ALP) and osteoclast activity (hydroxyproline) were also well correlated with the histomorphometric data, but the growth of the pigs was not affected.

These changes were due in part to the gradual secondary hyperparathyroidism that the pigs developed to maintain their plasma Ca concentration. PTH kept the plasma Ca in the normal range during the first 2 wk of the experiment in pigs fed the LCa and the VLCa diets by releasing Ca from the bone. Thereafter, PTH stimulated calcitriol synthesis, which helped to maintain plasma Ca by enhancing intestinal Ca absorption (Bouillon et al. 1995) and bone resorption (Marie et Travers 1983, Raisz et al. 1972, Stall et al. 1998). As Ca deficiency continued, the circulating concentrations of iPTH and calcitriol were further increased in the pigs fed the lowest Ca diet (VLCa). Nevertheless, the persistent lack of dietary Ca prevented these hormones from mobilizing enough Ca from intestinal absorption or bone resorption to maintain a normal serum Ca. The hormonal reactions appeared early, but not simultaneously because PTH began to increase (d 9) before calcitriol (d 16). This means that bones were first exposed to increased PTH and to increased calcitriol later. Consequently, minerals are mobilized from bone by PTH in growing pigs, as in rats (Stauffer et al. 1973).

Bone remodeling was severely altered. The documented effects of PTH on bone resorption (Fitzpatrick and Bileziakan 1996, Thompson et al. 1975, Wright et McMillan 1994) were observed, i.e., osteoclast surface areas were greater in the animals fed the VLCa diet as well as in animals fed the LCa diet. However, only pigs fed the LCa diet were able to maintain plasma calcium within the normal range.

The rate of bone formation was reduced in both Ca-deficient groups. The bone formation rate depends on osteoblast surface area and also on the rate of mineral apposition, reflecting the single osteoblast activity. This activity includes the synthesis and mineralization of bone matrix. Osteoblast surface areas and osteoblast-related markers (PICP, ALP and osteocalcin) were elevated in the VLCa pigs, suggesting an increase in the number and activity of the osteoblasts. Nevertheless, bone formation was subnormal. In fact, the mineral apposition rate was reduced, which led to the accumulation of undermineralized bone matrix as indicated by the elevated osteoid thickness. This defective bone mineralization was emphasized by the marked increase in the formation period (+60%) and mineralization lag time (+405%). These data indicate that mineralization was delayed, but even after its onset, mineral deposition was slower than normal. This response has also been described in Ca-depleted rats (Peterson et al. 1995, Stauffer et al. 1973), and in children showing evidence of dietary Ca deficiency (Marie et al. 1982). There was also a reduced mineral apposition rate in the LCa pigs, but there were no significant differences in the number or activity of osteoblast or osteoid thickness in these pigs. Thus, the impairment of mineralization is responsible for the reduced bone formation and is the direct result of the lack of Ca in the diet, and not of a lower osteoblast-related formation, because the number or the activity of the osteoblasts was either elevated or unaffected in both groups of Ca-deficient pigs.

The increase in osteoblast surface areas is probably due to the elevated plasma iPTH because PTH stimulates bone remodeling (Fitzpatrick and Bileziakan 1996, Parfitt 1976), and elevated osteoblast surface areas have been repeatedly observed in primary (Hock and Gera 1992, Tam et al. 1982) and secondary hyperparathyroidism in Ca-deficient children (Marie et al. 1982), baboons (Pettifor et al. 1984) and rats (Thompson et al. 1975). Calcitriol could also have a direct effect on osteoblast activity because these cells have receptors for this hormone (Kream et al. 1977, Liesegang et al. 1994). Short-term treatment with calcitriol modulates osteoblast activity in humans, as assessed by the elevated circulating concentrations of osteocalcin and PICP (Bollerslev et al. 1991, Gram et al. 1996). High doses of calcitriol stimulate bone formation in rats (Erben et al. 1997). However, the plasma concentrations of iPTH and calcitriol were increased in the LCa pigs, which showed no significant changes in osteoblast function. In fact, the plasma concentrations of calcitriol were elevated only at slaughter in these pigs, whereas iPTH was elevated from d 9, but not as markedly as in the VLCa pigs. Hence, these increases were either not sufficient, or have been going on for too short a time to significantly affect the osteoblasts in the LCa pigs.

Trabecular bone volumes in the LCa and VLCa pigs were lower than in control pigs, indicating marked osteopenia, which resulted from the higher bone resorption and the reduced bone accretion. This osteopenia was associated with a lower ash content and strength of bones, and was correlated with the severity of the Ca deficiency. These findings are also emphasized by the good correlations between the trabecular bone volume and the mineral contents relative to bone dry matter or volume. The drop in ash content relative to dry matter was less severe in the cortical than in the spongy metatarsal bone. This agrees with remodeling being more intensive in trabecular bone than in cortical bone (Parfitt 1984).

The good correlations between the plasma hydroxyproline concentration and the osteoclast surface areas on the one hand, and between the plasma hydroxyproline and PTH concentrations on the other hand, indicate that hydroxyproline reflects bone resorption. The plasma iPTH was inversely proportional to the amount of Ca in the diet; hence there may have been less bone resorption in the LCa than in the VLCa pigs. Thus, the unaffected plasma hydroxyproline levels in the LCa pigs suggests that this variable must not be sensitive enough to detect moderate stimulation of bone resorption. The good correlation between ALP, PICP and osteoblast surface areas reinforces the link between ALP and PICP and bone formation (Christenson et al. 1997, Delmas et al. 1996). The plasma osteocalcin concentrations at slaughter were not correlated with any of the histomorphometric or biochemical data related to bone formation. The elevated osteocalcin levels may reflect overall bone turnover rather than osteoblast activity, as reported in rats (Fries et al. 1992), humans (Parthemore et al. 1993) and pigs (Pointillart et al. 1995).

In conclusion, this study demonstrates that growing pigs, whose rapid growth rate is similar to that in humans during childhood, have an impressive capacity to counteract a severe lack of dietary calcium by altering the hormonal responses acting on bone and mineral metabolism. For instance, the plasma calcium remained normal in pigs fed 58% less calcium than the controls. Direct bone measurements detected bone loss, whereas only bone histomorphometry indicated that the defective bone mineralization resulted from both stimulated bone resorption and decreased mineralization rate, without any impairment of the capacity of the osteoblasts. These effects were essentially due to the secondary hyperparathyroidism and increased calcitriol production. Last, we showed that there are reliable correlations between the plasma bone-related markers, calciotropic hormones and direct bone measurements in this large, omnivorous mammalian species.

	FOOTNOTES

Manuscript received 10 July 1998. Initial reviews completed 26 August 1998. Revision accepted 5 October 1998.

	ACKNOWLEDGMENTS

We thank J. C. Bernardin and D. Besnard for animal care, X. Blanc for help in making the diets, and C. André and S. Renault for histomorphometry procedure. We also thank O. Parkes for editing the text.

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