Osteoporos Int (2014) 25:1905–1915 DOI 10.1007/s00198-014-2709-2
ORIGINAL ARTICLE
Calcium and vitamin D intake maintained from preovariectomy independently affect calcium metabolism and bone properties in Sprague Dawley rats C. Y. Park & W. H. Lee & J. C. Fleet & M. R. Allen & G. P. McCabe & D. M. Walsh & C. M. Weaver
Received: 12 November 2013 / Accepted: 3 April 2014 / Published online: 17 April 2014 # International Osteoporosis Foundation and National Osteoporosis Foundation 2014
Abstract Summary The interaction of habitual Ca and vitamin D intake from preovariectomy to 4 months postovariectomy on bone and Ca metabolism was assessed. Higher Ca intake suppressed net bone turnover, and both nutrients independently benefitted trabecular structure. Habitual intake of adequate Ca and ~50 nmol/L vitamin D status is most beneficial. Introduction Dietary strategies to benefit bone are typically tested prior to or after menopause but not through menopause transition. We investigated the interaction of Ca and vitamin D C. Y. Park : J. C. Fleet : C. M. Weaver (*) Department of Nutrition Science, Purdue University, 700 W State St, West Lafayette, IN 47907, USA e-mail: [email protected] W. H. Lee Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907, USA M. R. Allen Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA G. P. McCabe Department of Statistics, Purdue University, West Lafayette, IN 47907, USA D. M. Walsh School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland Present Address: C. Y. Park Department of Biochemistry and Cell Biology, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Korea Present Address: W. H. Lee Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Korea
status on Ca absorption, bone remodeling, Ca kinetics, and bone strength as rats transitioned through estrogen deficiency. Methods Sprague Dawley rats were randomized at 8 weeks to 0.2 or 1.0 % Ca and 50, 100, or 1,000 IU (1.25, 2.5, or 25 μg) vitamin D/kg diet (2×3 factorial design) and ovariectomized at 12 weeks. Urinary 45Ca excretion from deep-labeled bone was used to assess net bone turnover weekly. Ca kinetics was performed between 25 and 28 weeks. Rats were killed at 29 weeks. Femoral and tibiae structure (by μCT), dynamic histomorphometry, and bone Ca content were assessed. Results Mean 25(OH)D for rats on the 50, 100, 1,000 IU vitamin D/kg diet were 32, 54, and 175 nmol/L, respectively. Higher Ca intake ameliorated net bone turnover, reduced fractional Ca absorption and bone resorption, and increased net Ca absorption. Tibial and femoral trabecular structures were enhanced independently by higher Ca and vitamin D intake. Tibial bone width and fracture resistance were enhanced by higher vitamin D intake. Dynamic histomorphometry in the tibia was not affected by either nutrient. A Ca × vitamin D interaction existed in femur length, tibial Ca content, and mass of the soft tissue/extracellular fluid compartment. Conclusions Adequate Ca intake and serum 25(OH)D level of 50 nmol/L provided the most benefit for bone health, mostly through independent effects of Ca and vitamin D. Keywords Calcium . Histomorphometry . Kinetics . Ovariectomy . Vitamin D
Introduction In a healthy population, gradual bone loss occurs in older adults and is dramatically accelerated during the first 3–5 years of menopause [1]. Aging and menopausal estrogen loss is associated with decreased Ca absorption [2] and estrogen
1906
therapy increases Ca absorption and 1,25-dihydroxy vitamin D (1,25(OH)2D) status, the active vitamin D metabolite [3]. Therefore, awareness for osteoporosis prevention increases at this life stage, and most intervention research has introduced treatment after menopause when bone loss has stabilized. Ca supplementation with or without vitamin D has been successful to decrease the risk of osteoporosis and loss of bone mineral density (BMD) in women that are over 4-year postmenopausal [4, 5]. However, because eating habits and preferences, and consequently nutrient intake, gradually change and stabilize throughout adulthood before menopause, the consistent intake of nutrients are more likely to impact bone loss during and after menopause. The independent effects of Ca and vitamin D intake on adult trabecular bone have been reported in ovary-intact female rats 25 weeks of age [6] and male rats age 30 weeks [7], respectively. However, the independent and interactive effect of habitual Ca and vitamin D intake on Ca metabolism during the period of transition into estrogen deficiency has not been investigated. Older individuals are at risk for inadequate intakes of both Ca and vitamin D. In women over 50 years in the USA, the average Ca intake from diet is only 65 % (~770 mg/day) of the recommended intake, only 39 % exceed the recommended 1,200 mg Ca/day, and 35 % do not take any supplements [8]. This is of more concern, because women who do not consume Ca supplements are more likely to have lower Ca intake from diet as well [9]. Approximately 45 % of US women have serum 25-hydroxyvitamin D (25(OH)D), a vitamin D status marker, below the target level of 50 nmol/L [10], and mean serum 25(OH)D levels decrease with age [11]. In 2005–2006, less than 45 % of women 51–70 years and only 22 % of women over 70 years exceeded their previous vitamin D recommendations of 400 and 600 IU/day, respectively, through diet and supplements [8]. As Ca and vitamin D intake during adulthood can impact bone health and most women in the USA are not consuming the recommended intakes, the long-term effect of nutrient intake during adulthood through menopause needs to be understood. Ca and vitamin D supplementation has become standard dietary therapy to reduce the risk of osteoporosis [12]. Systematic reviews and meta-analyses show that Ca and vitamin D supplementation increases BMD or decreases loss of bone mass in older adults [13, 14]. The Institute of Medicine (IOM) set new recommendations for Ca and vitamin D intakes in 2011 [10], recognizing the needs of Ca and vitamin D are interdependent through coordinated action on Ca absorption and retention. At an adequate intake, Ca is absorbed through active and passive transport. Active Ca transport is known to be highly efficient and activated when 1,25(OH)2D elicits the transcription of Ca transport proteins such as transient receptor potential vanilliod family member 6 (TRPV6), vitamin Ddependent Ca-binding protein 9 kDa form (calbindin D9k) and plasma membrane Ca2+ ATPase 1b (PMCA1b) [15].
Osteoporos Int (2014) 25:1905–1915
Adequate serum concentrations of the precursor 25(OH)D is required for retention of bone volume and parathyroid hormone (PTH) suppression in older rats [7]. Some crosssectional studies in adult humans suggest that vitamin D status is a better indicator of bone health than Ca intake [11, 16]. Very low (100 nmol/ L, respectively) in order that the results be applicable to vitamin D status and dietary vitamin D intake for the general population. Phosphorus (P) was increased to 0.75 % in the 1.0 % Ca diets to keep the Ca/P ratio consistent with the basal diet and was 0.3 % in the 0.2 % Ca diets to prevent the deleterious effects on bone due to P deficiency [22]. OVX was performed at 12 weeks of age. Uterine weight was measured at harvest to determine proper OVX. Ca kinetics was performed 3–4 months post-OVX when bone loss was stabilized. Six rats (n=1/gp) were killed prior to OVX for analyses not included in this paper. The remaining rats were killed at 29 weeks of age by CO2 overdose. All bone imaging was performed on bones excised at killing.
Blood was drawn from a subset of 37 rats (5–7/gp) 1 week before transition to treatment diet (at 7 weeks of age) and from all rats at killing. Serum 25(OH)D was assessed by radioimmunoassay (RIA; IDS, Immunodiagnostic Systems Limited, UK). The inter- and intra-assay CV was 8 and 10 %, respectively. Serum total Ca was analyzed by atomic absorption spectrometry (AA; AAnalyst, Perkin Elmer) in duplicate. 45
Ca deep bone labeling
After 1 week of stabilization to the facility, rats were dosed with 47 μCi 45Ca as 45CaCl2 (Perkin Elmer, Boston, MA) through injection via the tail (Braintree Scientific Inc., Braintree, MA). The 45Ca is incorporated into bone, which enables assessment of net bone turnover over time through urinary 45Ca excretion. Twenty-four-hour urine was collected weekly starting 1 week after 45Ca dosing (5 weeks of age) till 24 weeks of age. Ca kinetics Analysis of Ca kinetics was performed between 25 and 28 weeks of age (13–16 weeks post-OVX), a time when OVX-induced rapid bone loss was in a relatively stable state [23]. Rats were randomized into six different kinetic groups. Jugular catheters were inserted 2 days prior to performing Ca kinetics on each rat. For Ca kinetics, 45Ca was administered intravenously (10 μCi; n=3/diet group) or orally (20 μCi; n= 10/diet group). Fifteen milligrams of Ca as calcium acetate was dissolved in ultrapure water and administered by oral gavage with a total volume of 1 mL. This Ca load was approximately 1/3–1/2 of the mean daily Ca intake of rats fed the 0.2 % Ca diets and therefore sufficiently low to assess active Ca transport [24, 25]. Blood was drawn at 0 (baseline), 2, 10, 20, 40, 60, 120, 180, 360, 720, 1,440, 2,160, and 2,880 min (200 μL/draw) after Ca load was provided. Collected blood was immediately centrifuged and 80 μL of plasma was transferred to scintillation vials. Fifteen milliliters of scintillation fluid (Ecolite+, MCP) was added, and samples were analyzed for 45Ca by liquid scintillation (LS6500, Beckman Coulter). At 3-h postdose, 2 mL saline and 5 % dextrose 50:50 solution was injected for fluid replacement. Water and food were also replaced at this time. Rats were housed in metabolic cages and 24-h urine and feces were collected for 4 days, and 4-day food intake was monitored. Kinetic models were developed and analyzed using WinSAAM software (NIH). Details in kinetic modeling are described elsewhere [26]. In our model, compartment 1 denotes serum, compartment 2 indicates intracellular and extracellular fluid and soft tissue, and compartment 3 refers to the exchangeable pool. The mass of these compartments (MC 13) are also reported.
1908
Urine and fecal 45Ca and Ca analysis All urine collections were centrifuged and diluted with ultrapure water to the nearest 0.5 mL and stored at 4 °C for future analysis. Feces were ashed, dissolved with 1 mL HNO3, and diluted with 24 mL ultrapure water. One milliliter of urine samples from deep bone labeling and both urine and fecal samples from kinetics were transferred to a scintillation vial with 15 mL scintillation fluid (Ecolite+, MCP) and analyzed for 45Ca by liquid scintillation (LS6500, Beckman Coulter). Results are reported as percent of dose to correct for decay. Kinetic samples were corrected for the initial 45Ca dosing (week 1 of the study) by subtracting the amount of 45Ca detected in 24-h urine and feces collected one day prior to Ca kinetics performance. Total Ca content of urine and feces were analyzed by AA. Histomorphometry Rats were injected IP with 0.3 mL of calcein (10 mg/kg) solution 10 and 3 days before sacrifice (age 28 weeks). Left tibiae were cleaned, soaked in 10 % neutral buffered formalin for 72 h then stored in 70 % ethanol for future analysis. Bones were randomly chosen for histomorphometry analysis (n=8–9/treatment group). These bones were also analyzed for μCT analyses. Tibiae were cut 1.25 cm from the proximal end for trabecular bone analysis, and midshaft was sampled 2.5 mm distal from the tibia-fibulae junction for cortical bone. Bones were infiltrated with 70, 95, and 100 % ethanol for at least 24 h, 50:50 solution of ethanol and methyl methacrylate (MMA) for at least 48 h, and 100 % MMA for 1 week. For dynamic histomorphometry, trabecular bone (proximal tibiae) were sectioned at 4 μm thickness, and cortical bone (tibia midshaft) was sawed with a diamond saw and ground to ~50 μm thickness. Sections were mounted on slides and analyzed for fluorescent labeling. Labeled surface and width between labels were assessed by BioQuant Osteo v10 (Nashville, TN) at ×10 and ×20 magnification, respectively. Two slides per site per rat were analyzed, and values were combined to calculate bone formation rate (BFR), mineral apposition rate (MAR), and mineralizing surface over bone surface (MS/BS). Trabecular bone was measured beginning 0.5 mm distal from the growth plate and encompassing a region of interest of 8 and 10 mm2. For static histomorphometry, tartrate-resistant acid phosphatase (TRAP) staining was performed on proximal tibiae sections. Osteoclast surface to bone surface ratio (OcS/BS) was analyzed with BioQuant Osteo v11.2.30 (Nashville, TN) at ×20 magnification. Bone microstructure Left femurs and tibiae were cleaned of muscle, soaked in 10 % neutral buffered formalin for 72 h and then stored in 70 % ethanol for 3 days or more. A subgroup of these bones (n=8– 10/treatment group) was randomly selected for trabecular and
Osteoporos Int (2014) 25:1905–1915
cortical bone microarchitecture measurement and scanned by microcomputed tomography (μCT; μCT 40 Scanner; Scanco Medical, Switzerland). Tibiae and femurs were scanned at voxel size of 12 and 16 μm3, respectively (X-ray tube potential (peak), 55 kVP; integration time, 190 ms). Trabecular bone was scanned in 63 continuous slices beginning at 18 % from the distal end in the femur, for a total length of 1 mm, and starting where spongiosa was not present in the tibial proximal end proceeding distally for 1 mm. Every ten scans of trabecular bone were manually contoured. Scans were auto-morphed and evaluated with segmentation values of sigma=0.8, support=1, and binarization threshold of 318. Parameters evaluated include tissue volume (TV), bone volume (BV), relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Cortical bone was scanned at the midshaft of the femur and tibia. Cortical bone was separated using a semiautomatic contour tracking algorithm to detect the outer boundaries of the cortex. Parameters evaluated for cortical bone include total area (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th), and polar moment of inertia (MOI). Bone density and Ca content BMD was measured by water displacement with a density determination kit (model AG204, Mettler Toledo) in right femurs and tibias. Bones were ashed in a muffle furnace at 600 °C for 5 days, dissolved in 1 mL of concentrated HNO3, diluted and analyzed by inductively coupled plasma optical omission spectrometry (ICP-OES; Optima 4300 DV, Perkin Elmer Instrument) to determine total bone Ca. Mechanical testing Right femurs and tibias were cleaned of muscle and stored in saline soaked gauze. Length and midshaft diameter were measured with a digital Vernier caliper. Bone mechanical properties were assessed by 3-point bending (Test Resources, Inc., Shakopee, MN). Bones were thawed to room temperature, presoaked in 0.9 % saline, and placed posterior side down on the bottom support. Bones were loaded to failure using a displacement rate of 0.5 mm/min with force vs. displacement data collected at 10 Hz. Structural mechanical properties were determined from the load-deformation curves using standard definitions. Material properties (ultimate stress, modulus, and toughness) were calculated with geometrical information from the microCT scans and structural mechanical properties using standard equations.
Osteoporos Int (2014) 25:1905–1915
1909
Table 1 Calcium kinetics in 29-week-old rats fed different levels of Ca and vitamin D (VD) Treatment diet Ca (%)
0.2
1.0
VD (IU/kg diet) 50 8
N
100 9
1,000 9
50 10
Ca effect VD effect Ca×VD 100 8
1,000 7
Body weight (g)
316±7
310±11
311±7
313±8
329±8
320±14
0.58
0.83
0.48
Food intake (g/day) Ca intake (mg/day) Urine Ca (mg/day) Fecal Ca (mg/day) Net Ca absorption (mg/day) Fractional Ca absorption Bone resorption (mg/day) Bone formation (mg/day) Bone balance (mg/day) Endogenous secretion (mg/day) MC1 (mg) MC2 (mg)
10.3±0.6 25.4±1.2 1.4±0.2 18.1±1.6 13.0±0.7 0.52±0.04 30.0±1.5 35.7±1.1 5.8±0.7 5.8±0.2 5.6±0.2 11.7±0.4 b
10.9±0.5 26.6±1.0 1.3±0.2 18.7±1.1 14.6±0.9 0.55±0.04 30.7±2.3 37.5±2.2 6.9±0.8 6.5±0.4 5.7±0.3
11.2±0.4 27.2±0.8 1.5±0.2 19.1±2.0 16.6±1.5 0.61±0.06 31.7±2.6 38.9±1.5 7.2±1.6 8.0±0.3 6.2±0.2
10.2±0.3 99.8±2.1 1.3±0.1 86.8±2.9 22.0±3.1 0.22±0.03 23.2±5.4 35.0±3.4 11.7±3.2 8.9±0.9 5.4±0.5
11.5±0.5 111.6±4.7 1.7±0.2 87.6±6.9 30.8±3.1 0.29±0.04 16.6±2.4 37.2±1.9 20.6±2.7 8.5±0.4 6.4±0.3
10.8±0.2 108.5±2.3 1.3±0.2 92.4±3.1 25.7±2.4 0.24±0.02 20.8±2.6 35.6±2.3 14.8±2.2 9.5±0.6 5.4±0.4
0.78
ORIGINAL ARTICLE
Calcium and vitamin D intake maintained from preovariectomy independently affect calcium metabolism and bone properties in Sprague Dawley rats C. Y. Park & W. H. Lee & J. C. Fleet & M. R. Allen & G. P. McCabe & D. M. Walsh & C. M. Weaver
Received: 12 November 2013 / Accepted: 3 April 2014 / Published online: 17 April 2014 # International Osteoporosis Foundation and National Osteoporosis Foundation 2014
Abstract Summary The interaction of habitual Ca and vitamin D intake from preovariectomy to 4 months postovariectomy on bone and Ca metabolism was assessed. Higher Ca intake suppressed net bone turnover, and both nutrients independently benefitted trabecular structure. Habitual intake of adequate Ca and ~50 nmol/L vitamin D status is most beneficial. Introduction Dietary strategies to benefit bone are typically tested prior to or after menopause but not through menopause transition. We investigated the interaction of Ca and vitamin D C. Y. Park : J. C. Fleet : C. M. Weaver (*) Department of Nutrition Science, Purdue University, 700 W State St, West Lafayette, IN 47907, USA e-mail: [email protected] W. H. Lee Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907, USA M. R. Allen Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA G. P. McCabe Department of Statistics, Purdue University, West Lafayette, IN 47907, USA D. M. Walsh School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland Present Address: C. Y. Park Department of Biochemistry and Cell Biology, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Korea Present Address: W. H. Lee Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Korea
status on Ca absorption, bone remodeling, Ca kinetics, and bone strength as rats transitioned through estrogen deficiency. Methods Sprague Dawley rats were randomized at 8 weeks to 0.2 or 1.0 % Ca and 50, 100, or 1,000 IU (1.25, 2.5, or 25 μg) vitamin D/kg diet (2×3 factorial design) and ovariectomized at 12 weeks. Urinary 45Ca excretion from deep-labeled bone was used to assess net bone turnover weekly. Ca kinetics was performed between 25 and 28 weeks. Rats were killed at 29 weeks. Femoral and tibiae structure (by μCT), dynamic histomorphometry, and bone Ca content were assessed. Results Mean 25(OH)D for rats on the 50, 100, 1,000 IU vitamin D/kg diet were 32, 54, and 175 nmol/L, respectively. Higher Ca intake ameliorated net bone turnover, reduced fractional Ca absorption and bone resorption, and increased net Ca absorption. Tibial and femoral trabecular structures were enhanced independently by higher Ca and vitamin D intake. Tibial bone width and fracture resistance were enhanced by higher vitamin D intake. Dynamic histomorphometry in the tibia was not affected by either nutrient. A Ca × vitamin D interaction existed in femur length, tibial Ca content, and mass of the soft tissue/extracellular fluid compartment. Conclusions Adequate Ca intake and serum 25(OH)D level of 50 nmol/L provided the most benefit for bone health, mostly through independent effects of Ca and vitamin D. Keywords Calcium . Histomorphometry . Kinetics . Ovariectomy . Vitamin D
Introduction In a healthy population, gradual bone loss occurs in older adults and is dramatically accelerated during the first 3–5 years of menopause [1]. Aging and menopausal estrogen loss is associated with decreased Ca absorption [2] and estrogen
1906
therapy increases Ca absorption and 1,25-dihydroxy vitamin D (1,25(OH)2D) status, the active vitamin D metabolite [3]. Therefore, awareness for osteoporosis prevention increases at this life stage, and most intervention research has introduced treatment after menopause when bone loss has stabilized. Ca supplementation with or without vitamin D has been successful to decrease the risk of osteoporosis and loss of bone mineral density (BMD) in women that are over 4-year postmenopausal [4, 5]. However, because eating habits and preferences, and consequently nutrient intake, gradually change and stabilize throughout adulthood before menopause, the consistent intake of nutrients are more likely to impact bone loss during and after menopause. The independent effects of Ca and vitamin D intake on adult trabecular bone have been reported in ovary-intact female rats 25 weeks of age [6] and male rats age 30 weeks [7], respectively. However, the independent and interactive effect of habitual Ca and vitamin D intake on Ca metabolism during the period of transition into estrogen deficiency has not been investigated. Older individuals are at risk for inadequate intakes of both Ca and vitamin D. In women over 50 years in the USA, the average Ca intake from diet is only 65 % (~770 mg/day) of the recommended intake, only 39 % exceed the recommended 1,200 mg Ca/day, and 35 % do not take any supplements [8]. This is of more concern, because women who do not consume Ca supplements are more likely to have lower Ca intake from diet as well [9]. Approximately 45 % of US women have serum 25-hydroxyvitamin D (25(OH)D), a vitamin D status marker, below the target level of 50 nmol/L [10], and mean serum 25(OH)D levels decrease with age [11]. In 2005–2006, less than 45 % of women 51–70 years and only 22 % of women over 70 years exceeded their previous vitamin D recommendations of 400 and 600 IU/day, respectively, through diet and supplements [8]. As Ca and vitamin D intake during adulthood can impact bone health and most women in the USA are not consuming the recommended intakes, the long-term effect of nutrient intake during adulthood through menopause needs to be understood. Ca and vitamin D supplementation has become standard dietary therapy to reduce the risk of osteoporosis [12]. Systematic reviews and meta-analyses show that Ca and vitamin D supplementation increases BMD or decreases loss of bone mass in older adults [13, 14]. The Institute of Medicine (IOM) set new recommendations for Ca and vitamin D intakes in 2011 [10], recognizing the needs of Ca and vitamin D are interdependent through coordinated action on Ca absorption and retention. At an adequate intake, Ca is absorbed through active and passive transport. Active Ca transport is known to be highly efficient and activated when 1,25(OH)2D elicits the transcription of Ca transport proteins such as transient receptor potential vanilliod family member 6 (TRPV6), vitamin Ddependent Ca-binding protein 9 kDa form (calbindin D9k) and plasma membrane Ca2+ ATPase 1b (PMCA1b) [15].
Osteoporos Int (2014) 25:1905–1915
Adequate serum concentrations of the precursor 25(OH)D is required for retention of bone volume and parathyroid hormone (PTH) suppression in older rats [7]. Some crosssectional studies in adult humans suggest that vitamin D status is a better indicator of bone health than Ca intake [11, 16]. Very low (100 nmol/ L, respectively) in order that the results be applicable to vitamin D status and dietary vitamin D intake for the general population. Phosphorus (P) was increased to 0.75 % in the 1.0 % Ca diets to keep the Ca/P ratio consistent with the basal diet and was 0.3 % in the 0.2 % Ca diets to prevent the deleterious effects on bone due to P deficiency [22]. OVX was performed at 12 weeks of age. Uterine weight was measured at harvest to determine proper OVX. Ca kinetics was performed 3–4 months post-OVX when bone loss was stabilized. Six rats (n=1/gp) were killed prior to OVX for analyses not included in this paper. The remaining rats were killed at 29 weeks of age by CO2 overdose. All bone imaging was performed on bones excised at killing.
Blood was drawn from a subset of 37 rats (5–7/gp) 1 week before transition to treatment diet (at 7 weeks of age) and from all rats at killing. Serum 25(OH)D was assessed by radioimmunoassay (RIA; IDS, Immunodiagnostic Systems Limited, UK). The inter- and intra-assay CV was 8 and 10 %, respectively. Serum total Ca was analyzed by atomic absorption spectrometry (AA; AAnalyst, Perkin Elmer) in duplicate. 45
Ca deep bone labeling
After 1 week of stabilization to the facility, rats were dosed with 47 μCi 45Ca as 45CaCl2 (Perkin Elmer, Boston, MA) through injection via the tail (Braintree Scientific Inc., Braintree, MA). The 45Ca is incorporated into bone, which enables assessment of net bone turnover over time through urinary 45Ca excretion. Twenty-four-hour urine was collected weekly starting 1 week after 45Ca dosing (5 weeks of age) till 24 weeks of age. Ca kinetics Analysis of Ca kinetics was performed between 25 and 28 weeks of age (13–16 weeks post-OVX), a time when OVX-induced rapid bone loss was in a relatively stable state [23]. Rats were randomized into six different kinetic groups. Jugular catheters were inserted 2 days prior to performing Ca kinetics on each rat. For Ca kinetics, 45Ca was administered intravenously (10 μCi; n=3/diet group) or orally (20 μCi; n= 10/diet group). Fifteen milligrams of Ca as calcium acetate was dissolved in ultrapure water and administered by oral gavage with a total volume of 1 mL. This Ca load was approximately 1/3–1/2 of the mean daily Ca intake of rats fed the 0.2 % Ca diets and therefore sufficiently low to assess active Ca transport [24, 25]. Blood was drawn at 0 (baseline), 2, 10, 20, 40, 60, 120, 180, 360, 720, 1,440, 2,160, and 2,880 min (200 μL/draw) after Ca load was provided. Collected blood was immediately centrifuged and 80 μL of plasma was transferred to scintillation vials. Fifteen milliliters of scintillation fluid (Ecolite+, MCP) was added, and samples were analyzed for 45Ca by liquid scintillation (LS6500, Beckman Coulter). At 3-h postdose, 2 mL saline and 5 % dextrose 50:50 solution was injected for fluid replacement. Water and food were also replaced at this time. Rats were housed in metabolic cages and 24-h urine and feces were collected for 4 days, and 4-day food intake was monitored. Kinetic models were developed and analyzed using WinSAAM software (NIH). Details in kinetic modeling are described elsewhere [26]. In our model, compartment 1 denotes serum, compartment 2 indicates intracellular and extracellular fluid and soft tissue, and compartment 3 refers to the exchangeable pool. The mass of these compartments (MC 13) are also reported.
1908
Urine and fecal 45Ca and Ca analysis All urine collections were centrifuged and diluted with ultrapure water to the nearest 0.5 mL and stored at 4 °C for future analysis. Feces were ashed, dissolved with 1 mL HNO3, and diluted with 24 mL ultrapure water. One milliliter of urine samples from deep bone labeling and both urine and fecal samples from kinetics were transferred to a scintillation vial with 15 mL scintillation fluid (Ecolite+, MCP) and analyzed for 45Ca by liquid scintillation (LS6500, Beckman Coulter). Results are reported as percent of dose to correct for decay. Kinetic samples were corrected for the initial 45Ca dosing (week 1 of the study) by subtracting the amount of 45Ca detected in 24-h urine and feces collected one day prior to Ca kinetics performance. Total Ca content of urine and feces were analyzed by AA. Histomorphometry Rats were injected IP with 0.3 mL of calcein (10 mg/kg) solution 10 and 3 days before sacrifice (age 28 weeks). Left tibiae were cleaned, soaked in 10 % neutral buffered formalin for 72 h then stored in 70 % ethanol for future analysis. Bones were randomly chosen for histomorphometry analysis (n=8–9/treatment group). These bones were also analyzed for μCT analyses. Tibiae were cut 1.25 cm from the proximal end for trabecular bone analysis, and midshaft was sampled 2.5 mm distal from the tibia-fibulae junction for cortical bone. Bones were infiltrated with 70, 95, and 100 % ethanol for at least 24 h, 50:50 solution of ethanol and methyl methacrylate (MMA) for at least 48 h, and 100 % MMA for 1 week. For dynamic histomorphometry, trabecular bone (proximal tibiae) were sectioned at 4 μm thickness, and cortical bone (tibia midshaft) was sawed with a diamond saw and ground to ~50 μm thickness. Sections were mounted on slides and analyzed for fluorescent labeling. Labeled surface and width between labels were assessed by BioQuant Osteo v10 (Nashville, TN) at ×10 and ×20 magnification, respectively. Two slides per site per rat were analyzed, and values were combined to calculate bone formation rate (BFR), mineral apposition rate (MAR), and mineralizing surface over bone surface (MS/BS). Trabecular bone was measured beginning 0.5 mm distal from the growth plate and encompassing a region of interest of 8 and 10 mm2. For static histomorphometry, tartrate-resistant acid phosphatase (TRAP) staining was performed on proximal tibiae sections. Osteoclast surface to bone surface ratio (OcS/BS) was analyzed with BioQuant Osteo v11.2.30 (Nashville, TN) at ×20 magnification. Bone microstructure Left femurs and tibiae were cleaned of muscle, soaked in 10 % neutral buffered formalin for 72 h and then stored in 70 % ethanol for 3 days or more. A subgroup of these bones (n=8– 10/treatment group) was randomly selected for trabecular and
Osteoporos Int (2014) 25:1905–1915
cortical bone microarchitecture measurement and scanned by microcomputed tomography (μCT; μCT 40 Scanner; Scanco Medical, Switzerland). Tibiae and femurs were scanned at voxel size of 12 and 16 μm3, respectively (X-ray tube potential (peak), 55 kVP; integration time, 190 ms). Trabecular bone was scanned in 63 continuous slices beginning at 18 % from the distal end in the femur, for a total length of 1 mm, and starting where spongiosa was not present in the tibial proximal end proceeding distally for 1 mm. Every ten scans of trabecular bone were manually contoured. Scans were auto-morphed and evaluated with segmentation values of sigma=0.8, support=1, and binarization threshold of 318. Parameters evaluated include tissue volume (TV), bone volume (BV), relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Cortical bone was scanned at the midshaft of the femur and tibia. Cortical bone was separated using a semiautomatic contour tracking algorithm to detect the outer boundaries of the cortex. Parameters evaluated for cortical bone include total area (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th), and polar moment of inertia (MOI). Bone density and Ca content BMD was measured by water displacement with a density determination kit (model AG204, Mettler Toledo) in right femurs and tibias. Bones were ashed in a muffle furnace at 600 °C for 5 days, dissolved in 1 mL of concentrated HNO3, diluted and analyzed by inductively coupled plasma optical omission spectrometry (ICP-OES; Optima 4300 DV, Perkin Elmer Instrument) to determine total bone Ca. Mechanical testing Right femurs and tibias were cleaned of muscle and stored in saline soaked gauze. Length and midshaft diameter were measured with a digital Vernier caliper. Bone mechanical properties were assessed by 3-point bending (Test Resources, Inc., Shakopee, MN). Bones were thawed to room temperature, presoaked in 0.9 % saline, and placed posterior side down on the bottom support. Bones were loaded to failure using a displacement rate of 0.5 mm/min with force vs. displacement data collected at 10 Hz. Structural mechanical properties were determined from the load-deformation curves using standard definitions. Material properties (ultimate stress, modulus, and toughness) were calculated with geometrical information from the microCT scans and structural mechanical properties using standard equations.
Osteoporos Int (2014) 25:1905–1915
1909
Table 1 Calcium kinetics in 29-week-old rats fed different levels of Ca and vitamin D (VD) Treatment diet Ca (%)
0.2
1.0
VD (IU/kg diet) 50 8
N
100 9
1,000 9
50 10
Ca effect VD effect Ca×VD 100 8
1,000 7
Body weight (g)
316±7
310±11
311±7
313±8
329±8
320±14
0.58
0.83
0.48
Food intake (g/day) Ca intake (mg/day) Urine Ca (mg/day) Fecal Ca (mg/day) Net Ca absorption (mg/day) Fractional Ca absorption Bone resorption (mg/day) Bone formation (mg/day) Bone balance (mg/day) Endogenous secretion (mg/day) MC1 (mg) MC2 (mg)
10.3±0.6 25.4±1.2 1.4±0.2 18.1±1.6 13.0±0.7 0.52±0.04 30.0±1.5 35.7±1.1 5.8±0.7 5.8±0.2 5.6±0.2 11.7±0.4 b
10.9±0.5 26.6±1.0 1.3±0.2 18.7±1.1 14.6±0.9 0.55±0.04 30.7±2.3 37.5±2.2 6.9±0.8 6.5±0.4 5.7±0.3
11.2±0.4 27.2±0.8 1.5±0.2 19.1±2.0 16.6±1.5 0.61±0.06 31.7±2.6 38.9±1.5 7.2±1.6 8.0±0.3 6.2±0.2
10.2±0.3 99.8±2.1 1.3±0.1 86.8±2.9 22.0±3.1 0.22±0.03 23.2±5.4 35.0±3.4 11.7±3.2 8.9±0.9 5.4±0.5
11.5±0.5 111.6±4.7 1.7±0.2 87.6±6.9 30.8±3.1 0.29±0.04 16.6±2.4 37.2±1.9 20.6±2.7 8.5±0.4 6.4±0.3
10.8±0.2 108.5±2.3 1.3±0.2 92.4±3.1 25.7±2.4 0.24±0.02 20.8±2.6 35.6±2.3 14.8±2.2 9.5±0.6 5.4±0.4
0.78
