Calcium (Ca) supplements, especially Ca carbonate (CaCO3), are the main alternative sources of di... more Calcium (Ca) supplements, especially Ca carbonate (CaCO3), are the main alternative sources of dietary Ca and an important part of a treatment regimen for osteoporosis, the most common metabolic bone disorder of aging and menopause. In a female ovariectomized (OVX) rat model for studying postmenopausal osteoporosis, we tested the hypothesis that a small compared with a large particle size of CaCO3 (13.0- vs. 18.5-mum geometric diameter) would result in increased Ca balance and subsequently bone mass and that this would be affected by dietary Ca level. We used 6-mo-old rats that were OVX either at 6 or 3 mo of age as models of early or stable menopausal status, respectively. The rats received semipurified diets that contained either 0.4 or 0.2% dietary Ca provided from CaCO3 of 2 particle sizes. A group of Sham-operated rats with intact ovaries served as control and were fed 0.4% dietary Ca from large particles. Estrogen deficiency as a result of ovariectomy had an adverse effect on bone density, mineral content, and bone mechanical properties (P < 0.001). Reducing dietary Ca from 0.4 to 0.2% resulted in significant adverse effects on bone density and mechanical properties (P < 0.001). The particle size of CaCO3 did not affect total Ca balance, bone dual energy X-ray absorptiometry and peripheral quantitative computed tomography indices, bone ash and Ca content, or the mechanical determinants of bone strength. We conclude that a decrease in particle size of CaCO3 to 70% of that typically found in Ca supplements does not provide a benefit to overall Ca metabolism or bone characteristics and that the amount of Ca consumed is of greater influence in enhancing Ca nutrition and skeletal strength.
Absorption, distribution and elimination of 14 C-labelled isoflavone-containing extracts from kud... more Absorption, distribution and elimination of 14 C-labelled isoflavone-containing extracts from kudzu (Pueraria lobata) root culture and red clover (Trifolium pratense) cell culture were investigated in an in vivo rat model. The predominant isoflavones in the kudzu extract were the glycosides puerarin, daidzin and malonyl daidzin, while in the red clover extract, the major isoflavones were formononetin and its derivatives, genistein and biochanin A, with radioactivities of 3·770 and 7·256 MBq/g, respectively. Male Sprague -Dawley rats, implanted with a jugular catheter and a subcutaneous ultrafiltrate probe, were orally administered with 14 C-labelled isoflavone extracts from either kudzu or clover cell cultures. Serum, interstitial fluid (ISF), urine and faeces were collected using a Culex w Automated Blood Collection System for 24 h. Analysis of bone tissues revealed that radiolabel accumulated in the femur, tibia and vertebrae at 0·04, 0·03 and 0·01 % of the administered dose, respectively, in both kudzu and red clover treatments. The liver accumulated the greatest concentration of radiolabel among the tissues tested, at 1·99 and 1·54 % of the administered kudzu and red clover extracts, respectively. Serum and ISF analysis showed that both extracts were rapidly absorbed, distributed in various tissues, and largely eliminated in the urine and faeces. Urine and faeces contained 8·53 and 9·06 % of the kudzu dose, respectively, and 3·60 and 5·64 % of the red clover dose, respectively. Serum pharmacokinetics suggest that extracts from kudzu may undergo enterohepatic circulation.
Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and with... more Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and without metabolic syndrome (MetS) using Ca tracer kinetics and coronary atherosclerosis measured with intravascular ultrasound. Pigs with MetS had higher Ca uptake into coronary arteries than lean pigs. Introduction Ca deposition into arteries is a common disease in humans. The Ossabaw pig develops MetS when fed an atherogenic diet. The aim of this study was to measure Ca deposition into arteries of lean vs. MetS pigs. Methods Male pigs were fed for 5 months with chow diet (healthy, lean; n07) or atherogenic diet (n08) consisting of chow supplemented with 2 % cholesterol, 43 % kcal from fat, and 20 % kcal from fructose. Pigs were verified to have MetS by obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. Two pigs received 50 nCi of 41 Ca i.v. and blood was drawn frequently for 24 h, and 2, 15, 20, and at sacrifice at 28 days after injection. Peripheral arteries were biopsied four times per pig over the 28th day and coronary artery sampled at sacrifice. Tissues were analyzed for 41 Ca:Ca. A compartmental model was used to estimate rates of Ca deposition into the arteries. Results The MetS swine had higher 41 Ca and atherosclerosis in coronary arteries than lean pigs. Conclusions This pig model is a suitable model for studying vascular calcification in humans.
The effects of honey and its carbohydrate constituents (glucose, fructose, and raffinose) on calc... more The effects of honey and its carbohydrate constituents (glucose, fructose, and raffinose) on calcium absorption in rats were investigated in acute and chronic feeding studies. In the acute study, rats (n ) 120) were gavaged with an oral solution consisting of (a) 10 µCi 45 Ca, (b) 25 mg of calcium as calcium acetate, and (c) one of the following: 0 mg of honey (control), or 200, 500, or 800 mg of honey, a glucose-fructose mixture, 10.75 mg of raffinose, or 200 mg of raffinose. Another group received 45 Ca intraperitoneally. Femurs were collected 2 days later and analyzed for 45 Ca content. Rats given 500 and 800 mg of honey showed 25.5 and 33.6% increases in calcium absorption (P < 0.05), respectively, over the control group. Groups given the glucose-fructose mixture or 200 mg of raffinose had a significantly higher increase in calcium absorption than the control group (17.1 and 25.6%, respectively). In the chronic study, rats (n ) 96) were fed for 8 weeks with either 0% honey (control), 5% honey, 10% honey, or a glucose-fructose-raffinose (GFR) mixture. Femurs of GFRfed rats had significantly lower calcium content, 45 Ca absorption, width, and BMD (at distal region) than control rats. Groups fed honey did not show the negative effects of GFR on bone, but had no advantage over the control group. No significant differences were observed in femur length, density, strength, or BMC among any treatment group compared to the control group. These results indicate that although a positive dose-response effect of honey and its carbohydrate constituents on calcium absorption was observed in the acute study, this effect disappeared upon long-term feeding in rats, implying adaptation had occurred.
New food sources are needed to bridge the gap between vitamin D intake and recommended intake. We... more New food sources are needed to bridge the gap between vitamin D intake and recommended intake. We assessed the bioavailability and efficacy of vitamin D in an 8 week dose-response study of bread made with vitamin D 2 -rich yeast compared to vitamin D 3 in growing, vitamin D-deficient rats. Plasma 25-hydroxyvitamin D (25OHD) levels increased in a curvilinear, dosedependent manner with both forms of vitamin D, but rats fed vitamin D 2 -rich yeast achieved lower levels than rats fed vitamin D 3 . Rats fed the highest doses of vitamin D had significantly greater (p < 0.05) trabecular BMC, BMD, bone volume, and connectivity density, and greater midshaft total cross-sectional area, compared to rats on the vitamin D-deficient diets, with no significant difference due to vitamin D source. Vitamin D 2 -rich yeast baked into bread is bioavailable and improves bone quality in vitamin D-deficient animals.
Hairy roots were induced in four genotypes from three kudzu species (Pueraria montana var. lobata... more Hairy roots were induced in four genotypes from three kudzu species (Pueraria montana var. lobata, P. lobata and P. phaseoloides) in vitro using Agrobacterium rhizogenes to stimulate rapid secondary metabolite synthesis. Hairy roots from P. montana var. lobata (United States Department of Agriculture no. PI 434246) yielded the highest puerarin and total isoflavone content and the greatest new biomass per growth cycle among the genotypes evaluated. Hairy roots from this genotype were selected for radiolabeling using 14 C-sucrose as a carbon source. Isoflavones from radiolabeled kudzu hairy root cultures were extracted with 80% methanol, partitioned by solvent extraction, and then subfractionated by Sephadex LH-20 gel filtration. Radiolabeled isoflavones were isolated in a highly enriched fraction, which contained predominantly puerarin, daidzin and malonyl-daidzin and had an average radioactivity of 8.614 MBq/g (232.8 mCi/g) dry fraction. The 14 Cradiolabeled, isoflavone-rich fraction was orally administered at a dose of 60 mg/kg body weight to male Sprague-Dawley rats implanted with a jugular catheter, a subcutaneous ultrafiltrate probe and a brain microdialysate probe. Serum, interstitial fluid, brain microdialysate, urine and feces were collected using a Culex w Automated Blood Collection System for 24 h. At the end of this period, rats were sacrificed and major tissues were collected. Analysis by a scintillation counter confirmed that a bolus dose of 14 C-radiolabeled, isoflavone-rich kudzu fraction reached bone tissues, which accumulated 0.011%, 0.09% and 0.003% of the administered dose in femur, tibia and vertebrae, respectively. Femurs extracted with 80% methanol were analyzed by high-performance liquid chromatography with electrospray ionization -mass spectrometry and were found to contain trace quantities of puerarin, daidzein and puerarin glucuronide. This study demonstrates that kudzu isoflavones and metabolites are capable of reaching bone tissues, where they may contribute to the prevention of osteoporosis and the promotion of bone health.
Dairy products provide most of the calcium in the diet, but consumption of milk has declined stea... more Dairy products provide most of the calcium in the diet, but consumption of milk has declined steadily over the last six decades. We determined (1) benefits to bone formation in growing female Sprague-Dawley rats when calcium is derived from dairy versus CaCO 3 and (2) residual benefits of calcium provided by dairy to rats subsequently receiving a low-calcium diet. During growth, femurs from rats fed nonfat dry milk solids (NFDM) had 8.4% higher peak breaking force, 6.4% greater Ca content, 4.8% greater weight, 4% greater width, 1.2% greater density, 13.1% greater midshaft cortical thickness, and 16.7% greater midshaft cortical area than from rats fed CaCO 3 . These effects were unrelated to differences in calcium absorption or serum IGF-1, but the NFDM group had higher rates of bone formation. If maintained on an adequate calcium diet, many of these advantages disappeared. However, rats fed adequate Ca as NFDM versus CaCO 3 during growth and subsequently switched to deficient Ca as CaCO 3 had significantly (p < 0.0001) higher femoral BMD (1.3%), total bone Ca (7.2%), Ca concentration (4.6%), and cortical thickness (9.4%) and a trend (p = 0.02) toward greater peak breaking force (17%). Thus, NFDMs improved bone measures during growth and protected bone against a subsequent period of calcium depletion compared with CaCO 3 .
Calcium (Ca) supplements, especially Ca carbonate (CaCO3), are the main alternative sources of di... more Calcium (Ca) supplements, especially Ca carbonate (CaCO3), are the main alternative sources of dietary Ca and an important part of a treatment regimen for osteoporosis, the most common metabolic bone disorder of aging and menopause. In a female ovariectomized (OVX) rat model for studying postmenopausal osteoporosis, we tested the hypothesis that a small compared with a large particle size of CaCO3 (13.0- vs. 18.5-mum geometric diameter) would result in increased Ca balance and subsequently bone mass and that this would be affected by dietary Ca level. We used 6-mo-old rats that were OVX either at 6 or 3 mo of age as models of early or stable menopausal status, respectively. The rats received semipurified diets that contained either 0.4 or 0.2% dietary Ca provided from CaCO3 of 2 particle sizes. A group of Sham-operated rats with intact ovaries served as control and were fed 0.4% dietary Ca from large particles. Estrogen deficiency as a result of ovariectomy had an adverse effect on bone density, mineral content, and bone mechanical properties (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). Reducing dietary Ca from 0.4 to 0.2% resulted in significant adverse effects on bone density and mechanical properties (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). The particle size of CaCO3 did not affect total Ca balance, bone dual energy X-ray absorptiometry and peripheral quantitative computed tomography indices, bone ash and Ca content, or the mechanical determinants of bone strength. We conclude that a decrease in particle size of CaCO3 to 70% of that typically found in Ca supplements does not provide a benefit to overall Ca metabolism or bone characteristics and that the amount of Ca consumed is of greater influence in enhancing Ca nutrition and skeletal strength.
Absorption, distribution and elimination of 14 C-labelled isoflavone-containing extracts from kud... more Absorption, distribution and elimination of 14 C-labelled isoflavone-containing extracts from kudzu (Pueraria lobata) root culture and red clover (Trifolium pratense) cell culture were investigated in an in vivo rat model. The predominant isoflavones in the kudzu extract were the glycosides puerarin, daidzin and malonyl daidzin, while in the red clover extract, the major isoflavones were formononetin and its derivatives, genistein and biochanin A, with radioactivities of 3·770 and 7·256 MBq/g, respectively. Male Sprague -Dawley rats, implanted with a jugular catheter and a subcutaneous ultrafiltrate probe, were orally administered with 14 C-labelled isoflavone extracts from either kudzu or clover cell cultures. Serum, interstitial fluid (ISF), urine and faeces were collected using a Culex w Automated Blood Collection System for 24 h. Analysis of bone tissues revealed that radiolabel accumulated in the femur, tibia and vertebrae at 0·04, 0·03 and 0·01 % of the administered dose, respectively, in both kudzu and red clover treatments. The liver accumulated the greatest concentration of radiolabel among the tissues tested, at 1·99 and 1·54 % of the administered kudzu and red clover extracts, respectively. Serum and ISF analysis showed that both extracts were rapidly absorbed, distributed in various tissues, and largely eliminated in the urine and faeces. Urine and faeces contained 8·53 and 9·06 % of the kudzu dose, respectively, and 3·60 and 5·64 % of the red clover dose, respectively. Serum pharmacokinetics suggest that extracts from kudzu may undergo enterohepatic circulation.
Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and with... more Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and without metabolic syndrome (MetS) using Ca tracer kinetics and coronary atherosclerosis measured with intravascular ultrasound. Pigs with MetS had higher Ca uptake into coronary arteries than lean pigs. Introduction Ca deposition into arteries is a common disease in humans. The Ossabaw pig develops MetS when fed an atherogenic diet. The aim of this study was to measure Ca deposition into arteries of lean vs. MetS pigs. Methods Male pigs were fed for 5 months with chow diet (healthy, lean; n07) or atherogenic diet (n08) consisting of chow supplemented with 2 % cholesterol, 43 % kcal from fat, and 20 % kcal from fructose. Pigs were verified to have MetS by obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. Two pigs received 50 nCi of 41 Ca i.v. and blood was drawn frequently for 24 h, and 2, 15, 20, and at sacrifice at 28 days after injection. Peripheral arteries were biopsied four times per pig over the 28th day and coronary artery sampled at sacrifice. Tissues were analyzed for 41 Ca:Ca. A compartmental model was used to estimate rates of Ca deposition into the arteries. Results The MetS swine had higher 41 Ca and atherosclerosis in coronary arteries than lean pigs. Conclusions This pig model is a suitable model for studying vascular calcification in humans.
The effects of honey and its carbohydrate constituents (glucose, fructose, and raffinose) on calc... more The effects of honey and its carbohydrate constituents (glucose, fructose, and raffinose) on calcium absorption in rats were investigated in acute and chronic feeding studies. In the acute study, rats (n ) 120) were gavaged with an oral solution consisting of (a) 10 µCi 45 Ca, (b) 25 mg of calcium as calcium acetate, and (c) one of the following: 0 mg of honey (control), or 200, 500, or 800 mg of honey, a glucose-fructose mixture, 10.75 mg of raffinose, or 200 mg of raffinose. Another group received 45 Ca intraperitoneally. Femurs were collected 2 days later and analyzed for 45 Ca content. Rats given 500 and 800 mg of honey showed 25.5 and 33.6% increases in calcium absorption (P < 0.05), respectively, over the control group. Groups given the glucose-fructose mixture or 200 mg of raffinose had a significantly higher increase in calcium absorption than the control group (17.1 and 25.6%, respectively). In the chronic study, rats (n ) 96) were fed for 8 weeks with either 0% honey (control), 5% honey, 10% honey, or a glucose-fructose-raffinose (GFR) mixture. Femurs of GFRfed rats had significantly lower calcium content, 45 Ca absorption, width, and BMD (at distal region) than control rats. Groups fed honey did not show the negative effects of GFR on bone, but had no advantage over the control group. No significant differences were observed in femur length, density, strength, or BMC among any treatment group compared to the control group. These results indicate that although a positive dose-response effect of honey and its carbohydrate constituents on calcium absorption was observed in the acute study, this effect disappeared upon long-term feeding in rats, implying adaptation had occurred.
New food sources are needed to bridge the gap between vitamin D intake and recommended intake. We... more New food sources are needed to bridge the gap between vitamin D intake and recommended intake. We assessed the bioavailability and efficacy of vitamin D in an 8 week dose-response study of bread made with vitamin D 2 -rich yeast compared to vitamin D 3 in growing, vitamin D-deficient rats. Plasma 25-hydroxyvitamin D (25OHD) levels increased in a curvilinear, dosedependent manner with both forms of vitamin D, but rats fed vitamin D 2 -rich yeast achieved lower levels than rats fed vitamin D 3 . Rats fed the highest doses of vitamin D had significantly greater (p < 0.05) trabecular BMC, BMD, bone volume, and connectivity density, and greater midshaft total cross-sectional area, compared to rats on the vitamin D-deficient diets, with no significant difference due to vitamin D source. Vitamin D 2 -rich yeast baked into bread is bioavailable and improves bone quality in vitamin D-deficient animals.
Hairy roots were induced in four genotypes from three kudzu species (Pueraria montana var. lobata... more Hairy roots were induced in four genotypes from three kudzu species (Pueraria montana var. lobata, P. lobata and P. phaseoloides) in vitro using Agrobacterium rhizogenes to stimulate rapid secondary metabolite synthesis. Hairy roots from P. montana var. lobata (United States Department of Agriculture no. PI 434246) yielded the highest puerarin and total isoflavone content and the greatest new biomass per growth cycle among the genotypes evaluated. Hairy roots from this genotype were selected for radiolabeling using 14 C-sucrose as a carbon source. Isoflavones from radiolabeled kudzu hairy root cultures were extracted with 80% methanol, partitioned by solvent extraction, and then subfractionated by Sephadex LH-20 gel filtration. Radiolabeled isoflavones were isolated in a highly enriched fraction, which contained predominantly puerarin, daidzin and malonyl-daidzin and had an average radioactivity of 8.614 MBq/g (232.8 mCi/g) dry fraction. The 14 Cradiolabeled, isoflavone-rich fraction was orally administered at a dose of 60 mg/kg body weight to male Sprague-Dawley rats implanted with a jugular catheter, a subcutaneous ultrafiltrate probe and a brain microdialysate probe. Serum, interstitial fluid, brain microdialysate, urine and feces were collected using a Culex w Automated Blood Collection System for 24 h. At the end of this period, rats were sacrificed and major tissues were collected. Analysis by a scintillation counter confirmed that a bolus dose of 14 C-radiolabeled, isoflavone-rich kudzu fraction reached bone tissues, which accumulated 0.011%, 0.09% and 0.003% of the administered dose in femur, tibia and vertebrae, respectively. Femurs extracted with 80% methanol were analyzed by high-performance liquid chromatography with electrospray ionization -mass spectrometry and were found to contain trace quantities of puerarin, daidzein and puerarin glucuronide. This study demonstrates that kudzu isoflavones and metabolites are capable of reaching bone tissues, where they may contribute to the prevention of osteoporosis and the promotion of bone health.
Dairy products provide most of the calcium in the diet, but consumption of milk has declined stea... more Dairy products provide most of the calcium in the diet, but consumption of milk has declined steadily over the last six decades. We determined (1) benefits to bone formation in growing female Sprague-Dawley rats when calcium is derived from dairy versus CaCO 3 and (2) residual benefits of calcium provided by dairy to rats subsequently receiving a low-calcium diet. During growth, femurs from rats fed nonfat dry milk solids (NFDM) had 8.4% higher peak breaking force, 6.4% greater Ca content, 4.8% greater weight, 4% greater width, 1.2% greater density, 13.1% greater midshaft cortical thickness, and 16.7% greater midshaft cortical area than from rats fed CaCO 3 . These effects were unrelated to differences in calcium absorption or serum IGF-1, but the NFDM group had higher rates of bone formation. If maintained on an adequate calcium diet, many of these advantages disappeared. However, rats fed adequate Ca as NFDM versus CaCO 3 during growth and subsequently switched to deficient Ca as CaCO 3 had significantly (p < 0.0001) higher femoral BMD (1.3%), total bone Ca (7.2%), Ca concentration (4.6%), and cortical thickness (9.4%) and a trend (p = 0.02) toward greater peak breaking force (17%). Thus, NFDMs improved bone measures during growth and protected bone against a subsequent period of calcium depletion compared with CaCO 3 .
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