Dietary calcium and phosphate restriction in guinea-pigs during pregnancy: fetal mineralization induces maternal hypocalcaemia despite increased 1 alpha,25-dihydroxycholecalciferol concentrations

Guinea-pig fetuses at term are mineralized to a degree comparable with human fetuses, which makes the guinea-pig an attractive animal model to study maternal-fetal interactions with regard to Ca and phosphate (P) homeostasis. We studied non-pregnant and pregnant (day 57) vitamin D-replete guinea-pigs, fed either a normal guinea-pig chow with 9.6 g Ca/kg and 4.9 g P/kg or a study diet with 2 g Ca/kg and 1 g P/kg (low-Ca-P diet) for 7-8 weeks. Both pregnancy and the low-Ca-P diet decreased plasma concentrations of 25-hydroxycholecalciferol (25(OH)D3), but increased total and free 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3), strongly suggesting an additive stimulation of 1 alpha-hydroxylase activity. Maternal and fetal 25(OH)D3 and 1,25(OH)2D3 levels were highly correlated (r 0.82 and 0.92 respectively, P < 0.001). Dual-energy absorption X-ray absorptiometry (DXA) showed that both pregnancy and the low-Ca-P diet decreased bone mineral density (BMD) of the maternal femur, particularly at the distal metaphysis. Despite higher 1,25(OH)2D3 concentrations and lower BMD, pregnant animals on the low-Ca-P diet were hypocalcaemic; blood Ca2+ levels were inversely correlated with the number of fetuses in this group (r -0.93, P < 0.001). Fetal growth as well as mineralization (assessed by whole-body and femoral DXA, bone histomorphometry and plasma-bone osteocalcin measurements) were unaltered in the low-Ca-P group. In conclusion, fetal mineralization proceeds normally but induces maternal hypocalcaemia in guinea-pigs with dietary restriction of Ca and P.     

Resection of rat small intestine: calcium, phosphorus, and fat balances and 1,25-dihydroxycholecalciferol

In rat with massive resection of mid-small intestine, calcium transport per segment, measured by in vivo perfusion 10 days after surgery, is decreased in duodenum and ileum but is the same in cecum and colon as compared with controls with transection and reanastomosis of mid-small intestine. To extend these findings, we measured balances of calcium, phosphorus, and fat from the 5th to 10th day after surgery and serum concentration of 1,25-dihydroxycholecalciferol on the 10th day after surgery in this experimental model. We found steatorrhea in the resected group, but balances of calcium and phosphorus and serum levels of 1,25-dihydroxycholecalciferol were the same in resected and control groups. We conclude that decreased transport defined by direct examination of membrane function may be undetectable when net transport is measured by balance. Calcium balance during early post-resection period provides no evidence for future calcium deficiency in this experimental model.     

Dietary protein and phosphorus: effect on calcium and phosphorus metabolism in bone, blood and muscle of the rat

Four- or 12-week-old rats were fed diets containing either 25 or 45% casein and either 0.35 or 0.8% phosphorus (P) for 7 weeks. Calcium (Ca) level of each diet was 0.9%. In the young, fast-growing rat (expt 1), plasma P was increased (P less than 0.01) when 0.8%, rather than 0.35%, P was fed. In the more mature rats (expt 2), increased dietary P depressed Ca in plasma but elevated P; high protein intake elevated whole-blood Ca, but depressed plasma P. Dietary treatment had little effect on muscle of mature rats, but wet weight and total P content of muscle were higher in young rats fed 0.8%, rather than 0.35%, P. Femur dry weight and P concentration were greater when 0.8%, rather than 0.35%, P was fed to young rats; dietary P did not affect any other parameter of bone (expt 1, 2). Excessive protein intake elevated femur P concentration in young rats, but depressed length and strength of femurs in mature rats. Some parameters of bone, blood and muscle can be influenced by dietary levels of protein and P. The response, however, is dependent on the physical maturity of the rat when dietary treatment begins.     

The effects of zinc and calcium deficiencies on the rate of bone resorption in the rat

An effect of zinc deficiency on bone resorption was assessed in rats, by measuring the rate of resorption in rapidly growing animals and a pathologically increased resorption secondary to calcium deficiency. Bone resorption was quantitated by the loss of ³H-tetracycline from previously labeled bone. There was no effect of zinc deficiency on bone resorption in either normally growing or calcium deficient animals. Calcium deficiency alleviated the effect of zinc deficiency on body weight. In calcium deficient animals, bone zinc concentration was almost double that of calcium supplemented animals, suggesting a partial substitution of zinc for calcium in bone mineral.     

Comparison of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 on the resorption of bone explants ex vivo.

26,27-Hexafluoro-1 alpha,25-dihydroxyvitamin D3 [F6-1,25-(OH)2D3] is more potent than 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in stimulating bone resorption in vitro and in vivo. The reason why F6-1,25(OH)2D3 is more active remains unclear. To clarify the relationship between the bone-resorbing activity of each vitamin D3 analogue and the metabolism of each analogue, in the present study, we used an ex vivo method that was established by Reynolds et al (Calcif Tissue Res, 1974, 15, 333-339). The effect of F6-1,25(OH)2D3 or 1,25(OH)2D3 on 45Ca release from parietal bones, prepared at 3, 14 and 24 h after injection of 1.9, 3.8, 7.6 or 15.2 pmol vitamin D analog/g body weight, was examined. F6-1,25(OH)2D3 was more potent than 1,25(OH)2D3 during each in vivo time period. 1,25(OH)2D3 at 3 h after the injection was more active compared to the control (no injection of 1,25(OH)2D3) but not at 14 and 24 h. The radioactivity of the bones after the injection of [3H]-F6-1,25(OH)2D3 was retained even at 24 h. In the case of [3H]-1,25(OH)2D3, the radioactivity of bones decreased with an increase in the in vivo period. In a HPLC analysis of the lipid extract of bone homogenate, [3H]-F6-1,25(OH)2D3 alone was detected at 3 h after the injection and both [3H]-F6-1,25(OH)2D3 and [3H]-26,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3 [F6-1,23,25(OH)3D3] were detected at 14 and 24 h after the injection. [3H]-1,25(OH)2D3 was highly detected at 3 h after the injection, but it decreased with an increase in the in vivo period. In the ex vivo test, the activity of F6-1,23,25(OH)3D3 was less than that of F6-1,25(OH)2D3 but similar to that of 1,25(OH)2D3. The present study indicates that F6-1,25(OH)2D3 is more active and more long-lasting than 1,25(OH)2D3 in the ex vivo method. A higher potency of F6-1,25(OH)2D3 is explained, at least partly, by the results that the amounts of both F6-1,25(OH)2D3 and its active metabolite, F6-1,23,25(OH)3D3, in the bones are higher than that of 1,25(OH)2D3, and that F6-1,25(OH)2D3 and its metabolite are retained in bones longer than 1,25(OH)2D3.     

血清钙、磷、锌元素及骨碱性磷酸酶在佝偻病患儿中的水平分析

【目的】 分析不同病情佝偻病(different rickets,DR)患儿血清中钙、磷、锌及骨碱性磷酸酶的水平。 【方法】 选择广州市地区DR患儿60例,并按不同病情分轻度、中、重度组,同时选择60例同期体检健康儿童一同进行血清钙、磷、锌及骨碱性磷酸酶的双盲检测。 【结果】 中、重度DR 组的血清钙、磷和锌水平均明显低于对照组,轻度DR组与对照组差异无统计学意义;DR组骨碱性磷酸酶均明显高于对照组(P均<0.05)。在DR组内,中、重度DR 组的外周血清钙、磷和锌水平均明显低于轻度DR 组,骨碱性磷酸酶亦明显高于后者(P均<0.05)。 【结论】 骨碱性磷酸酶结合血清钙、磷、锌水平是反映佝偻病病情的敏感指标。     

Effect of oligofructose or dietary calcium on repeated calcium and phosphorus balances,bone mineralization and trabecular structure in ovariectomized rats*

We investigated the effects of dietary oligofructose and Ca on bone structure in ovariectomized rats, using microradiography and histomorphometry. Ninety-six animals were allocated to seven experimental groups: G1, sham-operated; G2-G7, ovariectomized. Semi-purified diets containing 5 g Ca/kg (recommended content) without oligofructose (G1, G2) or with 25, 50 or 100 g oligofructose/kg (G3, G4, G5) or 10 g Ca/kg (high content) without oligofructose (G6) or with 50 g oligofructose/kg (G7) were fed for 16 weeks. At the recommended level of Ca, high oligofructose (G5) increased femur mineral levels in ovariectomized rats, while medium oligofructose did so at high Ca. Increasing Ca in the absence of oligofructose did not increase femur mineral content. Trabecular bone area (%) analysed in the tibia was 10.3 (sem 1.2) (G1), 7.7 (sem 0.6) (G2), 9.3 (sem 0.7) (G3), 9.4 (sem 1.0) (G4), 9.5 (sem 0.7) (G5), 10.2 (sem 0.8) (G6), and 12.6 (sem 0.8) (G7). At the recommended level of Ca, 25 g oligofructose/kg prevented loss of trabecular area due to increased trabecular thickness, while 50 or 100 g oligofructose/kg increased trabecular perimeter. At high Ca, oligofructose prevented loss of bone area due to increased trabecular number but similar thickness (G7 v. G6). When Ca was raised in the presence of oligofructose (G7), trabecular area and cortical thickness were highest, while loss of trabecular connectivity was lowest of all groups. At the same time, lumbar vertebra Ca was higher; 44.0 (sem 0.8) (G7) compared with 41.6 (sem 0.8) (G2), 41.4 (sem 0.7) (G4), and 40.5 (sem 1.0) mg (G6). We conclude that ovariectomy-induced loss of bone structure in the tibia was prevented but with different trabecular architecture, depending on whether dietary Ca was increased, oligofructose was incorporated, or both. Oligofructose was most effective when dietary Ca was high.     

1alpha,25-dihydroxycholecalciferol increases the expression of vascular endothelial growth factor in C3H10T1/2 mouse embryo fibroblasts

Evidence suggests that biologically active vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)], may inhibit carcinogenesis. Because angiogenesis is crucial to carcinogenesis, 1,25(OH)(2)D(3) regulation of proangiogenic vascular endothelial growth factor (VEGF) secretion was investigated in cellular models for multistage carcinogenesis. Conditioned media from 1,25(OH)(2)D(3)-treated C3H10T(1/2) mouse fibroblasts and their Harvey ras-oncogene transfected counterparts (rasneo11a cells) induced human umbilical vein endothelial cell (HUVEC) proliferation (1.3 and 0.3 times, respectively, P < 0.05), suggesting that 1,25(OH)(2)D(3) altered the angiogenic phenotype of the cells. Although rasneo11a cells secreted less VEGF than C3H10T(1/2) cells (97%, P < 0.005), 1,25(OH)(2)D(3) induced C3H10T(1/2) and rasneo11a cells to secrete 2 and 3 times, respectively, more VEGF than controls (P < 0.05). Similar effects on VEGF release occurred after 1,25(OH)(2)D(3) treatment of MCF10A and MCF10Aras cells, a human breast epithelial cell model for multistage carcinogenesis. In C3H10T(1/2) cells, 1,25(OH)(2)D(3) activated the VEGF promoter in a dose-dependent (5-100 nmol/L) manner (maximum 60%) and all doses induced VEGF secretion (P < 0.05). 1,25(OH)(2)D(3) induced VEGF mRNA expression ( approximately 50%) from 2 through 24 h; VEGF release was significantly increased at 8 h and sustained for 24 h. VEGF mRNA expression and release declined as C3H10T(1/2) cells grew more confluent, whereas the magnitude of 1,25(OH)(2)D(3)-stimulated changes in VEGF was greater in confluent (3.3 times RNA; 3.5 times release) than in subconfluent (50% RNA; 100% release) cultures (P < 0.05). Thus, 1,25(OH)(2)D(3) increases VEGF secretion, and in C3H10T(1/2) cells, this is likely through activation of the VEGF promoter and induction of gene expression. These data contribute to understanding the role 1,25(OH)(2)D(3) plays in regulation of angiogenesis in normal compared with disease states.     

"生长痛"患者骨密度与血钙、磷及碱性磷酸酶研究

目的探讨生长痛与钙营养的关系.方法检测生长痛患者骨密度及血钙、磷、碱性磷酸酶.结果生长痛患者中骨密度比峰值减少13%～24%者及比峰值减少25%以上者(即骨质疏松者)分别占26%和63%,表明生长痛患者普遍存在骨钙营养不良;但血钙正常,甚至偏高,90%生长痛患者钙磷乘积大于40,90%患者碱性磷酸酶增高,提示其骨基质形成活跃及钙沉积过程正常.结论生长痛是一种骨钙营养不良,骨矿物质含量下降引起的骨痛;其钙沉积过程不存在障碍,可能是钙营养不良,钙沉积量长期存在不足所致;骨密度检查可作为其灵敏确诊手段.     

Design and data quality of a mixed longitudinal study to elucidate the role of dietary calcium and phosphorus on bone mineralization in pre-, peri-, and postmenopausal women

The study design and data quality control of an ongoing study (10 yr duration) in a few hundred women are presented. Good variables with respect to their longitudinal usefulness are: body weight, body height, and span-width. Reasonable variables are the bone parameters of the radius (BMC, BW, and BMC/BW). Poor variables are: dietary calcium and phosphorus intake, dietary calcium-to-phosphorus ratio, urinary calcium-to-creatinine ratio, urinary sodium-to-creatinine ratio, hematocrit, serum alkaline phosphatase activity, serum gamma-GT activity, and serum parathyroid-hormone concentration. Bad variables are: urinary phosphorus-to-creatinine ratio, urinary hydroxyproline-to-creatinine ratio, creatinine clearance, hemoglobin, MCHC, serum calcium, serum ionized calcium, serum phosphorus, serum total protein, serum albumin, and serum creatinine. In conclusion, it is possible to relate bone loss to food intake and to changes in anthropometric variables on an individual basis. However, quantification of the metabolic process is not possible.     

Effects of calcium phosphate supplementation on calcium,phosphorus, and magnesium metabolism in the Wistar rat

Calcium supplementation has been used in humans and in experimental animals to retard bone loss and lower blood pressure, but there are few data in regard to the metabolic effects of supplementation. This study evaluated the impact of supplementing the AIN-76 diet with various amounts of CaHPO₄ on food intake, growth, Ca, Mg and P balance and serum and bone concentrations of these nutrients. Four groups of nine 5-wk old normal male Wistar rats were fed ad libitum for 12 wks the AIN-76 semi-purified diet either unsupplemented (nominally 0.5% Ca), or supplemented to approximately 1.0%, 1.4% or 2.2% Ca with CaHPO₄. Another group of five rats was fed Rat Chow^®. Addition of CaHPO₄ did not alter intake of other nutrients. Dietary Ca level had no effect on whole blood or plasma ionized Ca, plasma total Ca or plasma P in fasted animals, whereas plasma Mg tended to decline with increased Ca supplementation. Increased Ca balance followed CaHPO₄ supplementation, whereas Mg balance declined. P balance was positive and similar in all study groups. Femur dry weight and length did not differ with diet nor did bone Ca content increase with supplementation. Bone Mg content decreased with CaHPO₄ supplementation. The results indicate that ingestion of CaHPO₄ in supplementary amounts does not increase bone Ca in the young normal rat but it is accompanied by other metabolic changes, particularly a striking decrease in net absorption of Mg.     

葛根素联合雌二醇对去卵巢大鼠骨组织及血钙、磷和碱性磷酸酶的影响

目的:了解小剂量葛根素联合雌二醇对去卵巢大鼠的骨组织、血钙、磷和碱性磷酸酶的影响,为中西医结合治疗绝经后骨质疏松症提供实验依据。方法:5月龄健康雌性大白鼠120只,分成5个实验组(每组24只):①假手术组(sham);②去卵巢模型组(OVX);③葛根素组(Pr),皮下注射葛根素,50 mg/kg,1次/d;④雌二醇组(E2),皮下注射雌二醇200μg/kg,2次/周;⑤小剂量葛根素+雌二醇组(Pr+E2),皮下注射雌二醇100μg/kg,2次/周和葛根素25 mg/kg,1次/d。各实验组在第4、8、12和20周,随机取6只大鼠取股骨切片观察骨组织,采血测量血钙、磷和碱性磷酸酶,数据进行统计学分析。结果:OVX组第4、8、12、20周的骨组织呈骨质疏松病理改变,血钙、磷明显低于sham组(P﹤0.01),OVX组的第4、8、12、20周血碱性磷酸酶均明显高于sham组(P﹤0.01)。3个治疗组各时间的骨组织和血钙、磷和碱性磷酸酶与sham组无统计学意义(P﹥0.05)。小剂量的葛根素联合雌二醇治疗能使去卵巢大鼠骨组织和血钙、磷和血碱性磷酸酶基本恢复正常(P﹥0.05),与较大剂量的葛根素组或较大剂量的雌二醇组相比无统计学意义(P﹥0.05)。结论:小剂量的雌二醇与葛根素对去卵巢大鼠的骨质疏松症的治疗效果与单独使用较大剂量的葛根素或较大剂量的雌二醇相比治疗效果相近。