Regulatory effect of calcium-regulating hormones on the synthesis and/or secretion of bone gamma-carboxyglutamic acid-containing protein in chick embryonic calvaria in vitro.

Regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), 24,25-dihydroxyvitamin D3 (24,25-(OH)2-D3), parathyroid hormone (PTH) and calcitonin (CT) on BGP synthesis and/or secretion were studied using chick embryonic calvaria in vitro. BGP contents in calvaria and culture medium were determined by radioimmunoassay using antiserum to purified chick BGP. After 72 h culture, 1,25-(OH)2-D3 (10(-10)-10(-7) M) increased the BGP content in culture medium significantly, and the effect was maximum at 10(-8)M, while the BGP content in calvaria was not changed by 1,25-(OH)2-D3. 24,25-(OH)2-D3 increased the BGP content both in medium at 10(-6) M-10(-5) M after 24 h culture and in calvaria at 5 X 10(-7)-10(-5) M after 48 h culture. PTH increased BGP contents both in calvaria after 72 h culture and medium at 1 U/ml after 24 h culture, while it decreased them at 5-10 U/ml after 24 h culture. CT (0.5-10 U/ml) had no effect on BGP contents in calvaria and medium. 1,25-(OH)2-D3-induced increase of BGP in culture medium was observed from 24 to 72 h of culture and then reached a plateau at 120 h of culture. 1,25-(OH)2-D3-induced increase in the BGP level in medium after 72 h culture was 3-4 times the control value. On the other hand, the BGP content in calvaria was not affected by 1,25-(OH)2-D3 until 72 h of culture and then significantly increased at 120 h. The addition of 1,25-(OH)2-D3 (10(-8) M), 24,25-(OH)2-D3 (10(-6) M), and 1 U PTH (and 1 U CT added to those hormones) into culture medium of chick embryonic calvaria significantly increased BGP contents in calvaria and medium at 72 h after culture. The effect of three hormones was not synergistic, but had a tendency to be greater than that of a single addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 or PTH. Further addition of 1 U CT into culture medium did not affect the combined effects of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH except for the calcium content in calvaria. The addition of individual calcium-regulating hormones into culture medium of chick embryonic calvaria did not affect the calcium and phosphorus contents in calvaria, but the calcium content in calvaria was significantly increased by the addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Bone gamma-carboxyglutamic acid containing protein in the perinatal period.

We measured bone gamma-carboxyglutamic acid-containing protein (BGP), calcium (Ca), phosphorus (P), and alkaline phosphatase (Al-P) in paired maternal and cord sera, and urinary gamma-carboxyglutamic acid (gamma-Gla) in neonates. The circulating BGP was 41.21 +/- 2.47 ng/ml and 7.44 +/- 0.87 ng/ml in the cord (n = 15) and the maternal (n = 14) sera, respectively. The urinary gamma-Gla in the neonates was 147.68 +/- 10.75 mumol/g creatinine (n = 15). The cord serum BGP was significantly higher than the normal adult level. The maternal serum BGP was at the same level as in other adults. It is conceivable that the fetus may produce BGP during gestation, as the cord serum BGP level was significantly higher than the maternal level and there was no correlation between the cord and maternal serum BGP concentrations. The reason for the elevated circulating BGP level in the cord serum is not known, but increased bone turnover may be a factor. The cord serum BGP may include not only carboxylated but also non-gamma-carboxylated GP because of fetal vitamin K deficiency.

The effect of 1,25-dihydroxycholecalciferol on the changes of bone gamma-carboxyglutamic acid-containing protein in bone and serum of developing chick.

The effects of 1,25-dihydroxycholecalciferol(1,25-(OH)2-D3) injection into developing chick embryos on bone gamma-carboxyglutamic acid-containing protein (BGP) levels in bone and serum were observed in relation to those on calcium metabolism using chick embryos and chicks aged from 13 days' incubation to 7 days of age. Chick BGP was determined by radioimmunoassay using antiserum to purified chick BGP. The injection of 1,25-(OH)2-D3 into the developing chick embryos of 13 days' incubation resulted in the increases of bone BGP levels at hatching and at 2 days of age. However, the distribution of bone BGP at hatching was not influenced by 1,25-(OH)2-D3. On the other hand, serum BGP levels were significantly increased by 1,25-(OH)2-D3 at 14, 15, 18 days' incubation and at 2 days of age. The early changes of serum BGP levels after 1,25-(OH)2-D3 treatment were correlated with the increases of serum calcium concentrations and the decreases of inorganic phosphorus concentrations. These results suggest that 1,25-(OH)2-D3 may directly or indirectly mediate BGP synthesis or secretion associated with calcium metabolism.

The changes of bone gamma-carboxyglutamic acid-containing protein in bone and serum of developing chick.

The changes of bone gamma-carboxyglutamic acid-containing protein (BGP) levels in bone and serum were studied in relation to those in calcium metabolism using chick embryos and chicks aged from 13 days' incubation to 8 weeks old. Chick BGP was determined by radioimmunoassay using antiserum to purified chick BGP. BGP levels in bone and serum increased significantly at hatching and then decreased until 3-5 days of age. Thereafter, BGP levels in bone and serum increased gradually until 8 weeks of age. These changes of BGP levels were well correlated with those of serum calcium and inorganic phosphorus concentrations, serum alkaline phosphatase activity and bone calcium content. The molecular size of increased serum BGP at hatching was not different from that of bone BGP. These results suggest that BGP plays a role in hatching and bone formation during chick development.

Effect of aging and castration on the changes in the levels of bone gamma-carboxyglutamic acid-containing protein in bone and serum of female rat.

Using female rats from 2 to 64 weeks old, the changes in bone gamma-carboxyglutamic acid-containing protein (BGP) levels in bone and serum were studied in relation to the changes in calcium metabolism during aging. Intestinal calcium transport, serum phosphorus level, bone origin serum alkaline phosphatase activity and serum BGP content were high in rapidly growing animals and then decreased with aging. Serum BGP content correlated well to bone origin serum alkaline phosphatase activity. Bone density, bone ash content and bone BGP content increased during aging. Bone BGP content was correlated to bone density which indicates the level of bone calcification. Moreover, the effect of castration on calcium metabolism and bone and serum BGP contents were observed in young (10 weeks old) and aged (40 weeks old) female rat at 12 and 24 weeks after operation. Intestinal calcium transport in ovariectomized rat was significantly lower than in sham operated rat. Serum phosphorus level and serum BGP content were increased in castrated female rat. However, serum calcium level and bone origin serum alkaline phosphatase activity did not show a significant change in castrated female rat. Bone density was significantly decreased in aged rat at 24 weeks after operation. During aging or castration, serum BGP content changed more than bone BGP content. The determination of change in serum BGP levels in various disorders of calcium metabolism would be very informative in future study.

Synthesis and biological activity of 5,6-trans-vitamin D3 in anephric rats.

A modified procedure for the purification of 5,6-trans-D3 from a reaction mixture of vitamin D3 with iodine and its biological activity in sham-operated and anephric rats are described. When a solution of vitamin D3 in n-hexane was reacted with small quantities of iodine under influence of visible light, the reaction mixture gave four or five spots, including vitamin D3 and 5,6-trans-D3, on a thin layer chromatogram. After purifying the mixture by alumina column chromatography, a colorless oil from the separated 5,6-trans-D3 fractions was crystallized from n-hexane and snow white crystalline 5,6-trans-D3 was obtained. The purification method was thought to be better than the previously reported methods (14-16) because it gave good yield without the procedure of esterification using expensive p-phenylazobenzoyl chloride. A dose of 25 mug of 5,6-trans-D3 obtained thus gave significant elevation on intestinal calcium transport in both sham-operated and anephric rats, whereas the dose did not give positive effects on serum calcium levels in anephric rats. Serum phosphorous levels were extremely elevated by nephrectomy, but in both sham-operated and anephric rats they were unaffected by the administration of 25 myg of 5,6-trans-D3.