Supplementation with the antioxidant lycopene significantly decreases oxidative stress parameters and the bone resorption marker N-telopeptide of type I collagen in postmenopausal women.

UNLABELLED: To date, no intervention studies have been published demonstrating the effect of the antioxidant lycopene on bone. Postmenopausal women supplemented with lycopene had significantly increased antioxidant capacity and decreased oxidative stress and the bone resorption marker N-telopeptide (NTx). Lycopene decreases bone resorption markers and may reduce the risk of osteoporosis. INTRODUCTION: We have previously shown in vitro and in vivo that lycopene from tomato is associated with a protective effect on bone, but lycopene intervention studies have not been reported. Our aim was to carry out a randomized controlled intervention study to determine whether lycopene would act as an antioxidant to decrease oxidative stress parameters, resulting in decreased bone turnover markers, thus reducing the risk of osteoporosis in postmenopausal women. METHODS: Sixty postmenopausal women, 50-60 years old, were recruited. Following a 1-month washout without lycopene consumption, participants consumed either (N = 15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato Lyc-O-Mato lycopene capsules, or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30, and 0 mg/day respectively for 4 months. Serum collected after the washout, 2 and 4 months of supplementation, was assayed for cross-linked aminoterminal N-telopeptide, carotenoid content, total antioxidant capacity (TAC), lipid, and protein oxidation. RESULTS: Participants who consumed juice or lycopene capsules were analyzed in one group designated "LYCOPENE-supplemented". Repeated measures ANOVA showed that LYCOPENE-supplementation for 4 months significantly increased serum lycopene compared to placebo (p < 0.001). LYCOPENE-supplementation for 4 months resulted in significantly increased TAC (p < 0.05) and decreased lipid peroxidation (p < 0.001), protein oxidation (p < 0.001), and NTx (p < 0.001). These decreases in lipid peroxidation, protein oxidation, and NTx were significantly different from the corresponding changes resulting from placebo supplementation (p < 0.05, p < 0.005, and p < 0.02, respectively). CONCLUSIONS: Our findings suggest that the antioxidant lycopene is beneficial in reducing oxidative stress parameters and the bone resorption marker NTx.

Dog and rat kidney proximal tubule cells in culture: responses to parathyroid hormone.

We have described and compared culture systems for proximal tubule cell (PTC) preparations from dog and rat kidney. Cells were prepared from kidney cortex by enzyme digestion and purified on Percoll density gradient. The dog PTC and rat PTC differed in their growth characteristics in culture. Although the dog PTC tended to overgrow the contaminating fibroblastic cells, the rat PTC tended to be overgrown by the latter cells when cultured in medium containing 15% fetal calf serum (FCS). Cultures of rat PTC in serum-free medium or medium containing only 2% FCS yielded only epithelium-like cells exhibiting characteristics of cells that are proximal tubular in origin. These properties include protrusion of microvilli, high alkaline phosphatase activity, ability to transport sugar, and responsiveness to parathyroid hormone (PTH) in terms of cAMP production. The time course and dose-response curves of PTH-stimulated cAMP accumulation were studied in dog and rat PTC. The estimated half-maximal concentrations (Kact) for PTH in dog and rat PTC were 1.2 and 100 nM, respectively. Both values are within the range reported in the literature for the respective renal membrane preparations. In addition to our previously reported data on dog PTC, this study revealed the presence of PTH-inhibitable 25-hydroxyvitamin D3-24-hydroxylase in dog PTC. These two model cell culture systems should prove useful in studying PTH action in renal cells.

Pseudohypoparathyroidism with osteitis fibrosa cystica: direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone.

A young girl had tibial osteotomies at age 14 for genu valgum and then had recurrent tibial cysts over a number of years. Hypocalcemia and hyperphosphatemia were first noted at age 21. The diagnosis of pseudohypoparathyroidism was made at age 28, when elevated plasma PTH was detected. Clinical and biochemical features, including a PTH response test and assay of RBC Gs, established the diagnosis of pseudohypoparathyroidism type 1b. Failure to suppress plasma PTH with vitamin D therapy led to an exacerbation of her cystic bone disease; there were widespread lytic lesions radiologically, most of which took up [99mTc]diphosphonate on bone scan. Microradioscopy revealed evidence of resorption of phalangeal tufts. Bone biopsy showed osteitis fibrosa cystica. During an orthopedic procedure, trabecular bone fragments were taken from her right humerus, and bone-derived cells cultured using an explant technique. The cultured cells were osteoblast-like in morphology, fully responsive to PTH, cholera toxin, forskolin, and PGE1 in vitro, and had an alkaline phosphatase and osteocalcin response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Following this examination of skeletal responsiveness, attempts were made to suppress the elevated plasma PTH levels and symptomatic bone disease by optimizing therapy with oral 1,25-(OH)2D3. When bone pain associated with the cystic bone disease failed to resolve, the patient underwent total parathyroidectomy, following which the bone pain gradually resolved. This is the first direct demonstration of PTH responsiveness in cultured bone cells in the syndrome of pseudohypoparathyroidism with osteitis fibrosa cystica.

Carboxyl-terminal parathyroid hormone peptide (53-84) elevates alkaline phosphatase and osteocalcin mRNA levels in SaOS-2 cells.

Previous findings in our laboratory have shown that hPTH-(53-84) stimulates alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells. In the present study, we examined the effects of hPTH-(53-84) and hPTH-(1-34) on the expressions of alkaline phosphatase, osteocalcin, and collagen type I mRNA levels in the human osteosarcoma cell line SaOS-2. The effect of hPTH-(53-84) on alkaline phosphatase and osteocalcin message levels was dose dependent (ANOVA, p < 0.005 and p < 0.001, respectively), with significant stimulation observed at 10 nM. Treatment with 10 nM hPTH-(53-84) for 24 h resulted in significant 2- and 1.4-fold increases in mRNA levels for alkaline phosphatase and osteocalcin, respectively (p < 0.05), but had no effect on collagen type I expression. The increased alkaline phosphatase mRNA levels was associated with a 1.5-fold increase in enzyme activity (p < 0.05). In contrast, under similar incubation conditions, hPTH-(1-34) had no significant effects on alkaline phosphatase or osteocalcin mRNA levels. On the other hand, hPTH-(1-34) had dose-dependent stimulatory effects on collagen type I mRNA levels (ANOVA, p < 0.001), 10 nM hPTH-(1-34) stimulating collagen type I expression 1.6-fold (p < 0.05). The results indicate that carboxyl-terminal hPTH-(53-84) has direct and unique biologic effects in human osteoblast-like cells in culture.

Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells.

Bone cells derived from human trabecular explants display osteoblastic features. We examined the modulation of alkaline phosphatase activity and cAMP production as the result of exposing trabecular explants to physiologic concentrations of dexamethasone for 4 weeks during cellular outgrowth and subculture. Cells treated with dexamethasone were observed to grow generally more slowly than control cells. Cells appeared larger and more polygonal, and staining for alkaline phosphatase was more intense in the dexamethasone-exposed cultures. There was a progressive increase in cellular PTH responsiveness with increasing duration of exposure of cells to dexamethasone. Cells grown for 6 weeks in 3 x 10(-8) M dexamethasone had a 10-fold increase in PTH-stimulated cyclic AMP accumulation. Dexamethasone-treated cells also had a significantly increased alkaline phosphatase activity. 1,25-(OH)2D3-stimulated alkaline phosphatase activity was increased approximately 20-fold. cAMP responses were significantly increased to PTH (21.7-fold), PGE1 (2.67-fold), and forskolin (4.81-fold), but not to cholera toxin. Dexamethasone-treated cells also had a mean decrease in 1,25-(OH)2D3-stimulated osteocalcin production to 26.2% of control values (p less than 0.001). Hydrocortisone treatment gave rise to similar effects but of smaller magnitude than those of dexamethasone. Testosterone did not have a significant effect on alkaline phosphatase activity or cAMP production. Skin fibroblasts showed a significant enhancement of alkaline phosphatase activity in response to dexamethasone, but of a much smaller magnitude than in bone cells. The phenotypic changes induced by long-term culture in dexamethasone are consistent with the promotion of a more differentiated osteoblastic phenotype.

Normal parathyroid hormone responsiveness of bone-derived cells from a patient with pseudohypoparathyroidism.

Pseudohypoparathyroidism (PHP) is characterized by a lack of response to parathyroid hormone (PTH); however, normal skeletal responsiveness to PTH in some patients with PHP type Ia was previously suggested on the basis of clinical observations. To test this hypothesis, we measured cyclic adenosine monophosphate (cAMP) production in response to various agonists in bone-derived osteoblast-like (OBL) cells from trabecular explants obtained from an iliac crest biopsy of a 25-year-old woman with PHP. The patient was proved to have PHP type Ia on the basis of Albright's hereditary osteodystrophy and decreased activity of stimulatory guanine nucleotide-binding protein (Gs) in erythrocytes. Responsiveness of the patient's OBL cells was compared with OBL cells from eight subjects aged 18-39 years who had no evidence of metabolic bone disease. OBL cells from the patient responded to the following agonists (expressed in multiples of elevation of cAMP, stimulated/basal, mean +/- SE, n = 3): PTH, 3.8 +/- 0.3; forskolin, 8.2 +/- 0.2; and cholera toxin, 56.8 +/- 10.0. These responses were not significantly different from those of control OBL cells: PTH, 4.5 +/- 1.1 (range 2.4-7.5); forskolin, 7.7 +/- 1.4; and cholera toxin, 57.9 +/- 16.2. The normal cholera toxin response indicated the presence of functional Gs. Bone cells from patients with PHP type Ia may exhibit a normal PTH receptor-coupled adenylyl cyclase system in vitro despite clinical evidence of impaired hormone-responsive adenylyl cyclase in other tissues, including the kidney. Skeletal responsiveness to PTH may explain the long periods of spontaneous normocalcemia observed in this patient.

Binding of intact parathyroid hormone to rat osteosarcoma cells: major contribution of binding sites for the carboxyl-terminal region of the hormone.

Most studies of PTH receptor binding have been carried out with amino-terminal radioligands which only detect binding within that region of the hormone molecule. We studied the binding of electrolytically labeled intact bovine PTH [bPTH-(1-84)], and its amino-terminal fragment, bPTH-(1-34) to intact cloned rat osteosarcoma cells (ROS 17/2.8). We also measured the effects of these hormones on cell cAMP accumulation. Binding equilibrium for the two radioligands was reached by 2 h of incubation at 22 C. However, the cells had higher binding capacity (8-9% or 0.22-0.25 fmol/2 X 10(6) cells) for [125I]bPTH-(1-84) than for [125I]bPTH-(1-34) (4% or 0.11 fmol/2 X 10(6) cells). On the other hand [125I]bPTH-(1-34) bound to ROS cells with higher affinity [dissociation constant (Kd) = 19 nM] than did [125I]bPTH-(1-84) (Kd = 210 nM). Measurements of trichloroacetic acid precipitability and analysis of rebinding of previously incubated radioligand to fresh cells ruled out degradation of the tracer as an explanation for these differences. The maximum cell cAMP response to bPTH-(1-34) (Vmax = 780 +/- 32 pmol/2 X 10(6) cells X 5 min) was reached at 10(-7) M concentration with an affinity [Michaelis-Menten constant (Km)] of 3 nM. On the other hand, the Vmax with intact bPTH-(1-84) was lower (400 +/- 7 pmol/2 X 10(6) cells X 5 min), with a Km of 60 nM). Further studies with the bPTH-(1-84) tracer showed inability of hormonal fragments to compete completely for binding. At a concentration of 3 microM, bPTH-(1-84) reduced tracer binding by 82.5%, compared to 18% by bPTH-(1-34) and 10% by (Nle8,Nle18,Tyr34)bPTH-(1-34)amide, 60% by human PTH (hPTH)-(53-84), and 70% by the combination of bPTH-(1-34) and hPTH-(53-84). hPTH-(53-84) itself did not elicit a cAMP response after 5 min or 1 h of incubation nor did it significantly alter the cAMP response of the cells to bPTH-(1-84). These studies suggest that PTH binds to ROS 17/2.8 cells by sites carboxy-terminal (C-terminal) to position 34, in addition to sites within the amino-terminal portion of the hormone molecule; 72% of the binding of intact hormone to these cells was to the C-terminal 35-84 region of the PTH molecule. The significance of the C-terminal binding sites is presently unclear, but they do not appear to be coupled to adenylate cyclase. Further work is needed to determine the effects of C-terminal PTH fragments on bone cell metabolism.

Human parathyroid hormone carboxyterminal peptide (53-84) stimulates alkaline phosphatase activity in dexamethasone-treated rat osteosarcoma cells in vitro.

Previous studies in our laboratory have demonstrated relatively large numbers of cell surface binding sites for the carboxylterminal (53-84) region of PTH on ROS 17/2.8 rat osteosarcoma cells, a clonal osteoblast-like cell line. In order to gain insight into the significance of these carboxylterminal binding sites, we studied the effect of intact bovine PTH (1-84), its aminoterminal fragment bovine PTH (1-34), and the human PTH carboxylterminal fragment (53-84) on alkaline phosphatase activity in dexamethasone-treated rat osteosarcoma (ROS) 17/2.8 cells. While bovine PTH (1-84) and its aminoterminal 1-34 fragment inhibited alkaline phosphatase activity, we saw a dose-related stimulation of activity by human PTH (53-84), with maximal stimulation occurring after 120 hours, at a concentration of 10(-8) M. The effect was not seen in dexamethasone-untreated cells. To our knowledge, this is the first published demonstration of biological activity of this carboxylterminal PTH peptide, previously thought to be inactive. It is likely that dexamethasone caused differentiation of cells to a type more sensitive to human PTH (53-84). Further studies are necessary to elucidate the physiological significance of these findings.

Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C.

Five different cell populations, designated I to V, were isolated from minced newborn rat calveria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all five populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in cyclic-3'5'-adenosine monophosphate (cAMP) level. None of the five populations gave any response to calcitonin. Upon subsequent subcultures, all populations, except population IV, either lost or considerably decreased their response to PTH. Population IV gave a two to three-fold increase in cAMP concentration in response to PTH up to the third subculture. No morphological differences could be observed among the five populations. The third subculture of population IV cells that had been stored in 10% glycerol at -80C for four months was subsequently thawed and subcultured to the sixth subculture. These cells still responded to PTH with an increase in cAMP level. In a second experiment, 5 different cell populations designated I to V were isolated in a similar way by incubation with collagenase and DNAse. The maximum response to PTH was found in population 3. The preservation of the PTH-responsiveness of this population, after subculturing, freezing, storing in 10% glycerol at -80 C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the sixth subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U/ml to 5U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 microng/ml to 10 microng/ml. The time course of action on population IV of PTH was found to be different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.

Immunohistochemical demonstration of parathyroid hormone binding to specific cell types in fixed rat bone tissue.

Deparaffinized sections of fixed decalcified neonatal rat radii were incubated in bovine PTH (bPTH; 1-10 MRC units/ml) or in PTH-solvent. They were then stained for PTH by the peroxidase-antiperoxidase method using guinea pig antiserum to bPTH and the substrate 3,3'-diaminobenzidine-H2O2. Staining caused by nonspecific binding of PTH to the bone matrix and the glass slides supporting the sections was eliminated completely by preincubation of the sections in 100% normal goat serum. Cross-reactivity of the antiserum to erythrocytes was eliminated by preabsorption of the antiserum with fixed rat erythrocytes. After the cross-reactivity of the anti-PTH antiserum to erythrocyte components and the nonspecific binding of PTH to bone matrix were eliminated, we were able to demonstrate intense staining over the cytoplasm of the osteoclasts in rat radii sections incubated with PTH. Less intense staining was observed over the osteocytes, periosteal osteoblasts, and, possibly, the endosteal osteoblasts. An explanation for this differential staining could be that osteoclasts have a greater receptor number and/or a greater receptor affinity for the bPTH than do osteocytes and osteoblasts. This study demonstrates that binding of PTH to bone tissue can be localized in all identifiable osteoclasts, osteocytes, and osteoblasts, and thus suggests that all three cell types can interact directly with PTH.

17 beta-estradiol and parathyroid hormone potentiate each other's stimulatory effects on alkaline phosphatase activity in SaOS-2 cells in a differentiation-dependent manner.

We studied the effects of 17 beta-estradiol (E2) and PTH on alkaline phosphatase activity in SaOS-2 cells that had been passaged and grown continually in the presence (SaOS + Dex) or absence (SaOS - Dex) of 10(-8) M dexamethasone (Dex). We showed that the more differentiated SaOS + Dex cells had higher alkaline phosphatase activity and PTH-responsive adenylate cyclase than the less differentiated SaOS - Dex cells. In SaOS - Dex cells, E2 or PTH (1 x 10(-11)-1 x 10(-6) M) had no effect on alkaline phosphatase activity. On the other hand, in SaOS + Dex cells, PTH and E2 each had small but very significant stimulatory effects on alkaline phosphatase activity. The combined effects of PTH and E2 resulted in potentiation which was dose dependent for PTH and E2. 17 alpha-estradiol and tamoxifen (1 x 10(-11)-1 x 10(-6) M) had no effect on PTH-stimulated alkaline phosphatase activity in SaOS + Dex cells, but the latter inhibited the E2 effect, clearly demonstrating its specificity. In conclusion, we have shown that in more differentiated osteoblastic cells, E2 and PTH have interactive effects on alkaline phosphatase activity.

Immunohistochemical localization of the estrogen receptor in human osteoblastic SaOS-2 cells: association of receptor levels with alkaline phosphatase activity.

We have previously shown that the combination of estrogen (E2) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] enhanced alkaline phosphatase (ALP) activity in human osteosarcoma SaOS-2 cells which had been grown in the presence of 10 nmol/L dexamethasone (SaOS + DEX cells). To determine whether this increase in ALP activity was associated with changes in receptor protein levels for E2 (ER) in individual SaOS + DEX cells, a monoclonal antibody to ER and a histochemical stain for ALP were used localize the expression of these proteins in fixed cells. Western and Northern blot analyses were used to determine whether E2 and 1,25(OH)2D3 affected immunoreactive ER protein and mRNA levels, respectively. Our results showed that immunohistochemical staining for ER was primarily nuclear, whereas histochemical staining for ALP was cytosolic. Treatment of cells with 1,25(OH)2D3, E2, or E2 + 1,25(OH)2D3 increased the levels of both ER and ALP activity, as visualized by enhanced cellular staining. Western analyses showed that 1,25(OH)2D3 and E2, separately and in combination, significantly increased ER protein levels. 1,25(OH)2D3 enhanced ER levels in a dose-dependent manner [analysis of variance (ANOVA), F = 3.91, p < 0.05]; this effect was augmented by E2 (ANOVA, F = 5.98, p < 0.005). In comparison, 17 alpha-E2 + 1,25(OH)2D3 and tamoxifen + 17 beta-E2 + 1,25(OH)2D3 did not increase ER levels compared with those obtained with 17 beta-E2 + 1,25(OH)2D3. ER mRNA levels were not significantly increased by E2, 1,25(OH)2D3, or E2 + 1,25(OH)2D3 together. In contrast, in a population of SaOS cells which had been in culture longer (approximately 40 passages more) than the previous cells, E2 + 1,25(OH)2D3 did not enhance ALP activity or ER levels above those obtained with 1,25(OH)2D3 alone. These results showed that in responsive SaOS cells, E2 enhanced both the stimulatory effects of 1,25(OH)2D3 on ALP activity and the activation of ER. Thus changes in ALP activity are associated with changes in ER levels in SaOS + DEX cells.

Effects of 1alpha,25-dihydroxy-16ene, 23yne-vitamin D3 on osteoblastic function in human osteosarcoma SaOS-2 cells: differentiation-stage dependence and modulation by 17-beta estradiol.

We compared the separate effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analog, 1alpha,25-dihydroxy-16ene,23yne-vitamin D3 (1alpha25(OH)2-16ene,23yne-D3), as well as their interactions with 17-beta estradiol (E2) in our human osteosarcoma SaOS-2 cell models representing two stages of differentiation, the SaOS+DEX and SaOS-DEX cells. SaOS+DEX cells have been previously shown to express higher PTH-stimulated adenylate cyclase (PTH-AC) and basal alkaline phosphatase (ALP) activities compared with SaOS-DEX cells. ALP: In SaOS+DEX cells, 0.1 nmol/L analog, but not 1alpha,25(OH)2D3, increased ALP activity 1.7-fold (p < 0.05). Instead, 1 nmol/L 1alpha,25(OH)2D3 increased ALP 1.4-fold (p < 0.05). In these cells, E2 enhanced 1alpha,25(OH)2D3-stimulated ALP activity (ANOVA, F = 51.22, p <0.0001), while inhibiting the effect of the analog. [3H]-Thymidine uptake: In SaOS+DEX cells, 1alpha,25(OH)2D3 had biphasic effects (ANOVA, F = 13.08, p < 0.0001), which were not altered by E2. In contrast, the analog was stimulatory only with E2 (ANOVA, F = 3.59, p < 0.025). Osteocalcin (OC): 1alpha,25(OH)2D3 and its analog stimulated OC production in SaOS-DEX cells with smaller effects in SaOS+DEX cells. In SaOS-DEX cells, E2 enhanced the effect of 1alpha,25(OH)2D3, but not that of the analog. PTH-AC: In SaOS-DEX cells, 100 nmol/L analog inhibited PTH-AC activities by 50% (p < 0.01), whereas 1alpha,25(OH)2D3 had little effect. In SaOS+DEX cells, both compounds inhibited PTH-AC approximately 35%. E2 inhibited the effect of the analog in SaOS-DEX cells, but enhanced the effects of both compounds in SaOS+DEX cells. These results show that the analog 1alpha,25(OH)2-16ene,23yne-D3 was effective in regulating osteoblastic function; its effects were modulated by E2 and dependent upon the stage of osteoblast differentiation.

Elimination of cross-reactivity to some component of erythrocytes in an antiserum to parathyroid hormone by absorption with fixed erythrocytes.

Cross-reactivity to some component of rat erythrocytes with an antiserum to parathyroid hormone (anti-PTH) was detected in fixed demineralized sections of bone using the peroxidase-antiperoxidase method. The cross-reactivity was eliminated by the preabsorption of anti-PTH with fixed, washed rat erythrocytes. This technique provides an easy and rapid method with which to eliminate cross-reactivity to erythrocytes whenever such a situation is encountered in immunohistochemical procedures.

Specific immunohistochemical localization of type III collagen in porcine periodontal tissues using the peroxidase-antiperoxidase method.

Specific antibodies to porcine gingival type III collagen were raised in sheep. After purification on collagen affinity columns the antibodies were used for immunohistochemical localization of type III collagen in porcine periodontal and dental tissues employing the peroxidase-antiperoxidase (PAP) procedure. The extent of staining of the periodontal tissues was found to approximate the amount of type III collagen measured biochemically. A fairly uniform distribution of type III collagen was observed in the periodontal ligament and gingiva with more intense staining often being associated with blood vessels. A regular pattern of weakly staining fibers could be demonstrated throughout the cementum and in parts of the alveolar bone tissue. In addition, occasional sites in the cementum having a different morphological appearance from the rest of the cementum exhibited bundles of positively stained fibers. Although the bone tissue was essentially unstained, fibers in the endosteal spaces stained strongly. Sharpey's fibers passing from the soft connective tissues into alveolar bone and cementum also stained strongly. Three distinct arrangements of collagen fibers stained by the type III collagen antibodies could be identified: first, a reticular pattern, which was seen at the junction of the gingival epithelium and connective tissue, and in the endosteal spaces and dental pulp; second, a more diffuse pattern of fibers intermingled with type I collagen in the soft connective tissues; and third, a coating of some Sharpey's fibers, having a core believed to be type I collagen, and of fibers in the cementum inclusions.

17 beta-oestradiol enhances the stimulatory effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity in human osteosarcoma SaOS-2 cells in a differentiation-dependent manner.

We tested the effect of osteoblastic differentiation on the interactive effects of 17 beta-oestradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on alkaline phosphatase activity. As cell models we utilized the more differentiated human osteosarcoma (SaOS) cells that had been cultured for 6 days in medium containing 10 nM dexamethasone (Dex) (SaOS+Dex cells) and the less differentiated cells cultured in the absence of Dex (SaOS-Dex cells). The cells were challenged with 1,25(OH)2D3 in the presence or absence of Dex for 24 h and then with E2 for an additional 24 h. In SaOS-Dex cells, alkaline phosphatase activity remained constant over the 48-h period and was not significantly affected by E2, 1,25(OH)2D3 or 1,25(OH)2D3+E2 treatment. On the other hand, in SaOS+Dex cells, 1,25(OH)2D3 and E2+1,25(OH)2D3 stimulated alkaline phosphatase activity (ANOVA, F = 154.2, P < 0.0001) with the maximal response at 48 h (P < 0.01). In SaOS+Dex cells, 1,25(OH)2D3 had dose-dependent stimulatory effects which were strongly enhanced by 10 nM E2 (ANOVA, F = 46.0, P < 0.001). Studies on dose-dependent effects of E2, in the presence or absence of 100 nM 1,25(OH)2D3, revealed that in the presence of 1,25(OH)2D3, the E2 dose-response curve was biphasic in SaOS+Dex cells (ANOVA, F = 3.40, P < 0.005), with maximum stimulation at 10 nM E2 (P < 0.01). The specificity of E2 was verified using the inactive 17 alpha-oestradiol and the oestrogen antagonist, tamoxifen. These data indicate that E2 and 1,25(OH)2D3 have positive interactive effects on alkaline phosphatase activity in human osteoblasts, and suggest that the expression of this interaction is dependent on the stage of differentiation of the cells.

Calcium metabolism in a fatal case of sodium fluoride poisoning.

A patient was admitted to a district general hospital within an hour of ingesting a fatal dose of sodium fluoride. The results of laboratory investigations, together with some in vitro findings, support the hypothesis that the hypocalcaemia of fluoride poisoning is the result of fluorapatite formation and not calcium fluoride precipitation, and that its persistence reflects the severity of the calcium deficit and not an inhibitoin of normal homeostatic mechanisms. It is suggested that the role of renal clearance of fluoride may be more important than had been realised hitherto.

17Beta-estradiol stimulates mineralized bone nodule formation when added intermittently to SaOS-2 cells.

It is now well established that estrogen inhibits bone resorption. However, its effect on bone formation remains controversial. We studied the effect of 17beta-estradiol (E2) on mineralized bone nodule formation in long-term cultures of osteosarcoma SaOS-2 cells. We showed that SaOS-2 cells formed mineralized nodules which under electron microscopy revealed a bone structure with active osteoblasts, entrapped osteocytes, extracellular collagen fibrils and hydroxyapatite deposits, making this system a valid model to study bone formation in vitro. Intermittent addition of E2 for 6 hours during a 48-hour cycle of changes of medium, starting from day 3, resulted in a dose-dependent stimulation of mineralized bone nodule number and area, as well as alkaline phosphatase activity. In conclusion, we report for the first time a stimulatory effect of E2 on mineralized bone nodule formation in human osteoblasts in culture.

Antenatal diagnosis of placental sulphatase deficiency.

A dynamic test for the antenatal diagnosis of placenta sulphatase deficiency was examined in ten patients, selected on the basis of low urinary oestrogen levels (i.e. below the 10th centile at between 30 and 40 weeks gestation). The test consisted of measurement of plasma oestradiol levels over a period of 120 min after an i.v. injection of dehydroepiandrosterone sulphate. The oestradiol response was vigorous in seven patients, absent in two and small, but significant, in one. The three patients with reduced oestradiol response were also found to have raised levels of urinary steroid monosulphates and thus were probably placental sulphatase deficient. The overall clinical importance of placental sulphatase deficiency and the value of the dehydroepiandrosterone sulphate loading test in antenatal diagnosis are discussed.

Urinary oestrogen and plasma human placental lactogen as initial screening tests for a placental sulphatase deficiency.

As a result of routine screening of ante-natal patients by urinary total oestrogens and plasma human placental lactogen (HPL) from 35 weeks, a rare placental sulphatase deficiency was indicated which was later confirmed by in vitro studies of placental enzyme activities.