Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P <0.001 for each). In contrast to the effects of tunicamycin on N-linked glycosylation, the effects of mannosamine, which inhibits GPI-anchor glycosylation/formation, included (1) an increase in cell layer protein; (2) decreases in sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P <0.005 for each). These effects of mannosamine were, presumably, a consequence of inhibiting the insertion/attachment of sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction kinetics of sALP or on the apparent affinity (the value of KM) for the phosphoryl substrate.

Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that B1 and B2 have more (or more reactive) sialic acid residues compared with B/I, which mainly explains the apparent differences in molecular weight. Future investigations will focus on the clinical and functional significance of the revealed differences in sialic acid residues.

Quantitation of soluble and skeletal alkaline phosphatase, and insoluble alkaline phosphatase anchor-hydrolase activities in human serum.

BACKGROUND: The current studies were intended to compare the circulating levels of total and anchorless (soluble) skeletal and hepatic ALP isoenzyme activities, and insoluble ALP anchor-hydrolase activity in serum of postmenopausal women. METHODS: Preliminary studies of the insoluble ALP anchor-hydrolase activity in serum revealed a pH optimum of pH 5-6.5, a sensitivity to inactivation by heat at temperatures >45 degrees C (t(1/2)=8-9 min at 60 degrees C), and an apparent K(M) (at pH 7.5) of 40-45 mU/ml of insoluble skeletal ALP activity. RESULTS: Serum analyses showed that 94.5+/-0.5% (mean+/-SEM) of the ALP activity in serum was in the anchorless, soluble form. The data were also consistent with the notion that the amount of insoluble ALP anchor-hydrolase activity in serum, 52.8+/-0.8 U/l (mean+/-SEM), was sufficient for the conversion of anchor-intact (insoluble) ALP into the anchorless, soluble form, assuming activation by serum lipids and/or bile salts. Distributions of results for total, skeletal, hepatic, and insoluble ALP anchor-hydrolase activity were skewed toward the higher range and leptokurtotic (p<0.01 for each). Total ALP activity ranged from 42% to 208% of the group mean value; skeletal, hepatic, and insoluble ALP anchor-hydrolase activities ranged from 5% to 306%, 33% to 277%, and 2% to 325%, respectively. In contrast, the soluble ALP fraction only ranged from 71% to 106% of the group mean value. CONCLUSIONS: The correlations between the total and both skeletal (r=0.711, p<0.001) and hepatic (r=0.782, p<0.001) ALP isoform activities were predictive. Although correlations were also observed between insoluble ALP anchor-hydrolase activity and total (r=0.197, p<0.001), hepatic (r=0.184, p<0.001) and skeletal ALP activities (r=0.118, p<0.05), those relationships were not predictive (r(2)<0.04).

Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P <0.005 for each), with associated decreases in cell layer protein (P <0.05 for each) and concomitant increases in the release of sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P <0.001 for each). In contrast, caspase and calpain inhibitors reduced apoptosis, increased cell layer protein, and decreased the release of sALP activity (P <0.05 for each). Exposure to IGF-I also decreased apoptosis, in a time- and dose-dependent manner (e.g., r = 0.93, P <0.001 for IGF-I doses), with associated proportional effects to increase cell layer protein (P <0.001) and decrease the release of sALP activity (P <0.001). IGF-I also inhibited the actions of TNF-a and okadiac acid to increase apoptosis and sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

Exercise and mechanical loading increase periosteal bone formation and whole bone strength in C57BL/6J mice but not in C3H/Hej mice.

To identify the genes, and the mechanisms that account for the 53% higher peak bone density in C3H/HeJ (C3H) mice compared with C57BL/6J (B6) mice, we are performing quantitative trait locus and phenotypic analyses. The phenotypic studies revealed differences in bone formation and resorption, and showed that hindlimb immobilization (by sciatic neurectomy) caused a greater increase in endosteal resorption in the tibiae of B6 compared with C3H mice. The current studies were intended to examine the hypothesis that the bones of C3H mice are less sensitive to mechanical loading than the bones of B6 mice. To increase mechanical loading, 9-week-old female B6 and C3H mice (n = 10-13 mice/group) were subjected to a jumping exercise (20 jumps/day, 5 days/week, to heights of 20-30 cm) for a total of 4 weeks. Control mice did not jump. Osteocalcin, alkaline phosphatase (ALP) activity, and IGF-I were measured in serum. The left tibiae were used for histomorphometry (ground cross-sections prepared at the tibiofibular junction) and the right tibiae and femora were used for determinations of bone breaking strength (3-point bending). The results of these studies revealed (1) significant effects of both mouse strain (B6 and C3H) and the jumping exercise on tibial strength; (2) an exercise-dependent increase in serum IGF-I in C3H, but not B6 mice; and (3) no effects on serum ALP or osteocalcin. The histomorphometric analyses showed no effect of exercise on C3H tibiae, but significant exercise-dependent increases in total bone area, periosteal perimeter, periosteal mineral apposition rate (MAR), and periosteal bone formation (P < 0.02 for each) in B6 tibiae. There were no effects of exercise on periosteal resorption or any endosteal measurement in either C3H or B6 mice. Since the jumping exercise was designed to cause a two-three fold increase in muscular-skeletal loading at the tibio-fibular junction, and the calculated stress (g/mm2) at this sampling site was only 16% greater for B6 compared with C3H mice, we had anticipated that both strains of mice would show exercise-dependent increases in periosteal bone formation, with a greater response in the B6 mice. The lack of a response in the C3H tibiae demonstrates that the bones of C3H mice are less sensitive to mechanical loading (and unloading) than the bones of B6 mice.

Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities.

For the purpose of identifying genes that affect bone volume, we previously identified two inbred mouse strains (C57BL/6J and C3H/HeJ) with large differences in femoral bone density and medullary cavity volume. The lower density and larger medullary cavity volume in C57BL/6J mice could result from either decreased formation or increased resorption or both. We recently reported evidence suggesting that bone formation was increased in vivo and that osteoblast progenitor cells are more numerous in the bone marrow of C3H/HeJ compared with C57BL/6J mice. In the present study, we determined whether osteoclast numbers in vivo and osteoclast formation from bone marrow cells in vitro might also differ between the two mouse strains. We have found that the number of osteoclasts on bone surfaces of distal humerus secondary spongiosa was 2-fold higher in 5.5-week-old C57BL/6J mice than in C3H/HeJ mice of the same age (p < 0.001). Bone marrow cells of C57BL/6J mice cocultured with Swiss/Webster mouse osteoblasts consistently produced more osteoclasts than did C3H/HeJ bone marrow cells at all ages tested from 3.5-14 weeks of age (p < 0.001). Osteoclast formation was also greater from spleen cells of 3.5-week-old C57BL/6J mice than C3H/HeJ mice. The distribution of nuclei per osteoclast and the 1, 25-dihydroxyvitamin D3 dose dependence of osteoclast production from bone marrow cells were similar. Osteoclasts that developed from both C57BL/6J and C3H/HeJ marrow cells formed pits in dentin slices. Cultures from C57BL/6J marrow cells formed 2.5-fold more pits than cultures from C3H/HeJ marrow cells (p < 0.02). We compared the abilities of C57BL/6J and C3H/HeJ osteoblasts to support osteoclast formation. When bone marrow cells from either C57BL/6J or C3H/HeJ mice were cocultured with osteoblasts from either C57BL/6J or C3H/HeJ newborn calvaria, the strain from which osteoblasts were derived did not affect the number of osteoclasts formed from marrow cells of either strain. Together, these observations suggest that genes affecting the bone marrow osteoclast precursor population may contribute to the relative differences in bone density that occur between C3H/HeJ and C57BL/6J mouse strains.

Effects of zinc on human skeletal alkaline phosphatase activity in vitro.

Inorganic phosphate (Pi) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells by stabilizing the enzyme (without affecting transcription, ALP release from the cell surface, or the amount of ALP protein). These observations suggest that Pi determines the level of ALP activity by modulating a process of irreversible inactivation. The current studies were intended to examine the hypothesis that this inactivation of ALP activity is caused by the dissociation of an active center Zn and that Pi inhibits that dissociation. Initial studies showed that Zn, like Pi, could increase ALP specific activity in human osteosarcoma SaOS-2 cells in a time- and dose-dependent manner (e.g., a 50% increase at 0.2 micromol/liter Zn, P < 0.005). This effect was specific for Zn (i.e., no similar effect was seen with Ca, Fe, Co, Mg, Mn, or Cu), but not for SaOS-2 cells. Zn also increased ALP specific activity in (human osteosarcoma) MG-63 cells and in cells derived from normal human vertebrae (P < 0.001 for each). The effect of Zn to increase ALP activity was not associated with parallel increases in total protein synthesis, collagen production, or tartrate-resistant acid phosphatase activity (no change in any of these indices), net IGF-2 synthesis (a Zn-dependent decrease, P < 0.005), or PTH-dependent synthesis of cAMP (a biphasic increase, P < 0.02). Kinetic studies of Pi and Zn as co-effectors of ALP activity showed that Zn was a mixed-type effector with respect to Pi, whereas Pi was competitive with respect to Zn. Mechanistic studies showed that (1) Zn reversed the effect of Pi withdrawal to decrease ALP activity, but not by reactivating inactive ALP protein (the process required protein synthesis, without increases in ALP mRNA or the level of ALP immunoreactive protein); (2) Zn increased the half-life of ALP activity in intact cells and after a partial purification; and (3) Pi inhibited the process of ALP inactivation by EDTA (which chelates active center Zn). All these findings are consistent with the general hypothesis that Pi increases the half-life of skeletal ALP by preventing the dissociation of active center Zn and with a mechanistic model of skeletal ALP activity in which active center Zn participates in Pi-ester binding and/or hydrolysis.

Skeletal response to dietary zinc in adult female mice.

The current studies were intended to assess dose- and time-dependent effects of dietary zinc (Zn) on alkaline phosphatase (ALP) activity and tartrate-resistant acid phosphatase (TRAP) activity in adult female mice. In the first study, mice were given 0, 1x, 2x, 3x, or 4x normal dietary Zn for 2 weeks, 4 weeks, or 6 weeks. In the second study, mice were given 0, 1x, 2x, 3x, 4x, and 5x normal dietary Zn for 4 weeks. Sera were collected for measurements of ALP and (in the second study) osteocalcin. Tibiae and calvaria were extracted for measurements of ALP, protein, and TRAP. The first study showed positive correlations between dietary Zn and serum ALP (4 and 6 weeks, P < 0.001), Zn and tibial ALP (2, 4, and 6 weeks, P < 0.03), and Zn and tibial protein (2, 4, and 6 weeks, P < 0.001), as well as a negative correlation between dietary Zn and tibial TRAP (2, 4, and 6 weeks, P < 0.001). Covariant analyses showed that serum ALP, tibial ALP, tibial protein, and tibial TRAP were affected by the dose of Zn (P < 0.005) and by the treatment time (P < 0.03). Supplemental studies showed that (1) the dose-dependent effect of dietary Zn on serum ALP (at 6 weeks) was proportional to the effects on tibial ALP and calvarial ALP, but not to the effects of Zn on renal, hepatic, or intestinal ALP; (2) 6 weeks of dietary Zn caused dose-dependent increases in ALP specific activity in the tibia, calvaria, and liver, but not kidneys or intestines; and (3) Zn increased ALP activity and cell layer protein and decreased TRAP activity in monolayer cultures of the murine osteoblastic cell line, MC3T3-E1. The second dietary study confirmed the results of the first: 4 weeks of treatment with Zn caused significant increases in serum ALP, calvarial ALP, and tibial ALP activities, and a significant decrease in tibial TRAP (P < 0.05-0.005 for each). This study also revealed an effect of Zn to increase serum osteocalcin (P < 0.03 at 2x normal Zn). Together, these data indicate that incremental increases in dietary Zn are associated with increases in ALP activity in serum and in bone. The effect of Zn to decrease TRAP activity in osteoblast-line cells precludes the interpretation of a Zn-dependent decrease in tibial TRAP activity as evidence of decreased bone resorption.

Skeletal alkaline phosphatase activity is primarily released from human osteoblasts in an insoluble form, and the net release is inhibited by calcium and skeletal growth factors.

Skeletal alkaline phosphatase (ALP) is anchored to membrane inositol-phosphate on the outer surface of osteoblasts. Although skeletal ALP activity in serum is, essentially, all in an anchorless (soluble) form, in vitro studies indicate that ALP can be released in either an anchorless, soluble form (e.g., by a phospholipase) or an anchor-intact, insoluble form (e.g., by vesicle exocytosis). The current studies were intended to define the contributions of each of these putative processes of ALP release and to assess the significance of regulation by calcium (Ca) and skeletal effectors. ALP activity was measured in serum-free medium from replicate cultures of human osteosarcoma (SaOS-2) cells and normal human bone cells. Temperature-sensitive phase distribution (in Triton X-114) allowed separation of soluble from insoluble ALP activity. Our studies revealed that most of the ALP activity released from SaOS-2 cells was in an insoluble form (78% +/- 8%), a percentage that was constant between 2 and 96 hours. A similar result was seen for normal human bone cells. Calcium had a negative, biphasic dose-dependent effect on net release of ALP activity: r = -0.85, P < 0.001 at 24 hours, with KIapparent values for biphasic inhibition of 20 and 300 mumol/l Ca. Of the skeletal effectors tested, insulin-like growth factor-II (IGF-II) had the greatest effect, decreasing the net release of ALP activity in a dose-dependent manner (r = -0.82, P < 0.005). Neither Ca nor IGF-II affected the distribution of soluble/insoluble ALP activity by more than 9%. IGF-II had no effect on extracellular ALP stability, but the addition of Ca to Ca-free cultures resulted in parallel losses of extracellular ALP activity and ALP immunoreactive protein (P < 0.001 for each). A similar effect was seen when Ca was added to Ca-free, cell-free, conditioned medium, but not when Ca was added to purified ALP, which is consistent with the general hypothesis that a Ca-dependent protease might be present in the cell-conditioned medium. Together, these data suggest that most of the ALP activity released from osteoblasts is insoluble (and, presumably, anchorless), net release of ALP activity is negatively regulated by Ca and skeletal growth factors, the effect of Ca may reflect Ca-dependent protease activity, and an exogenous (e.g., serum) phospholipase may be responsible for releasing ALP from its insoluble anchor.

Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice.

Previous studies have shown that C3H/HeJ (C3H) mice have higher peak bone density than C57BL/6J (B6) mice, at least in part because of differences in rates of bone resorption. The current studies were intended to examine the alternative, additional hypothesis that the greater bone density in C3H mice might also be a consequence of increased bone formation. To that end, we measured two presumptive, indirect indices of bone formation and osteoblast number in these inbred strains of mice: alkaline phosphatase (ALP) activity in serum, bones, and bone cells; and the number of ALP-positive colony-forming units (CFU) in bone marrow stromal cell cultures. We found that C3H mice had higher serum levels of ALP activity than B6 mice at 6 (118 vs. 100 U/L, p < 0.03) and 32 weeks of age (22.2 vs. 17.2 U/L, p < 0.001). Tibiae from C3H mice also contained higher levels of ALP activity than tibiae from B6 mice at 6 (417 vs. 254 mU/mg protein, p < 0.02) and 14 weeks of age (132 vs. 79 mU/mg protein, p < 0.001), as did monolayer cultures of bone-derived cells from explants of 7.5-week-old C3H calvariae and femora (8.2 times more, p < 0.02, and 4.6 times more, p < 0.001, respectively). Monolayer cell cultures prepared by collagenase digestion of calvariae from newborn and 6-week-old mice also showed similar strain-dependent differences in ALP-specific activity (p < 0.001 for each). Our studies also showed more ALP-positive CFU in bone marrow stromal cell cultures from 8-week-old C3H mice, compared with B6 mice (72.3 vs. 26.1 ALP-positive CFU/culture dish, p < 0.001). A similar result was seen for ALP-positive CFU production at 6 and 14 weeks of age, and the difference was greatest for the CFU that contained the greatest numbers of ALP-positive cells. Because skeletal ALP activity is a product of osteoblasts and has been shown to correlate with rates of bone formation, and because the number of ALP-positive CFU is believed to reflect the number of osteoprogenitor cells, the current data are consistent with the general hypothesis that bone formation may be greater in C3H than B6 mice because of a difference in osteoblast number. Our data further suggest that peak bone density may be greater in C3H mice than B6 mice due to a combination of decreased bone resorption and increased bone formation.

Calcium deficiency in fluoride-treated osteoporotic patients despite calcium supplementation.

To test the hypothesis that the osteogenic response to fluoride can increase the skeletal requirement for calcium, resulting in a general state of calcium deficiency and secondary hyperparathyroidism, we assessed calcium deficiency, spinal bone density, by quantitative computed tomography, and serum PTH in three groups of osteoporotic subjects. Two of the three groups had been treated with fluoride and calcium (at least 1500 mg/day) for 32 +/- 19 months. Group 1 consisted of 16 fluoride-treated subjects who had shown rapid increases in spinal bone density (+ 3.8 +/- 2.6 mg/cm2 month), group II consisted of 10 fluoride-treated subjects who had shown decreases or only slow increases in spinal bone density (-0.05 +/- 0.6 mg/cm3 month), and group III consisted of 10 age-matched untreated osteoporotic controls. Calcium deficiency was assessed by measurement of calcium retention after calcium infusion. The results of our studies showed that 1) 94% of the subjects in Group I were calcium deficient compared with only 30% in groups II and III (P < 0.01 for each); 2) the subjects in group I retained more calcium (79%) than the subjects in group II (60%, P < 0.001) or the subjects in group III (64%, P < 0.005); 3) calcium retention was proportional to serum PTH (r = 0.37, n = 36, P < 0.03); and 4) calcium retention was proportional to the (previous) fluoride-dependent increase in quantitative computed tomography spinal bone density (in groups I and II, r = 0.48, n = 26, P < 0.02). To test the hypothesis that the calcium deficiency and the secondary hyperparathyroidism that were associated with the positive response to fluoride would respond to concomitant calcitriol treatment, a subgroup of 7 calcium-deficient subjects were selected from group I and treated with calcitriol (plus fluoride and calcium) for an average of 7 months. The calcitriol therapy reduced the calcium deficit in all 7 subjects, decreasing calcium retention from 80% to 62% (P < 0.02), and decreasing PTH from 50 to 28 pg/mL (P < 0.02). Together, these data indicate that fluoride-treated osteoporotic subjects may develop calcium deficiency in proportion to the effect of fluoride to increase bone formation, and this calcium deficit is responsive to calcitriol therapy.

Phosphate regulates the stability of skeletal alkaline phosphatase activity in human osteosarcoma (SaOS-2) cells without equivalent effects on the level of skeletal alkaline phosphatase immunoreactive protein.

Inorganic phosphate (P(i)) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells, but not by means of changes in transcription or release from the cell surface. The current studies were intended to determine whether (1) P(i) affected the inactivation of ALP activity in human osteosarcoma (SaOS-2) cells; and (2) P(i)-dependent changes in ALP-specific activity were associated with equal, concomitant changes in the level of ALP immunoreactive protein. The results of these studies revealed that P(i) increased the stability of skeletal ALP activity without equivalent effects on the level of ALP immunoreactive protein. An increase in P(i) (from 0 to 1.8 mmol/liter) caused a time-dependent increase in the amount of skeletal ALP activity in the SaOS-2 cells, without a parallel increase in the amount of skeletal ALP immunoreactive protein, and a decrease in P(i) (from 1.8 to 0 mmol/liter) caused a time-dependent decrease in the amount of ALP activity, without a significant decrease in the total cellular content of ALP immunoreactive protein. Together, these observations suggest that P(i) may alter the level of skeletal ALP activity in SaOS-2 cells by inhibiting a process of irreversible inactivation that does not effect equal, concomitant changes in the level of skeletal ALP immunoreactive protein.

An age-related decrease in the concentration of insulin-like growth factor binding protein-5 in human cortical bone.

The skeletal contents of insulin-like growth factor-2 (IGF-II), insulin-like growth factor binding protein-5 (IGFBP-5), and insulin-like growth factor binding protein-3 (IGFBP-3) were determined in duplicate samples of human femoral cortical bone obtained from 64 subjects (44 males and 20 females) between the ages of 20 and 64 years. The results of these quantitative measurements revealed an age-related decrease in the femoral cortical content of IGFBP-5 (r = -0.272, P = 0.031) in the total population. Although the femoral cortical content of IGF-II did not show a similar decrease with age, it could be correlated to the femoral cortical content of IGFBP-5 (r = 0.442, P < 0.001). In contrast, the femoral cortical content of IGFBP-3 did not decrease with age and could not be correlated to the femoral cortical contents of either IGFBP-5 or IGF-II. Comparisons of these results with previous measurements of insulin-like growth factor-1 (IGF-I) and transforming growth factor-beta (TGF-beta), in extracts of the same bones, showed significant cross-correlations between the femoral cortical contents of each of these growth factors and the femoral cortical contents of IGFBP-5 (r = 0.625 for IGF-I versus IGFBP-5, r = 0.554 for TGF-beta versus IGFBP-5, P < 0.001 for each) but not IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of a high prevalence of the BB vitamin D receptor genotype in severely osteoporotic women.

Studies of twins strongly suggest that more than 50% of the peak spinal bone density is determined by genetics. It was reported recently that this genetic effect is primarily determined by vitamin D receptor (VDR) alleles; specifically, a VDR genotype termed BB has been highly associated with low peak bone density. Homozygotes for the second VDR allele, bb, are associated with high peak bone density. If peak bone density is an important determinant of osteoporosis and if the VDR genotype is an important determinant of peak bone density, then patients with severe osteoporosis should have a high prevalence of the BB VDR genotype compared with that of control subjects. To test this hypothesis, we used Southern blot analysis to determine the VDR genotype of 41 Caucasian patients (72 +/- 14 yr) with severe osteoporosis (27 women with spinal bone densities below 50 mg/cm3 as determined by quantitative computed tomography; 14 women with spinal bone densities below 0.75 g/cm2 as determined by dual energy x-ray absorptiometry) and 23 Caucasian control subjects (68 +/- 7 yr) without osteoporosis (quantitative computed tomography values at or above the fracture threshold of 100 mg/cm3). Only 6 of the 41 individuals in the group with severe osteoporosis had the BB genotype, whereas 16 had the bb genotype. In the control group comprising 23 individuals, 7 had the BB genotype and only 6 had the bb genotype. We conclude that the BB VDR genotype is not a good predictor of risk for developing severe osteoporosis in our population.

Homogeneous sample preparation of raw shrimp using dry ice.

Sample homogeneity is critical to accurate and reproducible analysis of trace residues in foods. A method of uniform sample preparation using dry ice is described for shrimp. Other sample preparation techniques for raw shrimp produce nonhomogeneous samples. Sample homogeneity was determined through analysis of chloramphenicol added to intact tiger or white shrimp prior to sample preparation. Simulated chloramphenicol residue levels were 50, 15, 10, and 5 ppb. No significant differences were noted when analyses of shrimp inoculated with chlor-amphenicol prior to sample preparation with dry ice were compared with analyses of shrimp spiked after grinding with dry ice. Grinding shrimp with dry ice produced samples with homogeneous chloramphenicol residues. This technique should be applicable to other tissues and vegetable products.

Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells.

In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10(-13) to 10(-9) mol/liter, for 0-60 minutes at 37 degrees C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with 125I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and 125I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3 x 10(-12) mol/liter) to increase the level of tyrosylphosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P < 0.05 for each); and (2) dose-dependent effects of sCt (during 15 minutes of exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P < 0.05 for each). A supplementary study of calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses--time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P < 0.05 for each).(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in IGFBP-4 and IGFBP-5 levels in human serum and bone: implications for bone loss with aging.

Osteoporosis develops because of an age-dependent imbalance between the rates of bone formation and bone resorption (i.e. bone formation rate is inadequate compared with bone resorption rate to maintain bone volume). With regard to the mechanism for the deficiency in bone formation, we propose that age-associated changes in the IGF system components contribute to an age-related decrease in the skeletal capacity for osteoblast cell proliferation. As a means of testing this hypothesis, we have measured serum levels of IGFBP-4 and IGFBP-5 since our studies have shown that the mitogenic actions of IGFs in bone cells are modulated by inhibitory IGFBP-4 and stimulatory IGFBP-5. By using newly developed and validated radioimmunoassays for measurement of IGFBP-4 and IGFBP-5, we found that the circulating level of IGFBP-4 increases with age while that of IGFBP-5 declines with age. In subjects from 23-87 years, serum IGFBP-4 concentrations showed a significant positive correlation with serum PTH while serum IGFBP-5 concentrations showed a significant positive correlation with IGF-I. These age-related changes in the serum levels of IGF system components are consistent with our previous findings of age-related decreases in the femoral cortical contents of IGF-I, IGF-II and IGFBP-5. Although the biological implications of the sequestration of IGFs in bone are unknown, we have hypothesized that the level of the IGFs in bone is a reflection of their integrated local secretion by osteoblasts. Based on our data, we now propose a model in which (a) underproduction of the stimulatory components and overproduction of an inhibitory component of the IGF system occur as a consequence of aging, and (b) these changes lead to an age-related decrease in the local (autocrine/paracrine) as well as the hormonal (endocrine) actions of the IGFs, which in aggregate could contribute to the decrease in osteoblast proliferation and the deficiency in bone formation. In conclusion, although our findings provide indirect evidence that age associated changes in IGF system components could lead to a deficit in bone formation, further studies are needed to demonstrate a cause and effect relationship between changes in bone cell production of IGF system components and the age-related uncoupling of bone formation from resorption.