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J R Farley

Publications and source records attributed to J R Farley.

At least 19 recordsLinked to original sources
PubMed2000

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.

Alkaline Phosphatase↗
PubMed1999

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.

Alkaline Phosphatase↗
PubMed1999

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.

Animals↗
PubMed1998

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.

3T3 Cells↗
PubMed1998

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↗
PubMed1998

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.

Alkaline Phosphatase↗
PubMed1996

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.

Aged↗
PubMed1995

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.

Alkaline Phosphatase↗
PubMed1995

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)

Adult↗
PubMed1995

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)

Alkaline Phosphatase↗
PubMed1995

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.

Aging↗
PubMed1995

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.

Age Factors↗
PubMed1994

Specific activity of skeletal alkaline phosphatase in human osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase activity inversely regulated by calcium.

We assessed the significance of Ca and phosphate (P(i)) as determinants of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJb medium containing 0.25-2 mM P(i), the specific activity of ALP in SaOS-2 cells was proportional to P(i) concentration (r = 0.99, p < 0.001). The P(i)-dependent increase in ALP activity was time dependent (evident within 6 h) and could not be attributed to decreased ALP release, since P(i) also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25-2.0 mM) showed that the amount of ALP activity released from SaOS-2 cells was inversely proportional to the concentration of Ca (r = -0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) and could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinositol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol-glycan-anchored ALP, released by membrane vesicle formation). The range of Ca-dependent changes in ALP-specific activity was much smaller than the range of P(i)-dependent changes. The observed correlation between skeletal ALP-specific activity and P(i) was not unique to osteosarcoma cells or to P(i). Similar effects were seen in normal human bone cells in response to P(i) (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of P(i) esters and analogs (e.g., beta-glycero-P(i) and molybdate). Further studies indicated that the effects of phosphoryl compounds on ALP-specific activity could not be correlated with effects on ALP reaction kinetics, cell proliferation, or acid phosphatase activity and that the beta-glycero-P(i)-dependent increase in ALP activity was blocked by cycloheximide but not actinomycin D. Together these data suggest that the function of skeletal ALP may be regulated by P(i) and that Ca may be involved in ALP release.

Alkaline Phosphatase↗
PubMed1994

Age-related decreases in insulin-like growth factor-I and transforming growth factor-beta in femoral cortical bone from both men and women: implications for bone loss with aging.

We determined the skeletal content of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF beta) in human bone as a function of age, using 66 samples of femoral cortical bone obtained from 46 men and 20 women between the ages of 20-64 yr. We found a linear decline in the skeletal content of IGF-I (nanograms per mg protein) with donor age (r = -0.43; P < 0.001) in the total population. The skeletal content of TGF beta also decreased with age (i.e. 1/TGF beta vs. age; r = 0.28; P < 0.02) for the total population. We did not observe any difference in the skeletal growth factor content between male and female donors. IGF-I content, when analyzed by decade divisions of age, showed a reduction between the 20- to 29-yr-old and the 50- to 59-yr-old subjects (P < 0.02). The loss rate of IGF-I was 1.56 ng/mg protein.yr, corresponding to a net loss of 60% of skeletal IGF-I between the ages of 20-60 yr. The loss rate of TGF beta was 0.03 ng/mg protein.yr, corresponding to a net loss of 25% of the skeletal TGF beta between the ages of 20-60 yr.

Adult↗
PubMed1994

Quantification of skeletal alkaline phosphatase in osteoporotic serum by wheat germ agglutinin precipitation, heat inactivation, and a two-site immunoradiometric assay.

Three methods for quantifying skeletal alkaline phosphatase (ALP; EC 3.1.3.1) activity/immunoactivity in serum--heat inactivation, wheat germ agglutinin (WGA) precipitation, and an immunoradiometric assay--were tested for recovery and specificity and applied to 81 sera collected from 14 postmenopausal osteoporotic subjects. The heat-inactivation and WGA precipitation assays showed relative recoveries of 91-100% and 16-32%, respectively, for skeletal ALP with complete specificity (no cross-reactivity with hepatic or intestinal ALP); the IRMA showed a relative recovery of 86-100% and 11-14% cross-reactivity with hepatic ALP. There was a closer correlation between the heat-inactivation assay and IRMA (r = 0.833) than between the WGA precipitation assay and IRMA (r = 0.673) or between the heat-inactivation and WGA-precipitation assays (r = 0.568). The WGA precipitation assay failed to detect skeletal ALP in three serum samples that contained significant amounts as determined by the heat-inactivation assay and the IRMA.

Alkaline Phosphatase↗