Alkaline phosphatase as a marker of osteoinductive cells

Summary Epithelial cells with osteoinductive potential (KB and WISH cell lines, transitional epithelium of several species) are rich in alkaline phosphatase activity. In contrast, cells devoid of osteoinductive ability are low in this enzyme activity. However, there were no differences between the two classes of cells with respect to acid phosphatase activity.     

Alkaline phosphatase and ATPase activities of rat bone: Separation and characterization

Summary Extraction with Triton X-100 has proved effective in solubilizing alkaline phosphatase from rat bone particles, whereas ATPase with optimum activity at pH 8 remains attached to the bone particles. The kinetic characteristics of the ATPase activity of the Triton extracts are different from those of the same enzyme attached to bone particles, but the kinetic characteristics of the particle-bound and solubilized alkaline phosphatases are similar. The results suggest that the Triton extracts do not have true ATPase activity and provide a means of separating the ATPase and alkaline phosphatase activities.     

Purification of alkaline phosphatase from extracellular vesicles of fracture callus cartilage

Summary Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm³ band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm³ band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm³ band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm³ band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm³ fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation with-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.     

A comparative study of alkaline phosphatase in calcifying cartilage,odontoblasts and the enamel organ

The enzyme alkaline phosphatase (AP) (EC 3.1.3.1) in three different calcification areas was studied by means of a spectrophotometric micro method using p-nitrophenylphosphate as a substrate. Rat maxillary incisor odontoblasts and enamel organ from the zones of matrix formation and maturation and tissue from rabbit metatarsal cartilage were allowed to react with the substrate in glycine-NaOH buffer at room temperature. The reaction was found to be linear for a minimum of 20 min. The pH optima for AP from these tissues were in the pH range of 10.0–10.3. In order to compare AP from the four calcification areas different parameters were studied. Heating at 56°C or 60°C for varying times revealed that the enzymes were almost completely inactivated after 10 min. Mg²⁺ ions activated the enzymes by about 25% at concentrations of 2.5 mM (enamel organ 1.25 mM); while only higher concentrations of Mg²⁺ had an inactivating effect, Ca²⁺ and PO ₄ ^3– ions were inactivating at varying concentrations. F^– ions show no effect on AP activity at concentrations below 250 mM (enamel organ 125 mM) but caused inactivation of the enzymes at about 50% at 1 M. EDTA was found to be a very effective AP inactivator at concentrations above 0.06 mM, whereas urea did not noticeably affect the enzyme reactions at concentrations below 1 M. At higher concentrations, inactivation was observed. In order to determine AP localization in the epiphyseal plate successive 40-m-thick, freeze-sectioned slices were analyzed. The activity was highest nearest the zone of cartilage calcification and decreased towards the reserve cell zone.It was concluded that the same AP isoenzyme is present in these quite different calcification loci.     

Enzymatic characterization of the matrix vesicle alkaline phosphatase isolated from bovine fetal epiphyseal cartilage

Summary Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PP_i. Optimal activities forp-nitrophenyl phosphate, ATP, and PP_i are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations.p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg²⁺ and Hg²⁺ ions inhibit thep-nitrophenyl phosphatase activity at pH 10.5 while Mn²⁺ ions show no effect. P_i, levamisole, CN⁻, Zn²⁺, Ca²⁺ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. P_i is a linear noncompetitive inhibitor with a K_i of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca²⁺ ions inhibit noncompetitively with a K_i of 9.3 mM. Zn²⁺ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be²⁺ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromethanediphosphonate, L-cysteine, and N-ethylmaleimide. NaCl, KCl, and Na₂SO₄ at 0.5–1.0 M inhibit the enzyme. At pH 8.5, the cleavage of PP_i by the matrix vesicle enzyme is inhibited by Mg²⁺ and Ca²⁺ ions at concentrations greater than 0.5 mM. Mg²⁺ ions in the range of 0.1–4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca²⁺ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0. With 5′-AMP as substrate, the 3.5-fold decrease in the maximum velocity of the matrix vesicle relative to the chondrocyte enzyme may be accounted for in terms of a small excess enthalpy of activation (1160 cal/mole) partially compensated by an increased entropy of activation. Deceased May 8, 1975.     

Alkaline phosphatase inhibition by parathyroid hormone and isoproterenol in a clonal rat osteosarcoma cell line. Possible mediation by cyclic AMP

Summary The effect of parathyroid hormone (PTH 1–34 bovine) on alkaline phosphatase activity was investigated in an osteoblast-like clonal cell line derived from rat osteosarcoma (ROS 17/2). ROS 17/2 alkaline phosphatase resembled the bone enzyme in levamisole sensitivity and electrophoretic mobility but differed in heat stability. The specific activity of ROS 17/2 alkaline phosphatase increased with time in culture. This increase was inhibited by PTH (1–34) and (-)-isoproterenol in a dose-dependent manner starting at near-physiological hormone concentrations. The ID₅₀ values were 0.02 nM for PTH (1–34) and 1.7 nM for isoproterenol. The two hormones stimulated ROS 17/2 adenylate cyclase, albeit at higher concentrations: K_m values were 13 nM for PTH (1–34) and 16 nM for isoproterenol. The rise in alkaline phosphatase was also inhibited by dibutyryl cyclic AMP and 8-bromocyclic AMP (0.1 mM). These findings further document the osteoblastic properties of the ROS 17/2 osteosarcoma cell line, suggest that PTH inhibition of alkaline phosphatase represents a physiological response to the hormone in these cells, and implicate cyclic AMP as a mediator of this PTH effect.     

Alkaline phosphatase is independently associated with renal function in normoalbuminuric type 1 diabetic patients

Nonalcoholic fatty liver disease (NAFLD) is associated with an increased prevalence of chronic kidney disease in patients with type 1 diabetes. The aim of this study was to explore the relationship between markers of NAFLD, namely concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALK), γ-glutamyltransferase (GGT), bilirubin, and renal function in type 1 diabetic patients. This study included 313 normoalbuminuric type 1 diabetic patients with estimated glomerular filtration rate (eGFR) >60?mL/min/1.73?m², without clinical evidence of cirrhosis or other causes of chronic liver disease and before any interventions with statins, angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers. ALT, GGT, and bilirubin levels were significantly higher in subjects in the highest quartile of serum creatinine compared to those in lowest quartile (21 vs. 20 U/L, 18 vs. 14 U/L, and 14 vs. 10?µmol/L, respectively, for all p?0.05). ALK levels were significantly higher in subjects in the highest quartile of urinary albumin excretion rate compared to those in lowest quartile (71 vs. 69 U/L, p?=?0.03), as well as in hyperfiltrating subjects compared to those with normal or mildly impaired eGFR (81 vs. 68 and 64 U/L, p?0.001). In a multiple logistic regression model adjusted for age, sex, duration of diabetes, HbA1c, and body mass index (BMI), only ALK levels were significantly associated with disturbances in serum creatinine and eGFR in our subjects (p?≤?0.007), with odds ratios of 0.98–1.02. NAFLD associated markers, particularly ALK, are associated with renal function in normoalbuminuric type 1 diabetic patients.     

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.     

Ultrastructural localization and gradient of activity of alkaline phosphatase activity during rodent odontogenesis

Summary The ultrastructural localization and gradient of activity of alkaline phosphatase were studied with respect to cell differentiation, matrix synthesis, and matrix mineralization in the incisor and molar teeth of 4-day-old Sprague-Dawley rats. The animals were perfused intracardially at room temperature with 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4) with 3–4% sucrose. The jaws were dissected, immersion-fixed for 24 h, and the incisor and molar tooth germs removed. These were demineralized in 10% EDTA in NaOH (pH 7.4) with 7% sucrose. After reactivation of the enzyme with 0.1M MgCl in Tris-maleate buffer (pH 7.4) at 4°C, the teeth were incubated for alkaline phosphatase in a medium consisting of 6 ml 3% sodiumβ-glycerophosphate, 4 ml 0.2M Tris-HCl buffer (pH 9.2), 3 ml 1.6% MgSO₄, 12 ml 0.5% lead citrate (pH⋍12), and 2.1 g sucrose. The pH was adjusted to 9.2 with 0.2M HCl, the volume made up to 30 ml, and the solution centrifuged for 10 min at 5000 rpm. Control teeth were incubated in medium minus the substrate. Finally, the specimens were routinely post-fixed and embedded for sectioning and examination with a Philips 300 electron microscope. A gradient of alkaline phosphatase activity was mapped along the developing teeth in the cells of the stratum intermedium, the proximal borders of the ameloblasts, the early dentine matrix, the predentine-dentine border, matrix vesicles, and the plasma membranes of odontoblasts and subodontoblast cells. The gradient of alkaline phosphatase activity was evident in the forming tooth from the cervical loop to the crown apex and was related to the cellular events, matrix synthesis, and matrix mineralization occurring during odontogenesis.     

不同方法检测成骨细胞中碱性磷酸酶的比较

目的　探讨不同缓冲液和不同裂解方法对体外培养成骨细胞内碱性磷酸酶 (alkalinephosphatase,ALPase)活性的影响 ,建立较为理想的检测方法和条件 ,为研究骨代谢异常及骨质疏松症等骨病提供简便可靠的检测手段。方法　采取经体外培养 72h的成骨细胞 ,制备上清液、细胞裂解液和细胞反复冻融液样本 ,分别用二乙醇胺 (DEA)、碳酸盐 (CO=3)、2 氨基 2 甲基 1 内醇 (AMP)等 3种不同缓冲液种类的试剂 ,测定上述液体中的ALPase活性。结果　使用DEA缓冲液的 3种样本的测量值都为最高 ,且不精密度 (CV)较其他两种缓冲液小 ,各组比较分析 ,差异均有显著性 (P <0 .0 1 )。使用 3种缓冲液试剂 ,均以裂解液中ALP活性最高 ,而冻融液次之 ,上清液最低 ,分析结果 ,差异均有显著性 (P <0 .0 1 )。结论　用DEA缓冲液测量细胞裂解液体外培养成骨ALPase活性的效果较理想     

晚期妊娠妇女血清α-L-岩藻糖苷酶和碱性磷酸酶检测及临床意义

目的测定晚期妊娠妇女血清中α-L-岩藻糖苷酶和碱性磷酸酶的含量,以探讨其临床意义。方法实验组169例妊娠期妇女,对照组137例健康育龄妇女,分别检测血清AFU、ALP含量。结果实验组血清AFU、ALP含量较对照组高,差异有统计学意义（P〈0.01）,且随孕周数增加AFU、ALP也有所增加。结论血清AFU、ALP含量测定可以作为晚期妊娠妇女监测胎儿、胎盘的指标。     

Correlation between alkaline and acid phosphatase activities and age-related osteopenia in murine vertebrae

Summary Lumbar vertebrae (L₄) from CW-1 female mice were examined for age-related changes in alkaline and acid phosphatase activities from young to old age. Histochemically, both enzymes were encountered along the bony surfaces of both trabecular and cortical bones with no significant age-related changes in their distribution. Biochemical determinations of bone alkaline phosphatase (Alk'ase) activity revealed that for a given unit level of bone or the bone as a whole no significant changes took place, whereas acid phosphatase (Acid'ase) activity was found to have increased significantly with age. A high positive correlative relationship was noted between the calcium content and the trabecular bone volume of the same vertebrae. It may, therefore, be proposed that age-related bone loss in mice could be attributed to an enhanced resorption rather than to a substantial reduction in the formative potential of bone cells.     

Changes in alkaline phosphatase activity in periosteal cells in healing fractures

The quantitative changes in alkaline phosphatase activity in the periosteal cells close to the fracture in rat metatarsal bones has been measured during the first 5 days postfracture. This study has been made possible by two technological advances, firstly the development of cryostat microtomy for cutting unfixed, undemineralised bone, and secondly the use of scanning and integrating microdensitometry for quantifying the activity in each periosteal cell. The results showed a loss of alkaline phosphatase activity close to the fracture site, with activity rising to normal values 0.8–1.0 mm from the site. No alkaline phosphatase activity was found in the cells which proliferated from the periosteum. It is suggested that reduced glutathione could cause such inhibition.     

应用硷性磷酸酶组化法显示皮瓣微血管的研究

目的　探讨皮瓣微血管的酶组织化学染色效果。方法　应用酶组化法对人体皮瓣的微血管进行硷性磷酸酶染色。结果　组织用10%缓冲福尔马林(甲醛)4℃固定6～12小时,冰冻切片30～50μm,酶显示微血管的效果最好;皮瓣的微血管主要分布于真皮乳头层、网状层及皮下层;血管壁呈棕褐色,微动脉及毛细血管着色深,微静脉着色浅。结论　该方法不需灌注,对血管无扩张作用,无人为改变,适用于皮瓣微血管形态学研究和定量分析。     

应用硷性磷酸酶组化法显示皮瓣微血管的研究

目的　探讨皮瓣微血管的酶组织化学染色效果。方法　应用酶组化法对人体皮瓣的微血管进行硷性磷酸酶染色。结果　组织用 10 %缓冲福尔马林 (甲醛 ) 4℃固定 6～ 12小时 ,冰冻切片 30～ 5 0 μm ,酶显示微血管的效果最好 ;皮瓣的微血管主要分布于真皮乳头层、网状层及皮下层 ;血管壁呈棕褐色 ,微动脉及毛细血管着色深 ,微静脉着色浅。结论　该方法不需灌注 ,对血管无扩张作用 ,无人为改变 ,适用于皮瓣微血管形态学研究和定量分析     

Stimulation of alkaline phosphatase activity in cultured neonatal mouse calvarial bone cells by parathyroid hormone

Summary The effect of parathyroid hormone (PTH) on alkaline phosphatase activity was examined in confluent, serum-free primary cultures of neonatal mouse calvarial cells. It was found that synthetic bPTH-(1-34) caused an increase in the specific activity of skeletal alkaline phosphatase isoenzyme by 18 hours. Between 10 and 500 ng/ml, the mganitude of the change was directly related to peptide concentration. The change occurred in the absence of any effect on cell number, total cell protein, or DNA and was not the result of an effect on either proliferation or survival of a specific cell population. Results of histochemical studies indicate that bPTH-(1-34) caused an increase in the proportion of cells containing enzyme activity. The response was duplicated by intact bPTH-(1-84) and DBcAMP, but not by oxidized bPTH-(1-34) or insulin and did not require prostaglandin synthesis or hydroxylation of 25-hydroxyvitamin D₃. These results demonstrate that bPTH has a direct effect on osteoblast maturationin vitro, that the effect is specific for PTH, and suggest that it is mediated by cAMP.     

Pyrophosphatase and ATPase of isolated cartilage matrix vesicles

Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg²⁺, Ca²⁺, and PP_i. Further, the ATPase activity was not activated by Ca²⁺ in the presence of an optimal Mg²⁺ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PP_i, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.     

Calcium incorporation in matrix vesicles isolated from chicken epiphyseal cartilage

Summary Matrix vesicles isolated from chicken epiphyseal cartilage displayed an uptake of Ca²⁺ which was linear with time and the amount of vesicle protein. The matrix vesicles stimulated the incorporation of Ca²⁺ even at very low Ca × P, suggesting that they could bind Ca²⁺ and/or increase the local Ca × P to the metastable level. This uptake was abolished by EDTA or heating, and partially inhibited by cysteine, to the same extent as the hydrolysis of ATP. There was also a certain uptake of Ca²⁺ without added phosphate, this being stimulated by ATP up to 3 mmol, but diminishing again with higher concentrations. The presence of ATP failed to stimulate the uptake of Ca²⁺ more than an equimolar amount of phosphate in the form of inorganic KH₂PO₄. Mg²⁺ activated the hydrolysis ofp-nitrophenylphosphate at pH 10.5, and both Mg²⁺ and Ca²⁺ the hydrolysis of ATP from pH 7 to 9.5. Paradoxically, the omission of Mg²⁺ stimulated the uptake of Ca²⁺ several-fold.     

Monofluorophosphate is hydrolyzed by alkaline phosphatase and mimics the actions of NaF on skeletal tissues,In Vitro

Summary These studies were intended to assess the osteogenic activity of monofluorophosphate (MFP)in vitro, and to identify the enzyme(s) responsible for MFP hydrolysis—alkaline phosphatase (ALP) and/or acid phosphatase (AcP). ALP and AcP activities were determined by hydrolysis of p-nitrophenylphosphate (PNPP) at pH>8 and pH 5.5, respectively, and MFP hydrolysis was determined, between pH 5.5 and pH 9.0, from measurements of [fluoride ion], using an ion-specific electrode. We found (1) that MFP was an alternative substrate for purified ALP, but not for AcP; (2) that MFPase activity in the embryonic chick resembled ALP, but not AcP, with respect to pH-dependent hydrolysis, sensitivity to effectors (r=0.98,P<.001), and tissue distribution (r=0.96,P <.001); and (3) that intestinal MFPase activity in the embryonic chick co-purified with ALP activity (r=0.93,P<.01) and resembled ALP, but not AcP, in its distribution along the small intestine, being highest in the duodenum and lowest in the distal ileum (r=0.96,P<.001). We also found thatin vitro exposure to MFP increased (1) the proliferation rate of embryonic chick calvarial cells in serum-free monolayer cultures (i.e.,³[H]-thymidine incorporation into DNA,P<.001); (2) ALP activity in calvarial cells (P<.005) and in intact calvaria (P<.05); and (3) collagen production by intact calvaria (i.e.,³[H]-proline incorporation as³[H]-hydroxyproline,P<.05). Together these data indicate that ALP, and not AcP, is responsible for MFP hydrolysis, and that MFP can mimic the osteogenic actions of NaF,in vitro.     

The induction of alkaline phosphatase by extraskeletal implants of bone matrix

Implants of bone matrix, capable of inducing new bone formation, were prepared by decalcifying cortical bone in 0.6 N HCl at 2°C for 48 hours. The inductive property could be inactivated by decalcification in 0.6N HCl containing 66.5% ethanol. When implanted into muscle, both the active and inactive implants were invaded by mesenchymal cells. There was no significant difference in the DNA content of active and inactive implants, but only the active implants induced the synthesis of alkaline phosphatase by a new cell population. Alkaline phosphatase activity was present at 5 days, reached a peak at 20 days after implantation, then declined to 15% of the maximum value by the 40th day after implantation. The deposition of calcium in the implants, coinciding with the differentiation of new bone, began about 12 days after implantation, and gradually increased throughout the 40-day experimental period. Neither recalcification of old matrix nor differentiation of bone occurred in the acid-alcohol-inactivated implants. Induction of alkaline phosphatase synthesis occurred in the pre-osseous phase of morphogenesis 5 days before the appearance of calcium deposits and was not directly correlated with the mechanism of calcification.This investigation was supported in part by research grant DE-02103 from the National Institute of Dental Research and in part by a grant from the John A. Hartford Foundation.