Pentose-shunt oxidation in the periosteal cells in healing fractures

Summary The activity of pentose-shunt dehydrogenases is very low in periosteal cells of normal rat metatarsals, but increases one day post-fracture and rises linearly over the next two days. By four days postfracture, the distribution of this activity along the bone shows two centres of high activity: the first in the region of proliferation to form callus and the second at the site where new bone is first seen, one day later. The high rate of generation of NADPH would be expected to reduce glutathione; reduced glutathione has been shown to inhibit alkaline phosphatase activity in these cells.     

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.     

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.     

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.     

A phorbol ester induces secretion of alkaline phosphatase activity in human osteosarcoma cells

Summary The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) blocked the growth of, and induced the appearance of processes in the human osteosarcoma cell line U-2 OS. The phorbol ester decreased the intracellular level of alkaline phosphatase (APase) activity (as measured per mg cell protein) and caused a marked increase in the APase activity secreted from the cells into the culture medium. The secretion of APase appeared after a lag period of 4–6 hours of TPA treatment, and it could also be visualized with histological staining. Differential ultracentrifugation of the culture media showed that the APase was released to the media in the form of vesicles. The vesicles were studied by electron microscopy and appeared similar to matrix vesicles isolated from cartilage and chondrocytes. It is thus concluded that TPA is able to induce the primary steps of mineralization in these cells.     

Low-dose parathyroid hormone and estrogen reverse alkaline phosphatase activity suppressed by dexamethasone in mouse osteoblastic cells

Glucocorticoid (GC)-induced osteoporosis (GIO) is frequently seen in patients with excessive GC. Numerous questions remain to be clarified about the pathogenesis and treatment of GIO, and the mechanism of GC-inhibited bone formation is not well known. Several studies suggest that parathyroid hormone (PTH) and hormone replacement therapy are effective for GIO. We therefore investigated whether PTH and estrogen would affect cell proliferation and alkaline phosphatase (ALP) activity inhibited by dexamethasone (Dex) in mouse osteoblastic cell-line MC3T3-E1 cells. Low-dose (10⁻¹¹ M) PTH as well as 10⁻⁸ M 17-β-estradiol (17β-E₂) significantly attenuated Dex-inhibited ALP activity, although 10⁻⁸ M PTH did not affect it. ICI 182780 (10⁻⁸ M) antagonized the effects of 17β-E₂ on Dex-suppressed ALP activity. Neutralizing anti-IGF-I antibody (3 μg/ml) blocked the reverse effects of 17β-E₂ on ALP activity suppressed by Dex. PTH (10⁻¹¹ M), but not 17β-E₂, significantly attenuated [³H]thymidine incorporation inhibited by Dex. On the other hand, PTH and estrogen did not affect the level of 11-β-hydrosteroid dehydrogenase type I mRNA increased by Dex. In conclusion, the present study demonstrated that low-dose PTH and estrogen reversed Dex-inhibited ALP activity in the mouse osteoblastic cell-line.     

绝经后骨质疏松患者的骨特异性碱性磷酸酶变化及其临床意义

为了解骨形成的变化在绝经后骨质疏松发病中的作用，本文测定３７例绝经后骨质疏松患者和３１例健康绝经后妇女的骨特异性碱性磷酸酶，分析了这一变化与血总碱性磷酸酶及骨密度（用双能Ｘ线吸收仪测定）的相关性。结果发现绝经后骨质疏松患者骨特异性碱性磷酸酶和骨钙素较对照组明显降低，但这一变化与骨密度值无明显的相关性。提示骨形成减低并非绝经后骨质疏松的主要发病机理。     

氟化钠对人黄韧带细胞碱性磷酸酶活性及骨钙素合成的影响

目的 探讨氟化钠(NaF)对体外培养条件下人黄韧带细胞碱性磷酸酶(alkaline phosphatase,ALP)活性及骨钙素(bone gla protein,BGP)合成的影响.方法 依据手术标本来源不同,分为正常黄韧带细胞(NLF)组(取自急性外伤性胸腰椎骨折截瘫患者,7例)、退变黄韧带细胞(DLF)组(取自退变性腰椎管狭窄症患者,9例)及骨化黄韧带细胞(OLF)组(取自胸椎黄韧带骨化患者,8例).采用组织块贴壁法进行体外细胞培养,共获得24组传代细胞.取第五代细胞加入不同浓度的NaF,观察细胞形态变化,并测定NaF对各组人黄韧带细胞的ALP活性和BGP合成的影响.结果 不同来源的人黄韧带细胞均可在体外增殖并传代,DLF与OLF组人黄韧带细胞呈多形性表现,并可形成钙结节.体外培养条件下,高浓度(1.0mmol/L)NaF可导致人黄韧带细胞发生中毒反应;低浓度(0.01～0.125 mmol/L)NaF可促进DLF组黄韧带细胞增殖、钙结节形成,ALP活性上调,BGP合成明显增加,而对OLF与NLF组人黄韧带细胞则无明显效应.结论 体外培养条件下,低浓度(0.01～0.125 mmol/L)NaF可促进人退变黄韧带细胞的ALP活性增高及BGP合成增加,提示低浓度NaF可能促进人退变黄韧带细胞向成骨方向分化.     

The effects of prostaglandin E2, parathyroid hormone, 1,25 dihydroxycholecalciferol,and cyclic nucleotide analogs on alkaline phosphatase activity in osteoblastic cells

Summary Clone MC3T3-E1 cells isolated from newborn mouse calvaria had the same type of alkaline phosphatase (ALP) as that found in adult mouse calvaria (the liver-bone-kidney type), as judged by polyacrylamide gel electrophoresis as well as by heat lability and amino acid inactivation. The effects of prostaglandin E₂ (PGE₂), parathyroid hormone (PTH), 1,25 dihydroxycholecalciferol [1,25(OH)₂D₃], and adenosine-3′, 5′-cyclic monophosphate (cAMP) analogs on ALP were investigated. PGE₂ and 1,25(OH)₂D₃ increased ALP activity in dose-related manner with a maximal effect at concentrations of 10 ng/ml and 40 pg/ml, respectively. N⁶,O²-dibutyryl adenosine-3′, 5′-cyclic monophosphate (DBcAMP) also induced an increase in ALP activity in a dose-related fashion with a maximal effect at a concentration of 0.5 mM which was 2.2-fold over that of the controls. Induced ALP was of the “liver-bone-kidney” type. Antinomycin D and cycloheximide inhibited the increase in ALP activity induced by DBcAMP. The level of ALP was elevated by 8-bromo-adenosine-3′,5′-cyclic monophosphate and theophylline, but not by N⁶,O²-dibutyryl guanosine-3′,5′-cyclic monophosphate and sodium butyrate. Moreover, PGE₂ dramatically increased the level of cAMP in the cells with a maximal effect at a concentration of 10 ng/ml, indicating that PGE₂ and DBcAMP induce an increase of ALP activity in clone MC3T3-E1 cells by increasing the cAMP level; PTH did not affect enzyme activity and cAMP, level in the cells. These results suggest that PGE₂, DBcAMP, and 1,25(OH)₂D₃ are involved in bone formationin vivo as well.     

骨源性碱性磷酸酶预测骨质疏松骨折患者再骨折的意义

目的探讨骨质疏松患者初次骨折后发生再骨折的影响因素,分析血清骨源性碱性磷酸酶(BALP)对预测再发骨折的临床价值。方法前瞻性队列研究纳入我院178例确诊的50岁以上骨质疏松性骨折患者。初次骨折时检测患者血清BALP水平、骨密度值(BMD)、钙、磷及临床一般资料等,跟踪随访4年,以患者再发骨折为随访终点事件。采用Kaplan-Meier分析和多元Cox回归模型进行再发骨折的风险因素研究,受试者工作特征(ROC)曲线用于评判BALP的预测价值。结果 178例骨质疏松性骨折患者4年内30(16.9%)例发生再次骨折。Cox回归分析结果显示BALP、年龄、性别和BMD是患者再发骨折的独立影响因素。以BALP为预测标准,ROC曲线下面积为0.757,诊断临界点为29.0μg/L,灵敏度及特异度分别为84.3%、63.8%。Kaplan-Meier分析显示BALP29.0μg/L患者骨折再发率较BALP≤29.0μg/L更高(p=0.026)。结论骨质疏松骨折患者发生再骨折的风险较高,检测血清BALP水平是有效可行的预测指标。     

Elevation of alkaline phosphatase activity induced by parathyroid hormone in osteoblast-like cells from the spinal hyperostotic mouse TWY (twy/twy)

We have examined the alkaline phosphatase (AP) activity of primary calvaria-derived osteoblast-like cells from the twy (tip-toe walking Yoshimura) and normal ICR control mouse. The twy mouse displays elevated osseous formation particularly in the spine, and the pathophysiological features resemble that of human ankylosing spinal hyperostosis. In the proliferative stage of cultured bone cells, parathyroid hormone (PTH) stimulation induced the elevation of AP activity of both twy and ICR mouse-derived cells. When they reached confluence, the AP activity of ICR mouse-derived cells ceased to increase with PTH stimulation. The twy mouse-derived cells, however, continued to respond to PTH, with the enzyme activity increasing even in the confluent, stationary stage. PTH stimulation also increased the intracellular cAMP content of twy mouse-derived cells but it did not influence that of ICR mouse-derived cells in the stationary stage. Moreover, stimulation with dibutyryl cAMP, but not with phorbol myristate acetate, increased the AP activity of both twy and ICR-derived bone cells irrespective of culture conditions, either in the proliferative or in the confluent stage. These data suggest that the protein kinase A-mediated pathway plays a pivotal role in bone cells with PTH stimulation, and that the uninhibited AP activity observed in twy mouse-derived bone cells might be due to some deviating process between the PTH ligand/receptor interaction and cAMP generation.     

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     

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     

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.     

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.     

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。     

Effects of deferoximine on chondrocyte alkaline phosphatase activity: Proxidant role of deferoximine in thalassemia

The homozygous form of -thalassemia, the most common single gene disorder, is treated by red cell transfusion therapy. Following transfusion, the chelator, deferoximine, is administered to patients to remove excess iron. However, when this drug is given to young children, metaphyseal dysplasia and abnormalities of linear growth are frequently observed. To explore the notion that deferoximine mine interferes with endochondral growth by chelating zinc, we examined the effect of the drug on chondrocytes maintained in long-term culture. We found that deferoximine caused a dose-dependent inhibition of a wide range of functions including cell proliferation, protein synthesis (and possibly under-hydroxylation of type X collagen), and mineral deposition. Directly relevant to the mineralization process was the observation that the drug dramatically lowered the activity of alkaline phosphatase, a zinc-requiring enzyme. To test the hypothesis that enzyme inhibition was due to chelation of zinc by deferoximine, the cell culture medium was supplemented with excess zinc. However, this treatment did not overcome the deferoximine-dependent change in enzyme activity. We next examined the possibility that deferoximine, in the presence of ascorbate, could form a free radical system that would serve to inactivate the enzyme. Using alkaline phosphatase extracted from chick cartilage, we noted that the activity of the phosphatase was markedly reduced in the presence of deferoximine and ascorbate. These effects were consistant with the notion that deferoximine and ascorbate can act as a prooxidant couple. This conclusion was confirmed when we measured the oxidative activities of the system using nitroblue tetrazolium and cytochrome c. Indeed, we noted that deferoximine markedly activates the autocatalytic oxidation of ascorbate. We next investigated the possibility that the change in alkaline phosphatase activity was due to the formation of reactive oxygen radicals. Though oxygen radical scavengers and disproportionating agents did not change the activity of the enzyme, -tocopherol provided complete protection. In conclusion, the deferoximine-ascorbate couple inactivates chondrocyte alkaline phosphatase probably by generation of free radicals. As free radicals can damage cartilage as well as other tissues, clinical regimens that are directed at elevating ascorbate levels in thalassemia need to be carefully reviewed.     

Calcitonin has direct effects on3[H]-thymidine incorporation and alkaline phosphatase activity in human osteoblast-line cells

Summary Calcitonin had direct and dose-dependent actions on human osteoblast-line cells (in serum-free monolayer culture) to increase cell proliferation and alkaline phosphatase activity/mg cell protein. Salmon calcitonin increased (human osteosarcoma) SaOS-2 cell proliferation, as evidenced by dose-dependent increases in³[H]-thymidine incorporation into DNA (e.g., 153% of control after 20 h exposure at 0.1 nM,P<0.01), and MTT (thyzolyl blue) reduction/deposition (e.g., 161% of control after 72 h exposure at 0.03 nM). Continuous exposure was not required to elicit these proliferative responses. These effects were not unique to salmon calcitonin or to SaOS-2 cells. Similar effects were seen with human calcitonin (but not heat-inactivated human calcitonin) and with (human osteosarcoma) TE-85 cells and human osteoblast-line cells prepared from femoral heads. In addition to effects on cell proliferation, calcitonin also increased alkaline phosphatase-specific activity in SaOS-2 cells (e.g., 180% of control after 72 h of exposure to 0.1 nM salmon calcitonin,P<.005).     

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.