Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.     

The ultrastructural localization of adenosinetriphosphatase and alkaline phosphatase activity in eosinophil leukocytes

Summary The previously undescribed localization of reaction products of adenosinetriphosphatase and of alkaline phosphatase in eosinophil leukocytes was demonstrated by cytochemical studies of the rat intestine. Alkaline phosphatase reaction product was found only in minimal amounts on the plasma membrane but was distinct on the nuclear membranes and outer compartment of mitochondria but not on the cristae. The Golgi membranes and the endoplasmic reticulum reacted but less intensely. The specific granules showed no alkaline phosphatase activity.The adenosinetriphosphatase reaction, on the other hand, was found on the plasma membrane, vesicular or tubular profiles of the endoplasmic reticulum and on the matrix of the specific granules. The crystalloid of the granules did not show any reaction.Recipient of a postdoctoral fellowship from the muscular distrophy association of Canada.     

Ultrastructural localization of tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone.

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.     

Comparative study of the various methods for the ultrastructural localization of alkaline phosphatase activity

Summary Renal, intestinal and developing dental tissues were studied for alkaline phosphatase localization at the ultrastructural level using several metal salt modifications of the Gomori method and a new cadmium capture technic developed by Seligman. Except for particle size differences in the precipitates, localization both extra- and intracellularly were similar.This study was supported by USPHS Research Grant DE 2800-02, National Institutes of Health.     

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)     

New,improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity

Summary New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

New, improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity

New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.     

Ultrastructural localization of alkaline phosphatase activity in the normal and osteochondrotic joint cartilage of growing pigs

The present study aimed to describe the ultrastructural localization of alkaline phosphatase (AP) activity in articular-epiphyseal growth cartilage of the commercial pig and the minipig of wild hog ancestry, comparing areas with a normal endochondral ossification with those where the calcification of the matrix is insufficient, as in osteochondrotic cartilage. Intense AP activity was primarily present in the cytoplasm, the plasmalemmae, the long cellular processes and the matrix vesicles budding off from proliferative and hypertrophic chondrocytes in those areas of cartilage where normal calcification appeared. In the osteochondrotic cartilage, the only detectable AP activity was restricted to a few morphologically viable hypertrophic cells in the surroundings of the lesion. The lack of AP activity could partially explain the insufficient calcification of the osteochondrotic cartilage.     

Immunohistochemical localization of alkaline phosphatase in the chicken epiphyseal growth cartilage

Summary Alkaline phosphatase of chicken epiphyseal cartilage has been localized by two immunohistochemical methods. Double layer immunofluorescence and peroxidase anti-peroxidase (PAP) methods gave similar results. Alkaline phosphatase in epiphyseal cartilage is extracellular as well as intracellular in the localization. Extracellular reaction was strongest in the lower layers of growth plate and the most intense reaction was noted in the pericellular lacunae of hypertrophic chondrocytes. Also intracellular immunoreaction was noticed through the whole growth plate.     

Reduced alkaline phosphatase activity in diabetic rat bone: a re-evaluation

We found previously that human bone alkaline phosphatase (AP) was glycated by aseptic incubation with glucose, and partially broken down by reactive oxygen species. In this study, we examined whether selective in vivo glycation of AP molecules occurred in bone tissue, using experimental diabetic rats induced by streptozotocin and spontaneously diabetic rats. Additionally, the effects of hyperlipidemia on bone AP activity were examined. Serum AP activity was significantly elevated after incipient onset of diabetes, and the increased activity originated from the intestinal isozyme. High levels of intestinal AP activity were also observed in rats with hyperlipidemia induced by feeding high-fat or high-fructose chow, but the AP activity in bone tissues was maintained at a constant level. AP activity in bone was reduced after the onset of diabetes. The resulting bone AP molecule bound to an aminophenylboronic acid column, which had affinity for glycated proteins, and contained smaller molecular sizes than the native bone AP. These results suggest that elevated levels of serum AP activity originated from the intestinal isozyme accompanied with hyperlipidemia induced by diabetes. In contrast, the reduced serum levels of AP activity in diabetic rats might be dependent on inactivation of bone AP, which was glycated, followed by partial breakdown of bone AP molecules, possibly due to reactive oxygen species.     

Characterization and localization of alkaline phosphatase activity in rat testes

Alkaline phosphatase activity in extracts of testes of sexually immature (13 days old) and sexually mature rats has been characterized by its heat sensitivity, the extent of inhibition by homoarginine and phenylalanine, and by polyacrylamide gel electrophoresis. The testicular enzyme appears to be a liver-bone-kidney-type alkaline phosphatase. There are no significant differences in the properties of the enzyme from animals of these two ages. Spermatocytes and early spermatids contain very little alkaline phosphatase activity; the specific activity of a nonflagellate germinal cell suspension is only 1/20th that of the whole testis. Since the constant level of activity in immature and mature animals is not consistent with the enzyme activity being present only in late spermatids, we conclude that the majority of the testicular enzyme is present in nongerminal cells. The presence of alkaline phosphatase in plasma membrane purified from testes of adult rats was demonstrated.     

The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism.

For the purified alkaline phosphatase from bone, the ability to catalyze a phosphate transfer reaction from p-nitrophenyl phosphate to two different hydroxy acceptor compounds, ethanolamine and glycerol, was established by identification of the formed phosphorylated products, phosphoethanolamine and glycerol 3-phosphate, respectively. In addition, a steady-state kinetic analysis of the hydrolysis of p-nitrophenyl phosphate in the presence of an added nucleophile, diethanolamine, gave rise to the proposal of a simple model for the kinetic mechanism of the enzyme. This mechanism includes a covalent phosphoryl enzyme intermediate, the dephosphorylation of which by water (k3) or a nucleophile (k4) is rate-determining. According to this model, in the presence of diethanolamine, k3 and k4 were determined to be 4.44 s-1 M-1 and 1000 s-1 M-1, respectively. Therefore, in vitro a suitable nucleophile, such as diethanolamine, seems to be a better phosphate acceptor than water. These results may suggest that alkaline phosphatase from bone could be well suited for catalyzing phosphate transfer reactions in vivo as well.     

Imaging of alkaline phosphatase activity in bone tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.     

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.     

Cytochemical localization of alkaline and acid phosphatase in human vanishing bone disease

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.     

A method for determining alkaline phosphatase activity in marine phytoplankton using spectrofluorometry

A method for determining relative percent intensity alkaline phosphatase activity (APA) using enzyme labeled fluorescence coupled with spectrofluorometry is presented. Compared to traditional microscopy and flow cytometry, we increase statistical power and reduce sample-handling issues. Combined with a biological standard, our method can quantify APA of natural plankton assemblages.