Hypophosphatasia: a cytochemical study of phosphatase activities.

Skeletal abnormalities with defective formation of mature calcified bone are the most prominent clinical features of hypophosphatasia. Low concentrations of serum and tissue alkaline phosphatase and elevated plasma and urinary levels of phosphorylethanolamine (PEA) are also present. Although PEA is hydrolyzed by serum alkaline phosphatase, the relationship between PEA and the deficiency is unclear. PEA has not previously been tested as a cytochemical substrate for the in situ demonstration of human alkaline phosphatase activity. We have studied alkaline phosphatase activity in hypophosphatasia in tissue sections, utilizing PEA and adenosinetriphosphate (ATP) as well as the usual beta-glycerophosphate and naphthol phosphate substrates. Neutral and acid phosphatase activities were also examined. Our results demonstrate that PEA is a substrate for the localization of alkaline phosphatase in normal human tissue, but is not hydrolyzed in hypophosphatasia in the liver, brain or costochondral junction under alkaline conditions. In the kidney in hypophosphatasia only the straight segments of proximal tubules that rim the medullary rays are reactive with PEA. Similar results in hypophosphatasia were obtained at an alkaline pH with ATP, beta-glycerophosphate, and naphthol phosphate. However, the defect in hypophosphatasia is not a generalized deficiency of membrane-associated phosphatases because membranes that were deficient in alkaline phosphatase activity demonstrated normal reactivity with ATP at neutral pH. In addition, thiamine pyrophosphate was also split by Golgi membranes within the cytoplasm. Acid hydrolysis of beta-glycerophosphate by lysosomes was normal.     

Fermenthistochemische Untersuchungen des Lymphknotens

Summary The presence of alkaline phosphatase was investigated in slide preparations and smears of lymph nodes with the azo dye method.In the case of normal or hyperplastic lymph nodes, capillaries and arterioles are regularly and most strongly positive; the phosphatase activity in the endothelium of the venules, the larger blood vessels and the lymph vessels is irregular. One occasionally finds a few positive fibroblasts in the capsule and trabeculae. In addition, the pulpa sometimes shows a reaction in the region of the sinus, especially of the marginal sinus, and in the region of the germinal centers which appears to be associated with fibers. The activity in the region of the germinal centers is most fully developed with severe follicular lymphatic hyperplasia. A considerable degree of alkaline phosphatase activity can be noted at the edge of tubercles and tubercular caseous foci and in the scarified areas of the lympho-granulomatosis.In lymph node smears, elements interpreted as large reticulum cells were found to be about 16% phosphatase active; in part these cells are for certain vascular endothelium and fibroblasts.In the smears, we also found small lymphoid reticulum cells which gave a clear cut phosphatase reaction. No alkaline phosphatase activity could be demonstrated in the tissue mast cells, at least, that is, in their granula.Phosphatase activity could be clearly demonstrated in slide preparations as in smears in a part of the cells from 2 lymphosarcomas.     

Biocompatibility of a prolonged-action antialcohol preparation.

Acid and alkaline phosphatase activities in liver lysosomes and liver mitochondria, respectively, as well as in connective tissue capsule were studied for an antialcohol preparation (polyurethane carrier and disulfiram at 150, 300 and 625 mg/kg body weight) implanted subcutaneously. Increased acid phosphatase activity was observed in connective tissue capsule on days 14, 30 and 90 after implantation and in liver lysosome fraction on days 14 and 30 with preparation containing disulfiram at 300 and 625 mg/kg body weight. Alkaline phosphatase activity increased both in connective tissue capsule and in liver mitochondria up to day 30 only when sample with maximal disulfiram dose was implanted. Acid phosphatase activity is an adequate indicator for biocompatibility of prolonged-action medicine preparations. The data obtained show that the suggested medical form containing disulfiram 150 mg/kg body weight is biocompatible. Interaction between acid and alkaline phosphatase activity changes and biocompatibility and destruction of polymer implants is discussed.     

Biocompatibility screening of poly (vinyl chloride) implants

It was proposed to test the biocompatibility of a polymeric implant by assaying enzyme activity in the tissue homogenate. Medical grade poly(vinyl chloride) was implanted in rat gluteal muscle and muscle specimens taken from the implanted and non-implanted sites after 7, 14 and 21 days. In the muscle homogenate taken in suitable solvents, the activities of alkaline phosphatase, acid phosphatase, Ca2+ ATPase, leucine aminopeptidase (LAP) and succinic dehydrogenase (SDH) were tested and compared at implanted and non-implanted sites. Increased activity of acid phosphatase, alkaline phosphatase, LAP and Ca2+ ATPase were seen at the implanted site but the SDH activity was reduced. The enzyme activity at both sites was maximum around 14 days and the highest increase in activity at the implant site compared to the control site was observed for alkaline phosphatase and acid phosphatase. After 21 days, the activity decreased gradually. In case of SDH, the inhibition was more after 14 days and gradually decreased after 21 days.     

成人脂肪间充质干细胞的定向成骨诱导

背景：脂肪分离可获得大量的间充质干细胞并成功向骨、软骨、脂肪、心肌等多个方向诱导分化。 目的：建立成人脂肪间充质干细胞体外分离培养、成骨分化方法,并探讨脂肪间充质干细胞作为骨组织工程的种子细胞的前景。 方法：通过胶原酶消化法从成人脂肪中分离间充质干细胞,进行体外培养,流式细胞仪检测细胞表面标记物,CCK8检测细胞活性,成骨诱导液诱导干细胞向骨细胞分化,BCIP/NBT比色法染色检测碱性磷酸酶,茜素红染色检测钙结节形成,RT-PCR检测碱性磷酸酶,骨桥蛋白表达变化。 结果与结论：利用脂肪抽吸液成功培养出脂肪间充质干细胞,且能稳定传代,增殖能力旺盛;流式细胞仪检测证实有特定的间充质干细胞表面标记物表达;成骨诱导脂肪间充质干细胞后呈典型的成骨细胞形态;碱性磷酸酶染色阳性,茜素红染色后可见钙结节形成,成骨诱导培养0,3,7,14,21,28 d,RT-PCR定量检测结果证实碱性磷酸酶、骨桥蛋白阳性表达。说明用酶消化法可以从人脂肪中分离得到脂肪间充质干细胞;脂肪间充质干细胞可向骨细胞诱导分化,阳性表达骨桥蛋白,碱性磷酸酶,是一种优良的骨组织工程的种子细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Microvillus inclusion disease: specific diagnostic features shown by alkaline phosphatase histochemistry.

A technique using alkaline phosphatase histochemistry on routine sections of four jejunal biopsy specimens and one necropsy sample was applied to show that alkaline phosphatase activity, normally present in the brush border, occurs in the enterocytes of patients with microvillus inclusion disease. Sections were cut at 5 micron, mounted on to glass slides, and dried overnight at 37 degrees C before staining for alkaline phosphatase activity by the indoxyl phosphatase nitro blue tetrazolium method. Incubation periods amounted to 10 minutes for biopsy specimens and 30 minutes to one hour for necropsy samples. The demonstration of alkaline phosphatase activity in routinely processed biopsy specimens provides an effective, quick, and definitive test in the diagnosis of microvillus inclusion disease without recourse to electron microscopy.     

Effect of elastase on the histochemical demonstration of alkaline phosphatase activity in chick pecten

The effect of elastase on alkaline phosphatase activity in the chick pecten capillaries was studied electron histochemically. The pecten was treated with elastase before incubation in the medium for alkaline phosphatase. Inactive alkaline phosphatase in the chick pecten capillary under dark adaptation could be demonstrated electron histochemically after treatment with elastase. Under light adaptation, the elastase-treated pecten demonstrated more intense alkaline phosphatase activity in the same localization as in the untreated pecten. Biochemical data also supported histochemically demonstrated enhancement effect of elastase on alkaline phosphatase activity in the chick pecten oculi. Elastase is expected to be useful for demonstrating enzymatic activity which is otherwise hard to detect.     

Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies

Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).     

A semiquantitative method of determining alkaline phosphatase activity in human arterial wall

A new method of semi-quantification of the alkaline phosphatase enzyme activity in human vascular tissue is described. An index is derived from the number of alkaline positive cells and the different levels of intensity of the staining reaction. In the autopsy and surgical material of male and female subjects aged between 40 and 60 a, we evaluated the alkaline phosphatase enzyme reaction of normal and atherosclerotic arteries after various incubation intervals. Tissue samples were obtained from aorta, common carotid artery, renal artery, splenic and anterior tibial artery, and Sparks prosthetic graft.     

Differential alkaline phosphatase responses of rat and human bone marrow derived mesenchymal stem cells to 45S5 bioactive glass

Bioactive glass is used as both a bone filler and as a coating on implants, and has been advocated as a potential osteogenic scaffold for tissue engineering. Rat-derived mesenchymal stem cells (MSCs) show elevated levels of alkaline phosphatase activity when grown on 45S5 bioactive glass as compared to standard tissue culture plastic. Similarly, exposure to the dissolution products of 45S5 elevates alkaline phosphatase activity and other osteogenic markers in these cells. We investigated whether human MSCs grown under the same laboratory conditions as rat MSCs would exhibit similar responses. In general, human MSCs produce markedly less alkaline phosphatase activity than rat MSCs, regardless of cell culture conditions, and do not respond to the growth factor BMP-2 in the same way as rat MSCs. In our experiments there was no difference in alkaline phosphatase activity between human MSCs grown on 45S5 bioactive glass or tissue culture plastic, in samples from five different orthopaedic patients, regardless of culture media composition. Neither was there any consistent effect of 45S5 dissolution products on human MSCs from three different donors. These results suggest that the positive effects of bioactive glass on bone growth in human patients are not mediated by accelerated differentiation of mesenchymal stem cells.     

Alkaline Phosphatase in the Developing Bursa of Fabricius

The ontogeny of alkaline phosphatase in the bursa of Fabricius was studied by histochemical and biochemical methods. According to the quantitative determinations, the activity of alkaline phosphatase increased from the 11th to 17th day of incubation--that is, during the time of the lymphoid follicle formation in the developing bursa. The activity was localized in the mesenchymal tissue surrounding the lymphoid follicles. Testosterone given in ovo prevented the appearance of alkaline phosphatase in the bursal mesenchyme but had no effect on the activity of the embryonic liver. In contrast, in ovo treatment with cyclophosphamide had no effect on the alkaline phosphatase in the bursa. By using transplantation of embryonic bursal stem cells, it was further shown that, in contrast to cyclophosphamide, testosterone destroys the capacity of the bursa to serve as a differentiation site for the B-cell lineage. The results indicate that testosterone affects the stromal cells of the bursa, whereas cyclophosphamide destroys only the lymphoid population undergoing differentiation and leaves the bursal stroma intact.     

Improved double immunoenzyme labelling using alkaline phosphatase and horseradish peroxidase

Alkaline phosphatase and peroxidase complexes were applied to tissue sections simultaneously to label two antigens. An azo-coupling method was used to demonstrate the alkaline phosphatase activity. The final result gave clear contrast between the two antigens and allowed the use of haematoxylin as the nuclear counterstain.     

Alkaline and Acid Phosphatases in Bone Cells Serve as Phosphohydrolases at Physiological pH In Vivo: A Histochemical Implication

Net activity of tissue-nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) remains to be determined since enzyme histochemistry has adopted biochemically determined optimal pH, which is not likely to represent local pH in vivo. The present study aimed to evaluate TNAP and ACP activities associated with bone cells at physiological pH. At the physiological pH of tissue fluid, intense phosphatase reactions were demonstrable in osteoblasts and osteoclasts as well as the bone matrix associated with osteoclasts. In fresh-frozen and freeze-substituted specimens, intense phosphatase reactions appeared at both alkaline and neutral pH along the entire surface of osteoblasts including the osteoidal surface, where TNAP was shown to be absent by immunohistochemistry. Combined specificity tests suggested that TNAP and ACP in bone cells can serve as phosphohydrolases at pH 7.3 and that reactions along the osteoidal surface of osteoblasts differ from that of TNAP and represent novel enzyme.     

Alkaline and acid phosphatases in bone cells serve as phosphohydrolases at physiological pH in vivo: a histochemical implication

Net activity of tissue-nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) remains to be determined since enzyme histochemistry has adopted biochemically determined optimal pH, which is not likely to represent local pH in vivo. The present study aimed to evaluate TNAP and ACP activities associated with bone cells at physiological pH. At the physiological pH of tissue fluid, intense phosphatase reactions were demonstrable in osteoblasts and osteoclasts as well as the bone matrix associated with osteoclasts. In fresh-frozen and freeze-substituted specimens, intense phosphatase reactions appeared at both alkaline and neutral pH along the entire surface of osteoblasts including the osteoidal surface, where TNAP was shown to be absent by immunohistochemistry. Combined specificity tests suggested that TNAP and ACP in bone cells can serve as phosphohydrolases at pH 7.3 and that reactions along the osteoidal surface of osteoblasts differ from that of TNAP and represent novel enzyme.     

A new approach using tissue alkaline phosphatase histochemistry to identify Crohn's disease

In the present study, we investigate intestinal alkaline phosphatase activity in mucosal biopsies in patients with inflammatory bowel disease. Crohn's disease influences the alkaline phosphatase activity in the intestine, increasing its activity. We present a histochemistry-based method for alkaline phosphatase that is useful for the identification of Crohn's disease and the differentiation of ulcerative colitis.     

Quantitative method for determining serum alkaline phosphatase isoenzyme activity: estimation of intestinal component.

Intestinal alkaline phosphatase activity was measured using levamisole inhibition, and results were compared with a previously reported method using L-phenylalanine. Sixty two per cent intestinal, 39% placental, and 1.3% of either bone or liver alkaline phosphatase activity remained when alkaline phosphatase activity was inhibited in a 2-amino-2-methyl-1-propanol (AMP) buffer reagent system with 10 mmol/l levamisole (final assay concentration 8.1 mmol/l). The assay imprecision (SD) was 0.6 U/l compared with 3.9 U/l using L-phenylalanine for specimens with total alkaline phosphatase activity less than 250 U/l (reference range 30-120 U/l). In serum pools with raised total alkaline phosphatase activity errors in recovered intestinal activity were small (usually less than 3 U/l) when intestinal alkaline phosphatase was added. Much larger errors and many underestimated results were found using L-phenylalanine. For non-haemolysed specimens it is concluded that an assay based on levamisole inhibition provides a better measure of intestinal alkaline phosphatase activity than L-phenylalanine.     

Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents.

The use of alkaline phosphatase in an immunoenzymatic procedure for the localisation of antigens in paraffin sections or cell smears is described. The results of this method, when applied to the detection of immunoglobulins, lysozyme, or lactoferrin, were comparable in intensity and clarity to those obtained with the PAP immunoperoxidase procedure. Furthermore, double immunoenzymatic labelling (with alkaline phosphatase and peroxidase) of two cellular constituents in a tissue section is possible, the brown peroxidase reaction product contrasting well with the blue alkaline phosphatase product. Since the two antibody 'sandwiches' are applied simultaneously rather than sequentially the total duration of this double immunostaining procedure is only a few minutes longer than that required for detection of a single antigen. It was also found that the unlabelled antibody immunohistological procedure (whether used in conjunction with alkaline phosphatase or peroxidase) can be shortened without loss of sensitivity by carrying out two of the incubation steps simultaneously.     

Chosphatases of the human placenta

Summary Acid and alkaline phosphatase activity has been examined in chorionic and placental tissues of 123 women in the 4–5th to the 40th week of pregnancy, by the method of Gomori. Both enzymes could be detected over the whole duration of pregnancy. Most of the villi of preparations from early pregnancy showed trace or low acid phosphatase activity, and the proportion of such villi increased as pregnancy progressed. At the same time, there was a gradual rise in the acid and alkaline phosphatase activity of the structures, to a maximum at term. Alkaline phosphatase was present in the nuclei, but more abundantly in the cytoplasm of the syncytium; it was absent from the Langhans cells. Acid phosphatase was concentrated mainly in the nuclei, and was more widely distributed among the placental tissues than was alkaline phosphatase. The trophoblast showed the highest acid and alkaline phosphatase activities. The results reported are not in agreement with those of Thomsen.(Presented by Member AMN SSSR A. V. Lebedinskii) Translated from Byulleten Éksperimental'noi Biologii i Meditsiny, Vol. 56, No. 8, pp. 64–68, August, 1963     

Alkaline phosphatase activity in cultured skin fibroblasts from fibrodysplasia ossificans progressiva.

Alkaline phosphatase activity in four strains of cultured skin fibroblasts obtained from a patient with fibrodysplasia ossificans progressiva was at the low normal range. The enzyme activity in normal fibroblasts significantly increased at late confluency. It appears that the high levels of alkaline phosphatase activity reported in biopsies of lesions are not genetically determined but are secondary events of local tissue reaction.     

The Immunopathology of Amyloidosis

Abstract

A new methyl methacrylate embedding medium, Medim K-Plast, is used for histochemical demonstration of acid and alkaline phosphatase activities in undecalcified rat femurs. Fixation, dehydration, and embedding are completed within four days. The method requires no explosive initiator for polymerization, and there are no water bath temperature peaks exceeding 35°C. The method can be adapted to routine laboratory. conditions with a large number of tissue specimens. Short incubation times of 30 to 60 minutes are sufficient to demonstrate enzyme activities by simultaneous coupling azo dye methods. Enzyme activity is retained after storage of blocks for 3 months at 4°C or in sections at ?20°C for 4 weeks. Alkaline phosphatase activity for investigation of bone growth is unsatisfactory because the enzyme is associated not only with osteoblasts, but with reticular cells of bony trabeculae. Demonstration of newly formed bone matrix with Solochrome cyanine R staining, therefore, is done without prior deplasticizing of the sections. (The J Histotechnol 10: 103, 1987)