Demonstration Of Acid Phosphatase Activity Research Articles

Cytochemistry of the Acid Phosphatases

Cytochemistry of acid phosphatases has recently enjoyed an increase in clinical application. Better known among these methods is the tartrate-resistant acid phosphatase stain for the hairy cells of hairy-cell leukemia. The demonstration of prostatic acid phosphatase in tumor cells outside the prostate may further aid in the detection of metastatic prostate cancer. Aside from these methods for cytologic and histologic materials, it is now possible to cytochemically demonstrate acid phosphatase activity in subcellular organelles, extracts of whole tissue and cellular preparations, and in biological fluids and secretions. The methods for optimal staining of acid phosphatase activity in these various materials are necessarily different in one or more respects. The methods for cytochemical demonstration of acid phosphatase are reviewed in the context of the applications for which they may be used. Emphasis is placed on those methods for cytologic and histologic preparations. Applications where cytochemistry is frequently used in combination with biochemical, electrophoretic and immunologic techniques are also reviewed.

Cytochemistry of the Acid Phosphatases

Cytochemistry of acid phosphatases has recently enjoyed an increase in clinical application. Better known among these methods is the tartrate-resistant acid phosphatase stain for the hairy cells of hairy-cell leukemia. The demonstration of prostatic acid phosphatase in tumor cells outside the prostate may further aid in the detection of metastatic prostate cancer. Aside from these methods for cytologic and histologic materials, it is now possible to cytochemically demonstrate acid phosphatase activity in subcellular organelles, extracts of whole tissue and cellular preparations, and in biological fluids and secretions. The methods for optimal staining of acid phosphatase activity in these various materials are necessarily different in one or more respects. The methods for cytochemical demonstration of acid phosphatase are reviewed in the context of the applications for which they may be used. Emphasis is placed on those methods for cytologic and histologic preparations. Applications where cytochemistry is frequently used in combination with biochemical, electrophoretic and immunologic techniques are also reviewed.

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity.

The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO4-solution to which K4Fe(CN)6 had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M chi and L chi values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L chi values were reliable, whereas the M chi values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L chi values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.

Demonstration of acid phosphatase activity induced by 20-hydroxyecdysone in the fat body of Calliphora.

In the larval fat body of Calliphora erythrocephala, protein accumulation and autophagic activity occur prior to the onset of puparium formation. The involvement of the lysosomal system in the degradation of sequestered protein and cell organelles can be demonstrated by the electron-microscopical cytochemical localization of the lysosomal marker enzyme acid phosphatase in so-called protein granules. These granules contain not only newly synthetized or absorbed protein but also remnants of cell organelles such as mitochondria and endoplasmic reticulum. Ligation of the larvae behind the brain-ring gland complex prevents the appearance of these acid phosphatase-positive granules. They can be induced in ligated larvae by the injection of 20-hydroxyecdysone into the abdomen. These findings are briefly discussed in relation to the role of moulting hormones in normal development, especially with regard to the induction of autophagic activity.

A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. II. Effect of the composition of the incubation medium on the trapping of phosphate ions in acid phosphatase cytochemistry.

A model system developed for the study of the dynamics of capture reactions for diffusable compounds in cytochemistry served as a basis for the experiments reported in the present paper. The model was used to study the effect of the composition of the cytochemical medium on the trapping of phosphate ions by lead (II) ions in acid phosphatase cytochemistry. In this system a phosphate-containing solution and a lead-containing solution (cytochemical medium) are pumped along opposite sides of a polyacrylamide film. The phosphate concentration at which measurable precipitation starts in the film (critical phosphate concentration) was taken as a measure of the trapping efficiency of the cytochemical medium. The addition of beta-glycerophosphate and cytidine-5'-monophosphate to a buffered lead-containing solution resulted in a higher critical phosphate. The addition of chloride ions and acetone, as well as decreasing the molarity of the acetate buffer of the cytochemical medium, were found to lower the critical phosphate concentration, whereas the addition of fluoride ions, glucose, and sucrose had no effect. From the effect of variations in the composition of the cytochemical medium on the trapping efficiency and the turnover number of acid phosphatase in the medium, it was possible to predict which cytochemical medium would be the most suitable for the demonstration of acid phosphatase activity in guinea-pig peritoneal exudate cells. The results were in accordance with the localization of acid phosphatase activity: the higher the trapping efficiency and the turnover number, the higher the amount of precipitate and the number of positive enzymatic sites. In this way an improved cytochemical medium for acid phosphatase was developed.

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80-160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150-400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogeneous, some are lysosomes and the other are possibly the granules responsible for the secretion of pineal peptides.

A quantitative method for measurement of lysosomal acid phosphatase latency in cultured rat heart cells with 210Pb.

A method is described for measuring the latency of lysosomal acid phosphatase in cultured rat heart endotheloid cells. 210Pb was added to a medium used to demonstrate acid phosphatase activity by the Gomori lead method, and the amount of lead deposited was measured with a liquid scintillation counter. Deposition rates were measured after enzyme activation pretreatments with acetate buffer (pH 5.0) at various osmolalities, and after formaldehyde fixation. Formaldehyde, alloxan, or fluoride in the Gomori medium were evaluated for their differential effects on lysosomal and non-lysosomal acid phosphatase. The method was found to provide a sensitive, rapid and quantitative evaluation of acid phosphatase latency and should be useful for studying the integrity of lysosomes within cells.

Histochemical differentiation ofBrugia, Wuchereria, DirofilariaandBreinliamicrofilariae

Histochemical demonstration of acid phosphatase activity in microfilariae gives sufficiently characteristic and consistent results for the differentiation of even closely related species. No difference could be detected among nocturnally periodic, nocturnally subperiodic and diurnally subperiodic Brugia malayi, but they could readily be distinguished from B. pahangi. Similarly, Dirofilaria repens could be readily distinguished from D. immitis and B. booliati from B. sergenti. The enzyme distribution pattern of a Malaysian rural strain of Wuchereria bancrofti was different from those of other regions.

Lysosomal degradation of bile deposits in cholestasis

Deposits of bile in the cytoplasm of hepatocytes from six patients with cholestasis were studied by electron microscopy and ultrastructural cytochemistry. The deposits were identified in vacuoles of varying size and complexity. The demonstration of acid phosphatase activity in these vacuoles suggests that lysosomes participate in the elimination of bile deposits retained in hepatocytes.

Ultrastructural study of GERL in beige mouse alveolar macrophages.

Alveolar macrophages of the beige mouse mutant have a system of smooth-surfaced elements with the hallmarks of GERL. GERL also appears to produce residual bodies, and both organelles show cytochemically demonstrable acid phosphatase activity. When cells are exposed to colloidal silver, the tracer is endocytosed via pinocytic vacuoles to GERL.

Ultrastructural, Cytochemical, and Membrane Surface Marker Characteristics of the Atypical Lymphocytes in Infectious Mononucleosis

Abstract Atypical lymphocytes from nine young adults with acute infectious mononucleosis (IM) were studied for morphologic, ultrastructural, cytochemical, and membrane surface marker characteristics. There was an absolute increase in T lymphocytes in the patients. Atypical lymphocytes accounted for 83%-96% of the lymphocyte population. These lymphocytes contained cytoplasmic inclusions which ranged in size from 1000 to 6000 A, were usually membrane bound, and consisted of parallel arrays of microtubulelike structures. The inclusions, which have been referred to as parallel tubular arrays (PTA), were found in 15%-75% of the lymphocytes from the IM patients. Ultrastructural cytochemical methods demonstrated acid phosphatase activity within many of the membrane-bound PTA. The function of the PTA is unknown. Since they were observed only in the lymphocytes which appeared to correspond to the atypical lymphocytes on light microscopy, the majority of which typed as T cells, there appears to be an association between PTA and T lymphocytes. It is possible that PTA identify a specific subset of T lymphocytes which is expanded in IM. Alternatively, PTA may be a transient finding in lymphocytes appearing only in certain biologic states of the cell such as during T-lymphocyte activation.

Ultrastructural, cytochemical, and membrane surface marker characteristics of the atypical lymphocytes in infectious mononucleosis

Atypical lymphocytes from nine young adults with acute infectious mononucleosis (IM) were studied for morphologic, ultrastructural, cytochemical, and membrane surface marker characteristics. There was an absolute increase in T lymphocytes in the patients. Atypical lymphocytes accounted for 83%-96% of the lymphocyte population. These lymphocytes contained cytoplasmic inclusions which ranged in size from 1000 to 6000 A, were usually membrane bound, and consisted of parallel arrays of microtubulelike structures. The inclusions, which have been referred to as parallel tubular arrays (PTA), were found in 15%-75% of the lymphocytes from the IM patients. Ultrastructural cytochemical methods demonstrated acid phosphatase activity within many of the membrane-bound PTA. The function of the PTA is unknown. Since they were observed only in the lymphocytes which appeared to correspond to the atypical lymphocytes on light microscopy, the majority of which typed as T cells, there appears to be an association between PTA and T lymphocytes. It is possible that PTA identify a specific subset of T lymphocytes which is expanded in IM. Alternatively, PTA may be a transient finding in lymphocytes appearing only in certain biologic states of the cell such as during T-lymphocyte activation.

Histochemical and immunohistochemical techniques on acrylate embedded bone biopsies.

Till present the advantages of methacrylate embedded bone biopsies for the diagnosis of haemoblastic disorders have been somewhat restricted. This was a result of the impossibility to apply histochemical methods and other sensitive staining procedures to semithin sections. A routine method is described whereby enzyme activity, excellent fixation and good sectioning ability are retained. Fixation is carried out using 4% purified formaldehyde buffered in 0.1M sodium cacodylate. Dehydration is done with a water-miscible glycolmethacrylate. Naphthol-AS-D-chloroacetate esterase activity can be observed in granules of the entire neutropil cell lineage. By use of this method it becomes possible to demonstrate acid phosphatase activity and immunoglobulins in atypical plasma cells of multiple myeloma. A considerable decrease in processing time, as well as a preservation of enzyme activity during the postal mailing of fixed tissue samples from outside are further advantages.

Endocytosis, transfer tubules, and lysosomal activity in myeloid sinusoidal endothelium

The endocytosis and intracellular disposition of intravenously administered colloidal carbon and ferritin in the sinusoidal endothelial cells of the bone marrow have been studied. The carbon and ferritin tracer particles are initially removed from the circulation by newly formed large bristle-coated plasmalemmal vesicles. Subsequently these large coated vesicles (1) fuse with each other to form larger smooth-membraned storage vesicles (phagosomes) for the tracer particles, or (2) fuse with smooth-membraned “transfer tubules” that receive the tracer materials and in turn transfer the particles to dense bodies or multivesicular bodies. A number of the dense bodies in the sinusoidal endothelial cells show histochemically demonstrable acid phosphatase activity and may represent secondary lysosomes. Golgi cisternae and transfer tubules in the sinusoidal endothelium also contain reaction product for acid phosphatase and it is proposed that these transfer tubules arise from the Golgi cisternae and convey acid phosphatase to the dense bodies. In this circumstance, the transfer tubules would be the equivalent of primary lysosomes in the sinusoidal endothelial cells.

Light and Electron Microscope Study of Dunaliella primolecta Butcher (Volvocida)*

SYNOPSIS. Light and electron microscope observations on Dunaliella primolecta Butcher from logarithmic and stationary phases of batch cultures are correlated. Except for the lack of a cell wall the fine structure has typical volvocid features. The transition from logarithmic to stationary phase is marked by changes in content and size of cytoplasmic vacuoles, accumulation of cytoplasmic lipid, accumulation of starch in the plastid matrix, and by the formation of autophagosome‐like bodies. The organelles in stationary‐phase flagellates are closely packed together because of the cytoplasmic lipid and starch‐distended chloroplast. Organisms from logarithmic phase have an abundant ribosome‐packed groundplasm supporting the organelles. In the cytochemical demonstration of acid phosphatase activity, Golgi cisternae and smooth and coated Golgi vesicles contain Gomori reaction product. The possible roles of the Golgi apparatus in this flagellate are discussed.

Pathways of clearance in mouse lungs exposed to iron oxide aerosols.

The history of particle clearance was studied in lungs of mice serially sacrificed at intervals up to 14 months following single exposures to an aerosol of submicronic, particulate, iron oxide used as a similitude for atmospheric dust. Clearance was followed by light microscopy in unstained and Prussian blue stained frozen and plastic embedded sections, as well as by electron microscopy, where iron oxide can be recognized by its form. Related problems were investigated through histochemical demonstration of acid phosphatase activity in pulmonary lysosomes and Prussian blue staining of various tissues after administration of iron compounds by gastrointestinal and vascular routes. The iron particles settle extensively but not uniformly on pulmonary alveolar surfaces. Clearance is centripetal and involves two mechanisms, an extracellular mechanism fed by fluid currents sweeping across the surfcace, and cellular mechanism principally involving alveolar macrophages. In the early post exposure period both actively remove deposited particles predominantly through the pulmonary airways. By 24 hours uncleared residues have become ingested and clearance thereafter results mainly from cellular action. Macrophages enter bronchial passages where they sometimes continue to pursue normal activities. A chronic phase of clearance begins when deposited particles become sequestered in macrophages of pulmonary connective tissues. These cells are reached by several routes, not least by crossing the bronchial epithelium. Particle clearance from these macrophages is very slow, and residue-containg cells eventually congregate in lymphoid tissues surrounding major bronchi. These findings are discussed as they help to develop an overall picture of clearance from the lungs and as they bear on related topics, such as functional roles of alveolar and pulmonary connective tissue macrophages and the pathogenesis of chronic bronchial disease.

Unusual lysosomes in hamster hepatocytes

Residual bodies (lysosomes) in hepatocytes of the Syrian golden hamster possess a relatively large electron-lucent lipid inclusion. In males on normal chow the lipid inclusions are large enough, in periportal hepatocytes, to be visible by light microscopy when frozen sections are stained by Oil red O. Thus, the pericanalicular arrangement of the lysosomes may be revealed either by Oil red O staining or by incubation for acid phosphatase activity. Demonstrable acid phosphatase activity is restricted to the area surrounding the lipid inclusion and, usually, to a region at one pole of the lysosome that contains electron-opaque materials characteristic of residual bodies generally. Feeding either female or male hamsters cholesterol-supplemented chow results in a dramatic increase in the number of lipid-containing lysosomes visible by light microscopy. Chemical determinations demonstrate a rapid and sustained rise in liver cholesterol levels, and the presence of cholesterol in lysosomes is shown by electron microscopy of tissue fixed with digitonin-containing aldehydes. Polarizing microscopy reveals the pericanalicular arrangement of anisotropic lipid. Electron microscopic images suggest that the lipid inclusion forms in GERL or neighboring ER. Discontinuing the cholesterol diet leads to a gradual disappearance of the electron-lucent lipid; residual bodies now resemble lipofuscin granules. It is suggested that hamster liver may serve as a model for the study of inherited disorders of lipid metabolism such as Wolman's disease and cholesteryl ester storage diseases, and other diseases in which lysosomes are filled with electron-lucent lipid.

Anaphylactoid edema induced in rats by nickel and cobalt salts.

SummaryNickel and cobalt, especially in form of hydrosoluble compounds, injected either intraperitoneally or intravenously in doses of 5 mg caused regularly anaphylactoid edema in rats. The mast cells in the subcutaneous tissue of edematous ear lobes were often degranulated but were unreactive to selective metal stains. Unusual large granules with no demonstrable acid phosphatase activity were found. The edema reaction developed after bilateral nephrectomy indicating that it cannot be ascribed to the release of a renal erythropoietic factor after the parenteral administration of the cobalt or nickel salts. The present observations may provide an interesting model for further investigating histaminergic reactions in rats.

Erythrophagocytosis by the epithelial cells of the bladder.

ABSTRACT N-methyl-W-nitrosourea (MNU), methyl methane sulphonate (MMS), ethyl methane sulphonate (EMS) and cyclophosphamide (CP) are all cytotoxic to the transitional epithelium lining the urinary bladder, and also cause haemorrhage from the subepithelial blood capillaries. Erythrocytes enter the epithelium and are phagocytosed by the epithelial cells where they are subsequently digested. Two other compounds, dibutyl nitrosamine (DBN) and N-(4-(5-nitro-2-furyl)-2-thiazolyl)-formamide (NFTF) are only mildly cytotoxic but produce bladder epithelial tumours. Where haemorrhage occurs in these tumours, the erythrocytes are phago-cytosed and digested by the neoplastic epithelial cells. The ingested red cells appear to lie free in the epithelial cell cytoplasm, surrounded by only a single unit membrane. Densitometrie tracings, however, show that this membrane is derived from the epithelial cells and that the red cells lie within a phagocytic vacuole. Careful inspection reveals the red cell membrane within the dense content of the vacuole, indicating that the permeability of the red cell membrane changes rapidly as soon as it is engulfed, so that haemoglobin is released into the phagocytic vacuole. The contents of the vacuole disintegrate and myelin figures and small amounts of residual dense material appear, but at no time was it possible to demonstrate acid phosphatase activity associated with the vacuole.

Carrageenan-Induced Squamous Metaplasia of the Rectal Mucosa in the Rat

Degraded carrageenan, derived from Eucheuma spinosum, was administered to Sprague–Dawley rats as a solution in drinking water (5%; daily intake of carrageenan, 6 to 10 g per kg) or by stomach tube (0.5 or 5.0 g per kg per day). When the daily dose was 5 g per kg or more, the stools were soft to semi-fluid, and occult blood was detected in 3 to 7 days. Blood could occasionally be seen on the surface of the stool and on the anal margin. After 6 months, a gray-white coating was observed on the mucosal surface of the distal rectum. Histologically, this coating consisted of keratinizing stratified squamous epithelium. The epithelial transformation was preceded by acute and chronic inflammatory responses to carrageenan, in which aggregation of macrophages in the lamina propria and submucosa was a prominent feature. Loss of mucosal glands, atrophy of the luminal epithelium, and replacement of the columnar epithelium by a stratified squamous epithelium took place in close association with these macrophage aggregates. Focal mucosal ulceration, followed by an acute inflammatory response, resulted in more intense proliferation of squamous epithelium involving the glands as well as the luminal epithelium. Most of the macrophages contained material which stained metachromatically with toluidine blue, suggesting the presence of carrageenan. A striking increase in histochemically demonstrable acid phosphatase activity paralleled the accumulation of this material. An etiological role is postulated for these macrophages in the pathogenesis of the colonic changes reported here.