Optimization of the tartrate-resistant acid phosphatase detection by histochemical method

According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60ºC. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.     

Acid phosphatase localization in the fungus Whetzelinia sclerotiorum

Acid phosphatase was localized by light and electron microscopy in chains of vacuoles in hyphal tip cells of Whetzelinia sclerotiorum. The Enzyme was present in these vacuoles whether or not conditions favored extracellular acid phosphatase secretion. Apical vesicles, microbodies, woronin bodies, and lipid bodies did not contain acid phosphatase. The implications regarding terminology of organelles in filamentous fungi are discussed with special reference to the fungal spherosome concept.Abbreviations AP acid phosphatase     

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters. A quantitative histochemical study

The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Although the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.     

Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster.

The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.     

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.     

Acid phosphatase distribution and localization in the fungus Humicola lutea

Acid phosphatase activities in a culture liquid and mycelial extract were studied in submerged cultures of the filamentous fungus Humicola lutea 120-5 in casein-containingmedia with and without inorganic phosphate (Pi). The Pi-repressible influence on the phosphatase formation was demonstrated. Significant changes in the distribution of acid phosphatase between the mycelial extract and culture liquid were observed at the transition of the strain from exponential to stationary phase. Some differences in the cytochemical localization of phosphatase in dependence of Pi in the media and the role of the enzyme in the release of available phosphorus from the phosphoprotein casein for fungal growth were discussed.     

Preparation of histochemical sections for quantitative determination of acid phosphatase activity by means of laser microanalysis

Summary Based on former experiments dealing with a quantitative assay of acid phosphatase (acP) activity in histological sections by means of a laser microanalysis. The authors present results of investigations concerning optimal conditions needed for this purpose. The investigations were performed on rat liver sections. The realtion between incubation time and depth of acP reaction appearing in tissue blocks was investigated along with the dependence of photometric readings on thickness of histological sections and on incubation times. From results of experiments it appears that optimal conditions for quantitative determination of acP activity using the proposed laser method are provided by sections 30 to 50 in thickness which have been incubated in the substrate containing medium for 45 min. Sections thinner than 30 are not recommended for quantitative assay with this method because of low accuracy of readings caused by too low amounts of reaction product present there.     

Electron microscope histochemical localization of alkaline phosphatase(s) in Bacillus licheniformis.

Sites of alkaline phosphatase (APase) activity in a facultative thermophilic strain of Bacillus licheniformis MC14 have been localized by electron microscope histochemistry, using a lead capture method. The effects of 3% glutaraldehyde and 3.0 mM lead on APase activity were investigated, and these compounds were found to significantly inhibit enzyme activity, 68 and 18%, respectively. A number of parameters were varied in studies to localize APase activity, including: growth temperature (55 and 37 degrees C); substrate concentration in the histochemical mixture (0.06, 0.15, 0.30, 1.00 mM); fixatives; protoplast preparations and whole cells; phosphate-repressed and -derepressed cells; and age of vegetative cells (mid-log and late log). These variations affected the number but not the location of lead phosphate deposits, which appeared at discrete sites along the inner side of the cytoplasmic membrane. Control cells incubated in histochemical mixtures lacking substrate, lead, or both exhibited no lead phosphate depositis. The histochemical localization at membrane sites correlated well with biochemical localization data, which indicated that greater than 80% of the APase activity was associated with the membrane fraction in logarithmically growing cells.     

Acid phosphatase deactivation by a series mechanism

Acid phosphatase (E.C.3.1.3.2.) thermal deactivation at pH 3.77 has been investigated by monitoring the enzyme activity as a function of time in the hydrolysis of p-nitrophenyl phosphate. The experimental curves obtained show a two-slope behavior in a log (activity)versus-time plot, which indicates that deactivation occurs via a complex mechanism. From the dependence of the kinetic parameters on both deactivation and hydrolysis temperatures, it is inferred that the deactivation mechanism involves intermediate, temperature-dependent, less-active forms of the enzyme. This interpretation is confirmed by the results of additional tests in which the temperature was suddenly changed during the deactivation process.