Amount Of Final Reaction Product Research Articles

In Situ Localization of Transketolase Activity in Epithelial Cells of Different Rat Tissues and Subcellularly in Liver Parenchymal Cells

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.

Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity for the detection of (pre)neoplasm in rat liver.

Oxygen insensitivity of the histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PD) activity with NT as tetrazolium salt has been proved to be a powerful tool to discriminate various types of adenocarcinoma from normal tissues. Here we investigated whether this phenomenon can also be applied to differentiate between chemically induced hepatocellular (pre)neoplasms and normal liver tissue in rats. Residual activity (percentage of the amount of final reaction product that is generated in oxygen and that is generated in nitrogen) was 60% in (pre)neoplastic cells and 6% in normal liver parenchymal cells. This means that the oxygen insensitivity test is a useful tool to distinguish (pre)neoplasms from normal rat liver tissue. N-Ethylmaleimide, a blocker of SH groups, did not affect G6PD activity in (pre)neoplastic cells, whereas activity in normal cells was reduced by half. Therefore, the absence of essential SH groups in G6PD in (pre)neoplastic cells is held responsible for the oxygen insensitivity phenomenon. We conclude that oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap tool to diagnose (pre)neoplasms in rat liver. Discrimination is likely to be based on altered properties of the enzyme in (pre)neoplastic cells. (J Histochem Cytochem 49:565-571, 2001)

Effects of ischaemia and reperfusion on NADH coenzyme Q reductase activity in rat liver.

NADH coenzyme Q reductase (EC 1.6.5.3) has been suggested in the literature to be inactivated by ischaemia. In the present study, NADH coenzyme Q reductase activity was localized in unfixed cryostat sections of ischaemic rat livers and quantified using image analysis. In vitro ischaemia was induced by storage of rat liver fragments for 30, 60, and 120 min at 37 degrees C. In vivo ischaemia was provoked by clamping the afferent vessels of median and left lateral liver lobes for 60 min followed by 30, 60 and 180 min of reperfusion. NADH coenzyme Q reductase activity was demonstrated with the tetrazolium salt method in the presence of polyvinyl alcohol. Final reaction product was found in liver parenchymal cells and its distribution was homogeneous within liver lobules. Only low amounts of final reaction product were formed when the incubation was performed in the absence of the substrate NADH. A non-linear relation was found between the absorbance and incubation time when the reaction was performed in the presence of NADH. Therefore, the initial velocity was taken as the true rate of enzyme activity. A linear relationship was found for the initial velocity and section thickness up to 6 microm followed by a levelling off. Electron microscopically, NADH coenzyme Q reductase activity was localized at the outer and inner membranes of mitochondria. In vitro ischaemia up to 120 min did not affect NADH coenzyme Q reductase activity. At 30 min reperfusion after in vivo ischaemia for 60 min enzyme activity was slightly decreased in certain foci which also showed diminished lactate dehydrogenase activity. A further decrease of enzyme activities in foci was observed at 180 min reperfusion after ischaemia. It is concluded that NADH coenzyme Q reductase activity is not sensitive to ischaemia. Furthermore, it is likely that the enzyme leaks from liver parenchymal cells into the circulation during reperfusion after ischaemia.

Phosphohydrolytic activity in Paramecium caudatum at neutral pH

Phosphohydrolytic activity was cytochemically characterized in Paramecium caudatum, a ciliated protozoa, at neutral pH. We stained cells in the presence of several mononucleotides as substrates, namely adenosine 5'-monophosphate (5'-AMP), adenosine 2'-monophosphate, guanosine 5'-monophosphate (5'-GMP) and beta-glycerophosphate (beta-GLP) using a lead capture method at 37 degrees C. Cells were also incubated in the presence of 5'-AMP with the inhibitor for alkaline phosphatase, tetramisole. In all cases, varying amounts of final reaction product, lead sulfide, was observed in Paramecium cytoplasm. Tetramisole did not have any effect on Paramecium 5'-AMP hydrolytic activity. The phosphohydrolytic activity was measured as the increase in total absorbance of "test minus control" reactions at 440 nm per unit time after 20 min of incubation using a microphotometric system for image analysis that has been developed by us. From the relationship between the concentrations of 5'-AMP and activity, an apparent K(m) value was estimated to be 0.20 mM. These results suggest that mononucleotides and phosphate monoesters are hydrolyzed by one or more enzymes with wide substrate specificity in P. caudatum. All the activity distribution patterns in Paramecium cultures, that were tested, were monomodal. The mean activity for 5'-AMP hydrolysis widely varied in these cultures. To investigate substrate affinity, distribution patterns and mean activity with 5'-AMP as substrate were compared with those in the presence of 2 other substrates, 5'-GMP and beta-GLP. Affinity of the enzyme(s) was similar for 5'-AMP and 5'-GMP and lower for beta-GLP.

A quantitative histochemical study of alkaline phosphatase activity in isolated rat duodenal epithelial cells.

A simple separation method enabling the quantification of alkaline phosphatase activity in unfixed, isolated, individual, duodenal epithelial cells has been presented. The activity of intestinal brush border-bound alkaline phosphatase has been demonstrated using naphthol AS-BI phosphate as a substrate and hexazotized New Fuchsin as a simultaneous coupling agent. The amount of final reaction product, as measured cytophotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activity was obtained at pH 8.9. Variation of the substrate concentration revealed the kinetic parameters for naphthol AS-BI phosphate as Km = 0.17 i 0.015 and Vmax = 13.9 +/- 1.38. The specificity of the enzyme reaction was confirmed by the complete inhibition of the enzyme activity in the presence of L-cysteine (10 mm) and 80% inhibition with L-phenylalanine (30 mM). Comparison of alkaline phosphatase activity in 8-microm cryostat sections (beginning at the tip and proceeding to the cryptal part) along the villus axis, with the activity of individual cells obtained by successive separation, revealed similar values of the percentage quotient derived from the entire activities in these two different methods. This suggests that the presented separation procedure gives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valuable tool for the quantitative analysis of alkaline phosphatase activity along the length of the villus.

In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine.

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3'-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co(2+)-containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB-cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehyde oxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB-cobalt complex.

In situ detection of constitutive superoxide anion production in granules of mast cells.

3,3'-Diaminobenzidine, in the presence of manganese and cobalt ions, was applied for the detection of superoxide anions in unfixed cryostat sections of rat oesophagus, trachea, skin and intact mesenterium. In all connective tissues, a blue final reaction product was found in a granular form in mast cells. The amount of final reaction product formed after incubation with diaminobenzidine and cobalt ions was increased by the addition of manganese ions. Electron microscopical analysis revealed that the electron-dense final reaction product was exclusively present in the granules of mast cells and on elastin fibres. It was found that the constitutive spontaneous formation of final reaction product in mast cells was enzymatic and dependent on the presence of oxygen in the medium. Of all the enzyme inhibitors and free radical scavengers tested, only azide strongly reduced the amount of final reaction product. It was concluded that the reaction was partly caused by peroxidase activity, but that superoxide anions are also constitutively and spontaneously produced in mast cell granules. The exact enzymatic source could not be established. Whether this property of mast cell granules plays an antimicrobial role in connective tissues can only be speculated.

Quantitative enzyme- and immunohistochemistry of hexokinase and cytochrome c oxidase of spinal neurons in the zebrafish

A combined quantitative enzyme- and immunohistochemical procedure to demonstrate hexokinase (HK) was developed and tested on sections of spinal cord tissue of the zebrafish. In both procedures, the amount of final reaction product was linearly related with section thickness. When applied to serial sections of fish spinal neurons, the enzyme- and immunohistochemical activities appeared to correlate significantly (r = 0.61; p < 0.001). As HK and cytochrome c oxidase (COX) histochemistry have been used regularly to screen the average level of chronic activity of neurons, we subsequently analysed the relationship between HK and COX in fish spinal neurons, using previously published methods of quantitative enzyme- and immunohistochemistry for COX. The enzyme- as well as the immunohistochemical localisation patterns of HK showed a weak correlation with the enzyme- and immunohistochemical COX localisation respectively. Therefore, it is concluded that both enzyme- and immunohistochemical localisation of COX provide a poor estimate for the relative level of glucose utilisation in fish spinal neurons.

Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions.

The extinction coefficient is essential for the conversion of cytophotometric (mean integrated) absorbance values into absolute units of enzyme activity, for instance expressed in terms of moles of substrate converted per unit time and per unit wet weight of tissue. The extinction coefficient of polymerized diaminobenzidine (polyDAB) complexed with cobalt as the final reaction product of oxidase reactions was estimated at 575 nm by comparison of the amounts of final reaction products formed after incubation of serial unfixed cryostat sections of rat kidney to demonstrate D-amino acid oxidase activity with either the tetrazolium salt method or the cerium-DAB-cobalt-hydrogen peroxide method. Both procedures resulted in similar localization patterns of final reaction product in a granular form in epithelial cells of proximal tubules in rat kidney. The granules were peroxisomes. Linear relationships were found for both methods between the specific amounts of final reaction product generated by D-amino acid oxidase activity and incubation time. The cerium salt method gave rise to 7.4 times higher absorbance values of final reaction product generated per unit time and per unit wet weight of tissue than the tetrazolium salt procedure. The extinction coefficient of tetranitro BT-formazan is 19,000 at 557 nm. Therefore, the cytophotometric extinction coefficient of the polyDAB-cobalt complex as final reaction product of oxidase reactions was established to be 140,000.

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver

The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light- and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mM diaminobenzidine, 44 mM hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 microns. Catalase in rat liver showed a Km value of 2.0 mM for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mM. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.

Quantitative in situ analysis of xanthine oxidoreductase activity in rat liver.

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.

A quantitative histochemical study of xanthine oxidase activity in rat liver using the cerium capture method in the presence of polyvinyl alcohol.

A recently developed histochemical technique to demonstrate xanthine oxidase activity in milk globules of bovine mammary gland and in epithelial cells of rat small intestine using cerium ions and a semipermeable membrane was slightly modified. The semipermeable membrane method was replaced by the addition of 10% (w/v) polyvinyl alcohol to the incubation medium. This technically more simple procedure enabled detection of xanthine oxidase activity in unfixed cryostat sections of rat liver. Both methods gave qualitatively and quantitatively similar results. Activity was found in sinusoidal cells and in liver parenchymal cells, with 50% higher activity in pericentral than in periportal areas. The specificity of the reaction was proven by the generation of only small amounts of final reaction product on incubation either in the absence of the substrates hypoxanthine or oxygen or in the presence of hypoxanthine and allopurinol. Allopurinol is a specific inhibitor of xanthine oxidase activity. The amount of final reaction product, as measured cytophotometrically in rat liver, increased linearly with incubation time (15-90 min) and with section thickness (up to 12 microns). By varying the hypoxanthine concentrations, a Km value of 0.05 mM was found. Addition of dithiothreitol to the incubation medium reduced the amount of final reaction product by 85%, which was caused by conversion of reversible xanthine oxidase into xanthine dehydrogenase. This histochemical method can be used for quantitative analysis of in situ xanthine oxidase activity.

Combined quantitative immuno- and enzyme cytochemistry of cytochrome c oxidase in sections of neural tissue and cultured cells

Immuno- and enzyme cytochemical procedures were tested for their applicability to determine quantitatively the concentration and the activity of cytochrome c oxidase (COX) in tissue sections of fish spinal cord and sections of cultured human Molt-4 cells. Sections of gelatin gels containing known amounts of human skeletal muscle COX served as standard sections. The selected procedures fulfilled an important criterion for valid quantitative cytochemistry: the amount of final reaction product was linearly related with section thickness. When the immuno- and enzyme cytochemical methods were applied to sections of fish spinal neurons or cultured human cells, the COX concentration appeared to correlate significantly with COX activity. These cytochemical methods might therefore be valuable to detect differences in molecular COX activities between individual cell types as may occur in tissue sections of biopsies taken from patients with mitochondrial dysfunctions.

NADPH-dependent lipid peroxidation capacity in unfixed tissue sections: characterization of the pro-oxidizing conditions and optimization of the histochemical detection

Factors which influence the iron-stimulated lipid peroxidation in rat liver have been studied by incubating unfixed cryostat sections with a pro-oxidant system and using an optimized histochemical detection method for lipid peroxidation products with 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B. We used a method that was slightly different from the one described previously. The final reaction product was exclusively localized in the cytoplasm of liver parenchymal cells with a homogeneous distribution within the liver lobule. The absorbance maximum, as measured cytophotometrically, was found to be 550 nm. Maximum lipid peroxidation was observed when the pro-oxidant system contained 0.2 mM NADPH, 1 mM ADP and 15 microM FeCl2. Some reaction product was found when NADPH was omitted. Iron concentrations higher than 180 microM prevented the formation of lipid peroxidation products in certain areas of the sections, whereas ADP concentrations higher than 1 mM inhibited the reaction in the whole section. A pH dependency was also observed, with the highest lipid peroxidation at pH 7.2. Optimum lipid peroxidation was induced by incubating for 30 min at 37 degrees C with the pro-oxidant system. A linear relationship was found between the thickness of the sections (up to 20 microns) and the amount of lipid peroxidation products. The addition of scavengers of O2-. (superoxide dismutase), hydrogen peroxide (catalase) and OH. (mannitol) to the first step medium did not affect the amount of final reaction product. These findings appear to confirm the hypothesis proposed for events occurring in isolated microsomes, leading to the formation of hydroperoxides and ultimately lipid peroxidation-derived carbonyls.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5'-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 microns in thickness. For all the enzymes that could be detected, the 6 microns:3 microns ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.

A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.

A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme.

A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH 8.0, 0.5 mM inosine, 0.47 mM methoxyphenazine methosulphate and 1 mM Tetranitro BT. An enzyme film consisting of xanthine oxidase was brought onto the object slides before the section wa allowed to adhere. The specificity of the reaction was proven by the low amount of final reaction product generated when incubating in the absence of inosine. Moreover, 1 mM p-chloromercuribenzoic acid, a non-specific inhibitor of purine nucleoside phosphorylase, inhibited the specific reaction by 90%. The specific reaction defined as the test reaction, in the presence of substrate, minus the control reaction, in the absence of substrate was linear with incubation time at least up to 30 min as measured cytophotometrically. A high activity was observed in endothelial cells and Kupffer cells of rat liver and a lower activity in liver parenchymal cells. Pericentral hepatocytes showed an activity higher than that of periportal hepatocytes. In human liver, purine nucleoside phosphorylase activity was also high in endothelial cells and Kupffer cells, but the activity in liver parenchymal cells was only slightly lower than it was in non-parenchymal cells. The localization of the enzyme is in agreement with earlier ultrastructural findings using fixed liver tissue and the lead salt procedure.

Assessment of lysosomal function by quantitative histochemical and cytochemical methods

Quantitative histochemistry and cytochemistry enables a direct link to be made between metabolic functions such as the activity of lysosomal enzymes and the morphology of a tissue or a type of cell. Several approaches exist such as microchemistry based on (bio)chemical analysis of a single cell or a small piece of tissue dissected from a freeze-dried section. This technique has been routinely used for prenatal diagnosis of inherited enzyme defects and especially of lysosomal storage diseases. Other approaches are cytofluorometry or cytophotometry, which are based on the principle that a fluorescent or coloured final reaction product is precipitated at the site of the enzyme. The amount of final reaction product is analysed per cell or per unit volume of tissue using either a microscope cytofluorometer or flow cytometer for fluorescence measurements or an image analysing system or scanning and integrating cytophotometer for absorbance measurements. In principle, fluorescence methods are to be preferred over chromogenic methods because they are more sensitive and enable multiparameter analysis. However, only a limited number of fluorogenic methods are at hand that give a final reaction product which is sufficiently water-insoluble to guarantee good localisation. The best results have been obtained with methods based on naphthol AS-TR derivatives and with methods for the demonstration of protease activity using methoxynaphthylamine derivatives as substrates and 5'-nitrosalicylaldehyde as coupling reagent. Chromogenic methods are far better with respect to localisation properties and, therefore, most commonly used for quantitative histochemical analysis of lysosomal enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytofluorometric analysis of enzyme reactions based on quenching of fluorescence by the final reaction product: detection of glucose-6-phosphate dehydrogenase deficiency in human erythrocytes.

We developed a method for accurate cytofluorometric analysis of the final reaction product of enzyme reactions in individual cells. Glucose-6-phosphate dehydrogenase (G6PD) activity in human erythrocytes was demonstrated cytochemically, and the amount of final reaction product (formazan) per cell was detected indirectly by quenching of autofluorescence generated by glutaraldehyde fixation. Formazan quenches fluorescence in a dose-dependent manner. The method has been used for detection of G6PD deficiency. Heterozygous and homo(hemi)zygous deficiency could easily be established, even in cases of extreme "Lyonization" where microscopic inspection failed to discriminate between either normal individuals and heterozygously deficient patients or heterozygously and homozygously deficient patients. The principle of quenching of fluorescence by final reaction products of enzymes can be applied to flow cytofluorometric analysis of enzyme activity in individual cells in general.

Plateau absorbance measurements: an alternative approach to enzyme activity determination illustrated by the example of alkaline phosphatase.

A simple and reproducible method was used for the cytophotometric assay of alkaline phosphatase activity by end point measurements after incubation at 70 degrees C. Alkaline phosphatase was incorporated in polyacrylamide gel model films and its activity was demonstrated with a simultaneous coupling method. The initial reaction rate was 4.7 times faster than at 37 degrees C. At 37 degrees C, linear reaction rates were obtained up to 90 min incubation. Deviation from linearity occurred only when the amount of final reaction product precipitated inside the films was too high to be measured cytophotometrically. In that case, levelling off of the reaction rate was due to the out-of-range error of the cytophotometer. At 70 degrees C, reaction rates were distinctly non-linear from the onset of incubation. This was due to heat inactivation of the enzyme molecules. A plateau level was reached after approximately 60 min incubation, irrespective of the amount of enzyme incorporated, indicating that all enzyme molecules had become inactivated after this incubation period. The inactivation process followed first-order kinetics. The plateau value as well as the slope of the initial reaction were found to be linearly related to the amount of enzyme incorporated. Therefore, plateau absorbance values can be used as a relative measure of enzyme activity instead of initial reaction rates. This type of measurement could be valuable for routine applications of enzyme cytochemistry in diagnostic pathology, or when cytochemical reaction products are used as markers in immunocytochemistry or hybridocytochemistry. Precise control of incubation time is not necessary once the plateau value has been reached and preparations can be mounted and measured later.