Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 2. The biochemical investigation and comparison with the histochemical observations.

The reliability of enzyme histochemical semipermeable membrane techniques for the demonstration of acid hydrolases was investigated with a combined histochemical and biochemical study. In part 1 the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings. In m. soleus, m. plantaris, m. gastrocnemius and diaphragm of vitamin E deficient rabbits the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase and beta-glucuronidase is significantly increased. This increase in activity of the investigated acid hydrolases was equal for muscles with an aerobic or an anaerobic metabolism. By means of statistical calculations the activity of the enzymes demonstrated with histochemical techniques was compared with the enzyme activity determined with biochemical techniques. From the results of this investigation it can be concluded that the histochemical semipermeable membrane techniques for the demonstration of activity of acid hydrolases are very reliable. Considering the fact that these techniques are also tissue-saving, they are therefore extremely suitable for the study of catabolic wasting processes in skeletal muscle tissues of patients with inherited or acquired muscular diseases.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).     

The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation. I. The histochemical investigation.

The activity of acid hydrolases in skeletal muscles of normal rats and of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In this communication the histochemical findings are presented. After 4 days of DPPD treatment, coagulation necrosis, fragmentation and disintegration of fibres were seen in the muscles. An inflammatory infiltrate was seen between the muscle fibres. These pathological changes reached maximum intensity after 7 to 9 days. After 11 days the changes became less, despite continued treatment with DPPD. From the histochemical findings it appeared that the activity of acid phosphatase, beta-glucuronidase and E600 resistant non-specific esterase was increased in both a granular and a diffuse pattern in the skeletal muscles of the DPPD rats. The increase in activity of leucine aminopeptidase was much less pronounced and was mainly granular. The increase in the activity of acid hydrolases ran parallel to the severity of the pathological changes and reached a maximum after 7 to 9 days of DPPD treatment. The statistical calculations of the histochemical findings revealed that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second hydrolase. There was a moderate probability that the activity of all other histochemically studied acid hydrolases, with the exception of leucine aminopeptidase, was increased. There was no difference in activity and localization of the acid hydrolases studied in aerobic type I and anaerobic type II fibres. The localization of acid phosphatase and beta-glucuronidase activity in muscle fibres and in inflammatory infiltrate mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E600 resistant non-specific esterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the acid hydrolases examined were found to be present in the inflammatory exudate and in the connective tissue.     

A novel approach for enzyme histochemical and autoradiographic studies on single cells

Synopsis A simple technique that does not involve the use of heat has been developed to fix cells or cell organelles. The cells or organelles are mixed with a bovine serum albumin solution, gelled by the addition of a suitable fixative, and then either embedded or frozen. The gelled mixture contains well preserved cells or organelles that are evenly dispersed, thus eliminating the problems of pellet packing. The technique was excellent for ultrastructural autoradiography where radioactive materials bound to plasma membranes or cytoplasmic nucleotides were being studied. Histochemical tests could be applied to the fixed embedded material. Light and electron microscopy could be done on the same well-mixed sample. Fixed frozen albumin samples cut with ease on a cryostat but there was ice crystal formation.     

The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation

Summary The increase in activity of acid hydrolases in skeletal muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In part I the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings.In homogenates of m. biceps femoris, m. gastrocnemius and m. rectus femoris of DPPD-treated rats, the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase, and -glucuronidase was increased. This increase in activity was maximal after 7 to 9 days of DPPD treatment and ran parallel to the severity of the pathological changes. Statistical calculations clearly reveal that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second acid hydrolase. Moreover these calculations reveal that the biochemical activity findings correlated with the histochemical activity findings. However it was remarkable that in the histochemical study, the estimated increase in acid phosphatase activity was much more than the increase in acid phosphatase activity found biochemically, whilst on the other hand the histochemically estimated increase in -glucuronidase activity corresponded with the biochemical observations. The results of gel filtration techniques have shown that this discrepancy of acid phosphatase activity was caused by different substrate specificity of the different isoenzymes of acid phosphatase and that as a result of the DPPD treatment the isoenzyme pattern had been altered. The elution patterns showed three distinct isoenzymes of acid phosphatase of normal and of DPPD treated rats. These isoenzymes, termed I, II and III, have molecular weights of: 200,000 or more, 83,500–104,500 and 14,500–18,100. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of the DPPD treated rats. Isoenzyme III does not split naphthol AS-BI phosphate and the activity is not increased in the muscles of the DPPD treated rats. Considering the fact that it has been shown that the activity of isoenzyme III is high compared with that of the isoenzymes I and II, it is important to realise that by using naphthol AS-BI phosphate not all acid phosphatase can be demonstrated in sections of skeletal muscle.This study was partly supported by a grant from the Prinses Beatrix Fonds, 's Gravenhage, The Netherlands, and was mainly extracted from the Ph. D. thesis of D.E. Israël (1977).     

Autofluorescence of melanins induced by ultraviolet radiation and near ultraviolet light. A histochemical and biochemical study

The induction of autofluorescence of melanins by UV radiation (330–380nm) and near UV (400–440nm) light (jointly called UV light) was studied in tissue sections using three commercially available mounting media. Only Immu-Mount (Shandon) was found suitable for this purpose. UV irradiation of melanins in sections mounted in this medium induced strong yellow auto-fluorescence irrespective of the type of the polymer (eumelanin, neuromelanin, pheomelanin and ochronotic pigment). The phenomenon of auto-fluorescence induction was also observed with isolated natural and in vitro prepared melanins. It was inhibited by anhydrous conditions, sodium azide and catalase. In parallel experiments, rapid degradation of melanins with an intermediate fluorescent stage was achieved in UV-irradiated sections mounted in media artificially enriched with hydrogen peroxide, or directly in aqueous solutions of H₂O₂, Na₂O₂ or HIO₄. Oxidations not associated with UV light led to nonfluorogenic breakdown of melanins. These observations indicate that the common mechanism may be an oxidative attack resulting from a concerted action of hydrogen peroxide and UV light leading, through strongly fluorescent intermediates, to a complete bleaching and oxidative breakdown of melanin and melanin-like polymers. Reactive oxygen species (including ozone) are considered to be important reactants in these experiments. Lipopigments differ from melanin-like pigments by their primary auto-fluorescence, which mostly faded during continuous prolonged irradiation. The only regular exception was melanosis coli pigment, the auto-fluorescence of which was considerably augmented by UV irradiation. Our results demonstrate a novel type of fluorogen in auto-fluorescent pigment histochemistry. The implications of the results are discussed especially in the light of the possible presence of melanin-based fluorogens in lipopigments.     

The pentose phosphate pathway in skeletal muscle under patho-physiological conditions. A combined histochemical and biochemical study

Over the last 30 years, research into the neuromuscular apparatus, has expanded greatly. Multidisciplinary investigations have rapidly advanced our understanding both of diseases and of the basic neuromuscular mechanisms. The mode of pathological reaction of the neuromuscular apparatus is now quite well understood. The most notable aspect of the reaction of the injured neuromuscular apparatus is the remarkably stereotyped character of the resulting pathological changes as demonstrated by a wide variety of harmful causes, producing surprisingly similar effects. The findings of our combined histochemical and biochemical investigations presented in this monograph, are in complete harmony with the stereotyped character of the pathological changes. For example, it is particularly striking that many affected muscle fibres of patients with muscular dystrophies, congenital myopathies, inflammatory myopathies, metabolic myopathies, endocrine myopathies, or with diseases of the lower motor neuron, display an enhanced activity of both oxidative enzymes of the pentose phosphate pathway. Likewise, we found that experimental animals with disordered skeletal muscles, provoked by different types of agents or treatments, reveal the same marked rise in activity of GPDH and PGDH in the muscle fibres, with a positive correlation between the activity of both enzymes. Other findings of our investigations point to a positive correlation between the activity of GPDH and PGDH on the one hand and that of the non-oxidative enzymes of the pentose phosphate pathway, the enzymes TA, TK, RPI and RPE on the other hand. The rise in activity of PGDH and, in particular, of GPDH is regulated by two different mechanisms. The first represents a rapid control mechanism based on the stimulation of both oxidative enzymes of the pentose phosphate pathway by NADP+ and on their inhibition by NADPH. The other mechanism represents a long-term effect directed at the synthesis of the enzymes. It is this type of mechanism which is responsible for the rise in activity of GPDH and PGDH we observed. The findings obtained with the applied enzyme histochemical techniques clearly demonstrated that the rise in activity of both enzymes is not homogeneously distributed in the disordered skeletal muscles of man and experimental animals. For that reason, in order to obtain reliable quantitative information about enzyme activities in the muscle fibres themselves, the application of biochemical assays on a micro-scale was indispensable. The biochemical assay of enzyme activities was performed on histologically and histochemically selected dissected muscle specimens.(ABSTRACT TRUNCATED AT 400 WORDS)     

A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies

A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a 19F probe attached to the beta-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mM in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.     

Structural,histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles

Summary Horse milk fat glubules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 m. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealted by its binding of various lectins. In addition, the glycocalyx binds cationized ferritin, which indicates the presence of negative electric charges. Gel electrophoresis revealed the presence of high-molecular-weight glycoproteins in the MFG membrane of horse milk. Such glycoproteins are also present in human MFG membranes but are absent in the bovine MFGs. The casein micelles in horse milk are relatively large, their average volume-surface diameter being about 200 nm.Dedicated to Professor Dr. T. H. Schiebler on the occasion of his 65th birthdayS. Patton gratefully acknowledges the generous support of the Alexander von Humboldt Foundation for his research in scientific institutions in the Federal Republic of Germany     

Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles

Horse milk fat globules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 microns. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealed by its binding of various lectins. In addition, the glycocalyx binds cationized ferritin, which indicates the presence of negative electric charges. Gel electrophoresis revealed the presence of high-molecular-weight glycoproteins in the MFG membrane of horse milk. Such glycoproteins are also present in human MFG membranes but are absent in the bovine MFGs. The casein micelles in horse milk are relatively large, their average volume-surface diameter being about 200 nm.     

A computer program for selecting animals for control and experimental groups in biochemical studies

The standard method for selecting experimental animals for controland experimental groups is by a randomization process in whicheach animal is placed into one of the groups without lookingat any of its individual differences. An alternative, activematching process is proposed by which three to five animalsare chosen to form ‘experimental units’ that areclosely matched with respect to average weight and with respectto distribution of weights (standard distribution values). Severalsuch units are then randomly assigned to each control or treatmentgroup and, at the end of the experiment, the animals withineach unit are pooled prior to analysis. The merits of this approachand a computer algorithm for making the selections are described. ; accepted on March 10, 1986     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Rapid microspectrofluorimetry for biochemical and metabolic studies in single living cells

A rapid multichannel microspectrofluorometer (e.g., to NAD(P)H, fluorescent probes) can be operated on a topographic mode for the evaluation of intracellular metabolic topography or on a spectral mode for the individual or simultaneous intracellular spectral analysis of various fluorochromes. The fluorescence emission spectra of the living cells, as well as difference spectra (spectra after intracellular microelectrophoretic addition of substrate minus before)_are analyzed under various conditions, and provide a direct proof that the fluorescence observed is that of NAD(P)H. The spectral changes which accompany treatment with substrate (e.g., glucose-6-P) can be further followed in cells incubated with other probes (e.g., acridine orange). Repeated and quite reversible transients of NAD(P) reduction—reoxidation may be observed in cells having absorbed acridine orange following repetitive additions of substrate. The spectral response to substrate is also comparatively studied in cells grown in presence of agents affecting the cell cycle (e.g., dibutyryl cyclic AMP, bleomycin).     

Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach

The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gamma-glutamyl transpeptidase (GGT), beta-glucuronidase (beta-G1), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT50, PB3a, MC1, MC2) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and beta-G1 activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers.