Histochemical investigation of the activity of oxidoreductases in the skin lesions of lepromatous leprosy patients*

Bull. Org. mond. Sant( 1972, 46, 813-819 Bull. Wid Hith Org. Histochemical investigation of the activity of oxidoreductases in the skin lesions of lepromatous leprosy patients * G. F. 2URAVLEVA1 Thispaper reports an investigation ofthe activity ofthree basic groups ofoxidoreductases in lepromatous leprosy: specific dehydrogenases, flavoprotein enzymes, and cytochrome oxi- dase. The activity of the enzymes was studied before treatment, at various stages of treat- ment during exacerbations, and in the stage of regression. The data obtained are of impor- tance for evaluating metabolic process in the cells of the specific infiltrates and the dermal connective tissue in leprosy, for determining the nature and intensity of the inflammatory process, andfor control purposes in cases of regression. A determination of the activity of the oxido- reductases is useful in evaluating the level of metabo- lic activity of various organs. Highly complex enzyme systems are involved in tissue respiration, and deter- mination of their activity to some extent enables us to assess disturbances in the fine mechanisms of metabolism in the cells (Shelton & Schneider, 1952; Bamett & Palade, 1957, 1958; Portugalov & Jakov- lev, 1958; Scarpelli et al., 1958; Sedar & Rosa, 1958; Kaplan & Novikoff, 1958; Pearse, 1960; Holt & Hicks, 1961; Strukov, 1963; Strukov et al., 1967; and others). It has been established (Petrun', 1960; Hashimoto et al., 1964; Serbakov, 1966) that in the skin of persons suffering from certain dermatoses there is an increase in the activity of the aerobic tricarboxylic acid cycle and of the basic biochemical oxidation processes-tissue respiration and aerobic glycolysis. Using biochemical methods, Novosel (1968) and Golovcenko (1968) made the further discovery that there is intensified cytochrome oxidase activity in the affected areas of skin in persons with eczema and psoriasis. In their opinion, this is evidence of an acceleration of the oxidation-reduction processes in the skin, brought about by intensive oxygen absorp- tion. They consider that, at the same time, a certain * This investigation was financed by the World Health Organization. 1 Institute for the Study of Leprosy, Astrakhan, and Department of Morbid Anatomy, First Moscow Medical Institute, Moscow, USSR. degree of oxygen deficiency is detectable in the skin and an oxygen deficit is produced in the affected area. As the skin lesions are resorbed, a fall in the rate of tissue respiration is observed. In a biochemical study of oxidizing enzymes in leprosy, Naylor (1958) discovered that the amount of dehydrogenases in a leproma was noticeably greater before treatment than during the course of treatment. Visneveckij (1969) noted that active spe- cific infiltrates of the skin of patients with lepro- matous leprosy show a high cytochrome oxidase, " NAD diaphorase ", and " NADP diaphorase " content but no succinate dehydrogenase activity. Under the influence of treatment for leprosy, the activity of these enzymes in the skin decreases and tissue respiration is depressed. We have found no other data on the state of activity of oxidoreduc- tases in the skin of leprosy patients. MATERIALS AND METHODS We have studied the state of activity of all three groups of oxidoreductases in cases of lepromatous leprosy, i.e., (1) the specific dehydrogenases, includ- ing the basic oxidoreductases of the Krebs cycle (succinate, malate, isocitrate, glutamate, glycerol- phosphate, glucose-6-phosphate, lactate, and alco- hol dehydrogenases), (2) the flavoprotein enzymes " NAD diaphorase " and NADP diaphorase ", and (3) cytochrome oxidase. The general principle of the histochemical method for demonstrating dehydrogenase and " diaphorase " 2861 - 813 10 814 G. F. 2URAVLEVA activity is that colourless tetrazolium salts are, as it were, substituted for the substance to be reduced and become acceptors for the hydrogen, which is split off by enzyme action from the substance to be oxi- dized (Pearse, 1960). The tetrazolium salts are re- duced by addition of the hydrogen and converted into coloured formazans, which are precipitated at sites of high enzyme activity. The activity of oxidoreductases is best demonstrated with nitro-blue tetrazolium.1 The activity of enzymes was determined in non- fixed sections prepared in a cryostat at a temperature of - 20°C, using nitro-blue tetrazolium as the hydro- gen acceptor and appropriate substrates, following the methods described by Pearse (1960). The de- hydrogenases were determined by the methods of Nachlas (1957), Hess et al. (1958), and Burstone (1959), using inter alia substrates of sodium succinate, malate, isocitrate, and lactate. The " diaphorases " were demonstrated by the method of Farber et al. (1954) using directly reduced forms of the coenzymes NADH and NADPH. Cytochrome oxidase was determined by the method of Moog (1943) using p-aminodimethylaniline oxalate as the substrate. The sections were first treated with acetone for 30 se- conds to extract the fat droplets from the cell cyto- plasm. Control sections were incubated in solutions without substrates. The formation of dark-blue granules of diformazan and aminophenol blue were considered to be the index of enzyme activity, tak- ing into account their location, distribution, inten- sity, dimensions, and amount. Enzyme activity was estimated on the 5-point system: very high, high, medium, low, and nil. Besides the histochemical methods of investigation described, conventional histological staining tech- niques were also employed, i.e., haematoxylin-eosin for general orientation of the histological structure, C.I. solvent red 23 (Sudan III) and C.I. solvent black (Sudan black B) for lipids, and the Ziehl-Neelsen method for leprosy bacilli. Enzyme activitywas studied systematically through- out the course of the lepromatous process: before treatment, at various stages of treatment, during exacerbations, and in the regressive stage. A total of 69 fragrnents of skin obtained by biopsy from patients with lepromatous leprosy were examined together with a further 8 samples obtained during sur- gical operations on persons not suffering from leprosy as controls. The lepromatous cases were divided 1 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)bis[2-(p-nitro- phenyl)-S-phenyl-2H-tetrazolium]dichloride. into 5 groups according to treatment and clinical status: (1) untreated, with active manifestations of leprosy (9 cases); (2) treated, but still exhibiting clinically active changes (13 cases); (3) patients in the stationary stage of development of the leprosy pro- cess (14 cases); (4) patients with lepra reaction (erythema nodosum) (10 cases); and (5) patients at the regressive stage (23 cases). RESULTS AND DISCUSSION Histological investigation of the skin sections from affected areas in untreated active lepromatous pa- tients disclosed extensive areas of diffuse infiltration, consisting primarily of lepra cells containing lipids and large numbers of homogeneous mycobacteria. Histoenzymological studies of the lepra cells showed very high levels of activity of the Krebs' cycle en- zymes (succinate, malate, and isocitrate dehydro- genases), glutamate dehydrogenase, glycerophos- phate dehydrogenase, glucose-6-phosphate dehydro- genase, " NAD diaphorase ", " NADP diaphorase ", and cytochrome oxidase (Fig. 1-4). The dark-blue particles of diformazan, indicative of dehydrogenase and " diaphorase" activity, were fairly densely scattered and were present in large quantities through- out the cytoplasm of the lepra cells; they were mostly round and quite large. The diformazan granules were dispersed evenly throughout the cell. The cell nuclei were often difficult to distinguish since they were masked by the diformazan grains. Intense enzyme activity was noted in cells of the Malpi- ghian layer of the epidermis and in the vascular endothelium. A small number of fine particles of diformazan were scattered outside the areas of in- filtration, in the sweat-gland cells, in free fibroblasts, and in the fibrous structures of the dermis. In the same patients, lactate and alcohol dehydrogenase activity was low. Grains of aminophenol blue, an indication of cytochrome oxidase activity, were seen in the cytoplasm of lepra cells, where they appeared large and intensely coloured; often the grains had merged together, forming quite considerable conglo- merates. In other cellular structures, cytochrome- oxidase activity was lower. Histoenzymological investigation of lepromatous patients already under treatment but still showing histological signs of an active process disclosed in- tense persistent reactions for succinate, malate, iso- citrate, glutamate, glycerolphosphate, and glucose-6- phosphate dehydrogenases, " NAD diaphorase ", " NADP diaphorase ", and cytochrome oxidase. Gra- Fig. 1. Untreated lepromatous leprosy showing very high succinate dehydrogenase activity in the lepra cells of an infiltrate; x 1 00. Fig. 2. Active lepromatous leprosy showing high glutamate dehydrogenase activity in a granuloma; x 1 00 Fig. 3. Lepromatous leprosy lesion in the active stage, showing high NAD diaphorase " activity in an infiltrate; x 100 C~~~ ~~~~~or 1111iiiiZ Fig. 4. Untreated lepromatous leprosy; very high cytochrome oxidase activity in the lepra cells of the infiltrate; x 100 rr , [} ' Re I' t" 'S .' a::a - >^rt'9~~~~~~~~~~~~~~~~~~~~~~~;. Fig. 5. Regressing lepromatous leprosy showing small dermal infiltrates of lymphoid cells and fibroblasts; stained with haematoxylin-eosin; x 200 Fig. 6. Regressing lepromatous leprosy showing no succinate dehydrogenase activity in the residual dermal infiltrates; x 100 4 ..* -, ..,W .k 0,..:Z k ... i; .-. j1. -6 :. OXIDOREDUCTASE ACTIVITY IN LEPROMATOUS LEPROSY nules of diformazan and aminophenol blue were still evenly distributed in fairly large quantities throughout the cells in the areas of infiltration. Lactate and alco- hol dehydrogenase activity was low. In those cases in which the leprous process was in the stationary stage, histological examination reveal- ed moderately well-defined areas of infiltration in the skin, consisting of macrovacuolated lepra cells con- taining lipids and considerable numbers of granular leprosy bacilli. Histochemical examination of the lepra cells showed that the activity of all the de- hydrogenases investigated, except the lactate and alcohol dehydrogenases, and of " NAD diaphorase ", " NADP diaphorase ", and cytochrome oxidase was moderate or, more rarely, low. Blue-stained particles were found distributed evenly and in moderate num- bers in the lepra cells in the areas of infiltration. In the cells and fibres of dermal connective tissue, en- zyme activity was low. No cytochrome oxidase acti- vity was found in the epidermal cells. In skin sections from erythema nodosum lesions the following phenomena were observed when ordi- nary staining techniques were used: dilatation of the blood vessels, swelling of the vascular endothelium and thickening of the vessel walls, perivascular oedema and, in a number of cases, erythrocyte dia- pedesis. Small areas of infiltration consisting of lymphoid cells, fibroblasts, lepra cells and poly- nuclear cells were seen; the latter contained lipids and a small quantity of mainly granular or, more rarely, homogeneous mycobacteria. In some cases, homogenization and fibrinoid degeneration of the connective tissue had occurred. Histoenzymological studies indicated moderate or low activity of succi- nate, malate, glutamate, glycerolphosphate, and glu- cose-6-phosphate dehydrogenases in areas of skin infiltration and in the epidermis. Lactate and alco- hol dehydrogenase activity in lymphoid cells, lepra cells, and fibroblasts was low or completely absent. The cells and fibres of the granulomata showed a moderate level of " NAD diaphorase " and " NADP diaphorase ". A high level of cytochrome oxidase activity was seen in the lepra cells in the areas of infiltration. As the clinical symptoms of the reactive phase subsided, enzyme activity also decreased. As regressive changes advanced in the areas of dermal infiltration, there was a lowering of oxidore- ductase activity. Where there was marked regression of the lepromatous granuloma, the staining of skin sections from previously affected sites with haemo- toxylin-eosin, C.I. solvent black, C.I. solvent red 23, and by the Ziehl-Neelsen technique revealed regressing residual infiltrates consisting mainly of lymphoid cells and fibroblasts; occasional disrupted mycobacteria were seen (Fig. 5). The activity of the Krebs cycle enzymes, the dehydrogenases, the " dia- phorase ", and cytochrome oxidase was determined histochemically and found to be low or to have dis- appeared completely. The blue particles of difor- mazan in the infiltrate cells became paler in colour and finely granular, giving the appearance of detritus with an insignificant amount of stain (Fig. 6). Out- side the areas of infiltration, in the free fibroblasts, connective tissue fibres, skin appendages, nerves, and epidermis, the level of enzyme activity was relatively high. No cytochrome oxidase activity was found in the cells of the epidermis. These comparative data on the activity of the oxidoreductases, processed by the statistical method of Fisher (1952) as modified by Moncevi"jute-IEringene (1964), are shown in Table 1. The table shows that there is a very high and sta- tistically significant level of activity of the oxidore- ductases investigated, except for lactate dehydrogen- ase and alcohol dehydrogenase, in the cells of active lepromatous granulomata of the skin. Since lactate dehydrogenase activity is an important index of the anaerobic consumption of glucose and the production of lactic acid, it can be concluded that biological oxidation plays the most important role in the meta- bolic processes that occur in skin granulomata in leprosy. These results indicate that lepra cells are biologically highly active, and this is in agreement with the conclusions reached by Chatterjee (1958), Kosolapkina (1962) and Vdovina (1964) on the high ribonucleoprotein and mucopolysaccharide content of lepra cell protoplasm. The depression of dehydro- genase, " diaphorase ", and cytochrome oxidase acti- vity, and the simultaneous intensification of their activity in the connective tissue cells and the skin appendages, is evidence of a relative depression of tissue respiration in the areas of infiltration and of a certain normalization of the oxidation-reduction processes in the skin of leprosy patients as a result of treatment with dapsone. Determination of enzyme activity (in conjunction with a number of other clinical and laboratory indices) is of importance in evaluating the nature, intensity, and duration of the inflammatory process and in following up the regressive changes that occur. In this connexion, the data obtained can be of value in both diagnosis and prognosis. The change in the level of activity of the respiratory enzymes in lepro- matous leprosy may be regarded as the result of a disturbance in the metabolism of the cells of specific 817 G. F. IURAVLEVA Table 1. Levels of activity of respiratory enzymes in patients with untreated active lepromatous leprosy compared with levels in the regressive stage of the disease Enzyme Ni Ml ± mib N2a M2 ± m2b tc p succinate dehydrogenase 9 3.4 ± 0.27 23 0.6 ± 0.12 9.6 <0.1 malate dehydrogenase 9 3.4 ± 0.28 23 0.7 ± 0.11 9.0 <0.1 isocitrate dehydrogenase 8 3.2 ± 0.24 21 0.6 ± 0.13 9.6 <0.1 lactate dehydrogenase 8 0.8 ± 0.12 23 0.5 ± 0.13 1.6 12 glucose-6-phosphate dehydrogenase 9 3.3 + 0.25 23 0.7 ± 0.11 9.6 <0.1 alcohol dehydrogenase 9 1.4 ± 0.22 22 0.6 ± 0.15 2.8 1.1 glycerolphosphate dehydrogenase 9 3.4 ± 0.28 23 0.6 ± 0.13 9.0 <0.1 glutamate dehydrogenase 9 3.0 ± 0.64 23 0.6 ± 0.12 3.6 0.1 ' NAD-diaphorase 9 2.9 ± 0.16 23 0.7 ± 0.11 11.0 <0.1 - NADP-diaphorase - 9 3.3 ± 0.33 23 0.7 ± 0.11 7.0 <0.1 cytochrome oxidase 9 3.6 ± 0.2 21 0.6 ± 0.14 12.0 <0.1 a Ni = No. of observations on untreated lepromatous patients; N2 = No. of observations on treated lepromatous patients in the regressive stage. b MI, M2 = arithmetic mean; ml, m2 = standard error of the artithmetic mean. c t = Statistic for the difference between Mi and M2. infiltrates. From the nature and intensity of enzyme activity, it is possible to form some idea of the state of tissue respiration and determine the nature of the metabolic activity of the cells; this is helpful in studying the pathogenesis of skin changes occurring in leprosy patients undergoing treatment with modem drugs. Consequently, in the pathogenesis of skin infec- tions in leprosy, disruption of the activity of the enzymes investigated is of considerable importance; it would appear to be caused by metabolic disturb- ances occurring not merely in the foci of infection but in the body as a whole. The use of modem histoenzymological techniques considerably broadens our ideas on the nature of the morphogenesis and pathogenesis of the specific leprous involvement of the skin at the various stages of treatment. Conclusions (1) A high degree of oxidation-reduction activity can be demonstrated histochemically in the cells of active lepromatous granulomata of the skin. It takes the form of a high level of activity of succinate, malate, glutamate, glycerolphosphate, and glucose- 6-phosphate dehydrogenases, " NAD diaphorase", "NADP diaphorase", and cytochrome oxidase. (2) The low level of activity of lactate and alcohol dehydrogenases in the cells of active lepromatous infiltrates points to a fall in anaerobic oxidation processes and a predominance of biological oxi- dation. (3) Regressing lepromatous infiltrates show low levels of dehydrogenase, " diaphorase ", and cyto- chrome oxidase activity, which indicates that the oxidation-reduction processes in the cells of these infiltrates are inhibited. (4) The data obtained from histoenzymological investigations are significant in evaluating the inten- sity of the specific inflammatory process and in following the regressive changes that occur inleproma- tous patients. 818 OXIDOREDUCTASE ACTIVITY IN LEPROMATOUS LEPROSY 819 RESUME INVESTIGATIONS HISTOCHIMIQUES SUR L'ACTIVITE DES OXYDOREDUCTASES DANS LES LESIONS CUTANEES CHEZ DES MALADES ATTEINTS DE LEPRE LEPROMATEUSE On a etudie a I'aide de methodes histochimiques I'activite des deshydrogenases specifiques - dont notam- ment les principales oxydoreductases du cycle de Krebs , des enzymes flavoproteiniques et de la cytochrome-oxy- dase chez des patients atteints de lepre lepromateuse a divers stades d'evolution de la maladie. Les examens ont porte sur 69 fragments de peau preleves par biopsie chez des malades et sur 6 fragments preleves chez des sujets non 1epreux. L'activit6 enzymatique a e mesur6e sur des substrats appropries grace a l'emploi de bleu de tetrazolium nitre. Chez les patients atteints de lepre lpromateuse evolu- tive et non traitee, on note une forte activite de la cyto- chrome-oxydase, de la # NAD diaphorase #, de la #NADP diaphorase >) et des diverses deshydrogenases, a l'exception de la lactate deshydrogenase et de l'alcool deshydrog&- nase. Dans les cas d'erythema nodosum, l'activite enzy- matique est moderee ou faible dans les zones d'infil- tration. Au fur et a mesure de la regression des lesions, l'inhibition ou l'abolition complete de l'activite des oxy- doreductases dans les cellules des infiltrats residuels contraste avec le maintien d'une activite enzymatique elevee dans les fibroblastes, le tissu conjonctif, les annexes de la peau, les nerfs et l'epiderme. Selon l'auteur, l'etude de l'activite des oxydoreductases dans les lesions cutanees peut fournir des indications sur l'evolution de la lepre lepromateuse et faciliter le diagnostic et le pronostic. REFERENCES Barnett, R. J. & Palade, G. B. (1957) J. biophys. bio- chem. 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