A valid quantitative histochemical technique for assaying cytochrome c oxidase in single cells.

A quantitative histochemical method for assaying cytochrome c oxidase (COX) has been validated with two new findings concerning the optimal tissue thickness and a suitable substrate. The kinetics of a COX-catalysed reaction coupled to the oxidation of diaminobenzidine (DAB) were followed at 37 degrees C in single muscle fibres in unfixed sections of mouse gastrocnemius using a real-time image analysis system. The optimum composition of the substrate medium for the reaction was 0.1 mM reduced cytochrome c, 4 mM DAB, 2% dimethylsulphoxide, 2% polyvinyl alcohol and 0.1 mM HEPES buffer, final pH 7.5. The absorbances at 451 nm of the final reaction products, DAB polymer oxides, deposited in the intermyofibrillar mitochondria increased linearly as a function of incubation time for at least 80 s after the start of incubation. The initial velocities (v(i)) of the COX reaction calculated from the gradients of the linear regression best fits for times between 40 and 60 s were reproducible. The v(i) determined in single muscle fibres at a saturated concentration of cytochrome c (0.1 mM) were proportional to section thickness for thicknesses less than 3 microns, but they decreased exponentially when the thickness was greater than 4 microns. Thus, for the quantitative assay, unfixed sections 3 microns thick must be used. The Michaelis constants (Km) determined for commercial cytochrome c in the range of 20-26 microM for COX in three types of skeletal muscle fibres of mouse gastrocnemius were higher than the corresponding in situ Km (12-13 microM) for reduced cytochrome c. However, the Km values for commercial cytochrome c were in good agreement with the value previously determined with homogenates of rat hind limb muscle. Therefore, reduced cytochrome c is a more suitable substrate for the kinetic study and assay of COX in situ.

Most apoptotic cells in mdx diaphragm muscle contain accumulated lipofuscin.

An age-related pigment, lipofuscin (LF), which accumulates in postmitotic, long-lived cells, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals. In the present study we show that LF is accumulated in some myofibres, myosatellite cells and interstitial cells in the diaphragm muscles of the X chromosome-linked muscular dystrophic (mdx) mice at the age of 10 weeks when repetitive cycles of de- and regeneration of myofibres occur. In contrast, LF is virtually absent in diaphragm muscles of age-matched C57BL/10 (C57) normal control mice. Therefore, mdx muscle is more susceptible to oxidative stress than normal muscle. We hypothesise that gene-regulated cell death (apoptosis) occurs in dystrophic muscle cells that accumulate LF as a consequence of either oxidative stress or injury. We found that 74-79% of apoptotic myosatellite cells, interstitial cells and myofibres in mdx diaphragm contain accumulated or dotted LF granules, but only 12-20% of non-apoptotic cells contain LF. Apoptotic cells are very rare in the diaphragm of age-matched C57 control mice. This suggests that the regeneration of mdx diaphragm muscle initiated from myosatellite cells is impaired by their apoptosis as the result of either oxidative stress or a product of oxidative injury.

Localisation and quantification of dehydrogenase activities in single muscle fibers of mdx gastrocnemius.

The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate sub-sarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions.

The everchanging advances in enzyme histochemistry, 1986-1996.

The principal developments in the field of enzyme histochemistry in the ten years since 1986 are reviewed briefly. They include the replacement of catalytic histochemistry by immunohistochemistry as the principal means of localising enzymes in situ, including isoenzymes and classes of enzymes that were not possible to visualize hitherto; the development of in situ hybridization and reverse transcriptase-polymerase chain techniques; and the quantification of enzyme distribution and kinetic parameters.

Effects of tissue protectants on the kinetics of lactate dehydrogenase in cells.

We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. the kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation vi-v = a0A, where vi is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and 0 A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed (vi-v) against 0 A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12-43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

Kinetic parameters of lactate dehydrogenase in liver and gastrocnemius determined by three quantitative histochemical methods.

We determined the Michaelis constant (K(m)) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on L-lactate concentration (s). The Km values obtained with three assay methods were similar and in the range of 21.1-21.9 mM for pure LDH, 8.62-13.5 mM for LDH in mouse periportal hepatocytes, and 13.3-17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K(m) values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.

The kinetics of enzymes in situ, with special reference to lactate and succinate dehydrogenases.

The kinetics of two enzyme systems in situ that have been studied with real-time image analysis systems are reviewed in detail. The enzymes are a structurally-bound mitochondrial enzyme, succinate dehydrogenase (SDH) and a soluble cytoplasmic enzyme, lactate dehydrogenase (LDH). The image analysis system is used to capture successive images of a tissue section at constant time intervals whilst it is being incubated on a substrate-containing gel film. The increasing absorbances of the final reaction products in each cell are measured in the successive images as a function of incubation time. The absorbances of the formazan reaction products formed by SDH, for example, in sections of liver determined by such means increase linearly during the first minute of incubation, but non-linearly afterwards. The initial velocities of SDH in single hepatocytes in sections incubated on gel substrate films are calculated from the activities during the first 20 s of incubation. In contrast, the activities of LDH measured in various cell types, including hepatocytes, with the gel film technique increase nonlinearly during the first minute of incubation, but linearly for incubation times between 1 and 3 min. The initial velocities (vi) of LDH in single cells can be, calculated, however from the activities during the first interval, 10 s, of the image capturing sequence. Unfortunately, the experimental errors of the initial velocities of LDH determined in this way are relatively high. To overcome this problem, we have found empirically that the equations vi = a1oA and vi = v + a2oA enable reliable initial velocities (vi) of the LDH reaction in single cells of various types to be calculated using the data of the linear activities for incubation times between 1 and 3 min. Dependence of the initial velocities of the SDH and LDH reactions on substrate concentrations gave the Michaelis constants (Km) and maximum velocities (Vmax). The Km values determined in situ for SDH in hepatocytes and for LDH in various cell types with the gel film technique are in the same order of magnitude as the corresponding values determined biochemically. The constants a1, a2 and Km of LDH are characteristic for each cell type and seem to be related to the intracellular localization of the enzyme and to its ligand-binding rather than to the different isozyme compositions in various cell types.

The initial reaction velocities of lactate dehydrogenase in various cell types.

The initial reaction velocities (vi) of lactate dehydrogenase in hepatocytes, cardiac muscle fibres, skeletal (gastrocnemius) muscle fibres, gastric parietal cells, ductal epithelial and acinar cells of the parotid gland, and oocytes were determined, by computer-assisted image analysis, in unfixed sections of these tissues incubated at 37 degrees C on substrate-containing agarose gel films. They were found to fit the equations vi = a1 zero A (equation 1) and vi-v = a2 zero A (equation 2) reported previously for mouse hepatocytes (Nakae & Stoward, 1993a, b), where v and zero A are, respectively, the gradients (or steady-state velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) of the final reaction product on incubation times between 1 and 3 min, and a1 and a2 are constants. Both equations 1 and 2 fitted the observed vi closely for mouse (a1 = 2.7, a2 = 2.2) and human (a1 = 3.0, a2 = 1.9) hepatocytes. However, equation 2 fitted the observed vi better than equation 1 for mouse cardiac muscle fibres (a1 = 1.5), skeletal muscle fibres (a2 = 1.2), gastric parietal cells (a2 = 1.7), acinar (a2 = 1.4) and striated ductal (a2 = 2.2) epithelial cells of the parotid gland, and oocytes (a2 = 1.6). The values of vi calculated from the two equations agreed with the observed vi to within about 11%. They ranged from 105 mumole hydrogen equivalents/cm3 cell/min units in hepatocytes to 24 units in parotid acinar cells, but for other cell types they were between 46 and 61 units. These are all considerably higher than values reported previously.

The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell.

The kinetics of lactate dehydrogenase in mouse cardiac muscle fibres, skeletal muscle fibres, gastric parietal cells, parotid gland ductal and acinar cells, oocytes and mouse and human hepatocytes were studied as a function of substrate concentration in sections of unfixed mouse and human tissues incubated at 37 degrees C on lactate agarose gel films. The absorbances of the final reaction products deposited in single cells of various types were measured continuously as a function of incubation time using an image analysis system. The initial velocities (vi) of the dehydrogenase were calculated from two equations deduced previously by us, vi = a1 zero A (equation 1) and vi = v + a2 zero A (equation 2), where v and zero A are, respectively, the gradient (steady-state velocity) and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min, and a1 and a2 are constants characteristic for each cell type. Hanes plots using vi calculated from equation 2 gave more consistent estimates of the Michaelis constant (Km) and the maximum reaction velocity (Vmax) than those employing either steady-state velocity measurements or vi calculated from equation 1. The Km thus found for mouse skeletal muscle fibres (10.4-12.5 mM) and hepatocytes (14.3-16.7 mM) agreed well with values determined previously in biochemical assays. However, the Km for cardiac muscle fibres (13.4 mM) was higher. The Km of the enzyme in gastric parietal cells, parotid gland cells and oocytes was in the range 7.6-9.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimating the initial reaction velocity of a soluble dehydrogenase in situ.

The initial reaction velocities (vi) of lactate dehydrogenase in single hepatocytes were determined, by microdensitometry or computer-assisted image analysis, in sections of unfixed mouse liver incubated at 37 degrees C on substrate-containing agarose gel films. They were found to fit the equations vi = 2.82 degrees A and vi = vi + 2 degrees A, where vi and degrees A are, respectively, the gradients (or steady-state linear velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) on incubation time plots for incubation times between 1 and 3 min. Both equations were independent of section thickness between 4 and 14 microns. The observed and calculated values of vi agreed within 11.5% (n = 71). The validity of the equations for vi was confirmed by showing that the calculated vi was proportional to the thickness of the section and hence the amount of enzyme present. Thus, vi can be determined from measurements of either degrees A alone or vi and degrees A.

Kinetic analysis of lactate dehydrogenase in situ in mouse liver determined with a quantitative histochemical technique.

The kinetics of lactate dehydrogenase in situ were studied in sections of unfixed liver of the male mouse using a quantitative histochemical technique. The sections were incubated on substrate-containing gel films. The absorbance of the final reaction products deposited in a single hepatocyte was measured continuously during the incubation as a function of incubation time using a scanning microdensitometer. The absorbance increased non-linearly during the first minute of incubation, but linearly for at least the next 3 min afterwards. The initial velocity (vi) of the dehydrogenase was calculated from two equations proposed previously by us, vi = 2.82 degrees A and vi = vi + 2 degrees A, where vi and degrees A are, respectively, the gradient and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min. The dependence of vi on lactate concentration gave the following mean kinetic constants. For periportal hepatocytes, the apparent Km = 14 mM and Vmax = 80 mumoles hydrogen equivalents formed cm-3 hepatocyte cytoplasm min-1. For pericentral hepatocytes, Km = 12 mM and Vmax = 87 mumoles hydrogen equivalents cm-3 min-1. The Km values are very similar to those determined previously from biochemical assays. The concentrations of the enzyme in single hepatocytes calculated from the Vmax values are in good agreement with those obtained by another method. These data substantiate the validity of our equations.

Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system.

The initial reaction kinetics of succinate dehydrogenase in situ were investigated in sections of mouse unfixed liver using an ARGUS-100 image analyser system. The sections were incubated on substrate-containing agarose gel films. Images of a section, illuminated with monochromatic light (584 nm), were captured with the image analyser in real time at intervals of 10 s during the incubation. The absorbances of selected hepatocytes in the successive images were determined as a function of time. In every cell, the absorbance increased nonlinearly after the first minute of incubation. The initial velocity of the dehydrogenase was calculated from the linear activities during the first 20 s of incubation. Hanes plots of the initial velocities and succinate concentration yielded the following mean kinetic constants. For periportal hepatocytes, the apparent Km = 1.2 +/- 0.8 mM and Vmax = 29 +/- 2 mumol hydrogen equivalents formed/cm3 hepatocyte cytoplasm per min. For pericentral hepatocytes, Km = 1.4 +/- 1.0 mM and Vmax = 21 +/- 2 mumol hydrogen equivalents/cm3 per min. The Km values are very similar to those determined previously from biochemical assays. These results, and the observed dependence of the initial velocity on the enzyme concentration, suggest that the technique reported here is valid for the histochemical assay of succinate dehydrogenase.

Secretory pathway of vitellogenesis in the liver of the cockerel as revealed by immuno-gold and computer-assisted digitization techniques.

The protein A-gold immunocytochemical technique was used to localize the secretory pathway of oestradiol-induced vitellogenin in hepatic parenchymal cells of the cockerel. Liver was removed from experimental birds on the 1st, 4th and 8th day following oestradiol-treatment, and embedded in Lowicryl K4M resin cured at -20 degrees C. In selected electron micrographs the fractional surface area of each of the intracellular compartments was measured by the computer-assisted digitization technique. Labelling was detected over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the immature secretory vacuoles (ISV) including condensing vacuoles and the mature secretory vacuoles (MSV). Counts of the gold particles demonstrated an increasing concentration which progressed in the order RER less than Golgi less than ISV less than MSV and identified the secretory pathway of the protein. The highest density of labelling was obtained on the 4th day, when vitellogenin reaches its peak activity. Autophagic activity (or crinophagy) was also found in lysosomes and its labelling intensity increased daily. A hypothesis concerning the secretory pathway of non-stored proteins by the liver is discussed further.

Immunoelectron microscopical demonstration of egg-yolk plasma precursor vitellogenin in the hepatocyte of oestradiol-treated cockerels.

Vitellogenin has been localized at the electron microscopical level in the liver of the cockerel using a colloidal gold technique. White leghorn cockerels were treated with 17 beta-oestradiol to induce vitellogenesis. Pieces of liver were removed from control and experimental birds on the 4th and 8th days following hormone treatment, and embedded in Lowicryl K4M. Vitellogenin was isolated from the plasma of oestradiol-treated cockerels, and the antibody to it elicited in rabbits and made vitellogenin-specific by affinity chromatography on lipovitellin-Sepharose columns. At the light microscopical level, the intensity of immunohistochemical staining was considerably above background levels in oestradiol-treated cockerels. At the electron microscopical level, gold particles indicating antigenic sites of vitellogenin were largely confined to the rough endoplasmic reticulum, Golgi apparatus, immature and lysosomes and phagosomes within hepatocytes and sinusoidal cells respectively. These observations strongly suggest that the intracellular pathway of vitellogenin secretion in chicken hepatocytes under the experimental conditions studied involves external stimuli and secretory vacuoles. The labelling of lysosomes may reflect catabolic turnover (crinophagy).

In situ lactate dehydrogenase patterns as markers of tumour oxygenation.

The histochemical patterns of lactate dehydrogenase, LDH, are here proposed as indicators of the local levels of oxygenation of malignant tissue. This parameter has outstanding importance in determining the tumour aggressiveness and response to treatment. The tetrazolium salt reaction previously proposed for the mapping of hypoxia has been improved by the use of polyvinyl alcohol as a tissue stabilizer. The intracellular coloured products of this reaction appear in two distinct forms, diffuse and granular, which we previously postulated to be indicative of LDH isoenzymes soluble and bound, respectively. Solubility is promoted by H-LDH subunits preferentially synthesized under good oxygenation; binding to membranes is favoured by the presence of M-LDH subunits preferentially active under poor oxygeneration. A reversible shift between the two forms apparently regulates the cells' metabolic adaptation to different stress situations. We assume that the anoxic shock protein LDHk exists exclusively in the bound form. In the Ehrlich carcinoma model previously employed, we verify a drift towards the exclusive presence of the granular form as the section's depth increases and/or when the cuff width decreases. This trend is ascribed to a progressive worsening of the local oxygenation levels. At the tumour interface, a chronic inflammatory tissue (notoriously highly hypoxic) is characterized by a granular LDH activity. New models of hypoxia are proposed and discussed for explaining the patterns here described and observed also in other studies, namely those derived from hyperviscosaemia, damaged endothelia, fibrosis, anaemia, poor ventilation and impaired cardio-vascular system.

Stroma formation in Ehrlich carcinoma. I. Oedema phase. A mitosis burst as an index of physiological reoxygenation?

Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels leads to diffuse oedema. It is here reported that cell loosening and a remarkably high mitotic burst were observed in Ehrlich carcinoma in regions in contact with the exudate, particularly at the perinecrotic (hypoxic) region. This suggests both an enhanced cell detachment from the tumour parenchyma and an improvement of the microenvironment, the exudate thus appearing as beneficial to the malignant cells contributing to the reoxygenation of formerly hypoxic regions. The temporary and well-localized concentration of mitoses in inner tumour areas has perhaps been disregarded by the pathologists engaged in mitosis counting for tumour grading. Peripheric and intraparenchymal concentrations of mast cells, lipid pools and platelets were seen in apparently key geometric disposition for controlling fibrin deposition and angiogenesis. Hypoxia is known to cause resistance to oxygen-dependent treatments and to facilitate cell detachment; normal fibroblasts respond and survive under hypoxic conditions by exhibiting features of the malignant phenotype. During reoxygenation, gene instability, cellular heterogeneity and increased drug resistance and metastatic spread have been reported. A reoxygenation process can also be deduced from several other histochemical and morphological patterns observed in this study. The findings here reported thus suggest that the oedema phase is a crucial phase regulating growth, invasion and dissemination of tumour cell populations, that should be specifically addressed therapeutically.

Effects of therapeutic percutaneous electrical stimulation of atrophic human quadriceps on muscle composition, protein synthesis and contractile properties.

The effects of percutaneous electrical stimulation (70 V, 300 microseconds pulses at 30 Hz) on muscle composition and rate of protein synthesis were studied in seven patients with quadriceps atrophy secondary to unilateral osteoarthritis of the knee (stimulated group). Quadriceps were stimulated on the affected side for 1 h per day. The results were compared to those from seven patients who did not use a muscle stimulator (control group), in whom muscle biopsy at surgery provided evidence of wasting of tissue protein on the side of osteoarthritis (normal leg 608 +/- 266 micrograms protein micrograms-1 DNA, affected leg 256 +/- 100 micrograms protein micrograms-1 DNA, means +/- SD, P less than 0.05; type I fibre diameters: normal 53.2 +/- 6.7 microns, affected 43.8 +/- 4.0 microns, P less than 0.05). In patients who had received stimulation there was no residual difference between the legs in either muscle protein concentration (normal 411 +/- 168 micrograms protein micrograms-1 DNA, affected 373 +/- 112 micrograms protein micrograms-1 DNA) or fibre diameter (type I diameters: normal 56.1 +/- 7.8 microns, affected 58.0 +/- 10.7 microns). Stimulation did not influence the ratios of muscle force elicited by acute stimulation at 20 and 50 Hz (normal 75 +/- 15%, affected 79 +/- 15%), or rates of muscle relaxation (percentage losses of tetanic force 10 ms-1: normal 7.66 +/- 1.2%, affected 8.67 +/- 2.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous histochemical assay of two dehydrogenases in the same cell.

A new approach has been developed for the simultaneous assay of the activities of two enzymes (lactate and succinate dehydrogenases) in the same cell in sections of unfixed liver. The sections, mounted on coverslips, were placed on top of 0.6-mm thick 0.8% low gelling-temperature agarose films containing the substrates of both enzymes (70 mM lactate and 50 mM succinate, respectively) plus 80 mM Tris-HCl buffer (pH 7.5), 5 mM EDTA, 10 mM NaN3, 1.5 mM NAD+, 1.2 mM Nitro BT and 0.26 mM phenazine methosulphate. The integrated absorbance (A) at 585 nm of the final reaction product formazans deposited by the two enzymes in a selected hepatocyte was measured continuously at 37 degrees C as a function of incubation time, using a Vickers M85 microdensitometer. The intercept A0 on the A-axis of the linear regression line of A on time was determined. After a known incubation time t, the absorbance A1, was noted and the section placed on another gel film lacking the substrates in order to estimate the final reaction product either formed in the gel film or lost from the cell. The absorbance A2 of the hepatocyte was remeasured. The reaction velocities (activities) vL and vS of lactate and succinate dehydrogenases, respectively, were calculated from the following equations: vL = [(A1-A2) - A0(1- alpha L)]/(1-alpha L)t and vS = (A2-alpha LA1)/(1-alpha L)t where alpha L = A2/A1 for hepatocytes incubated on gel films containing only lactate as the substrate. This parameter was found to be virtually constant (0.44) over a wide range of vL.(ABSTRACT TRUNCATED AT 250 WORDS)