ARTICLE |
Correspondence to: Yoshiko Nakae, Dept. of Oral Anatomy 1, Tokushima Univ. School of Dentistry, 3 Kuramoto-cho, Tokushima 770, Japan.
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Summary |
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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 = a°A, where vi is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and °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 °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. (J Histochem Cytochem 45:1417-1425, 1997)
Key Words: lactate dehydrogenase, enzyme kinetics, quantitative histochemistry, image analysis, skeletal muscle, liver, intracellular interactions
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Introduction |
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Lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the so-called "soluble" glycolytic enzymes present in the cytoplasm of cells. Previously we reported that, in histochemical assays of LDH based on the reduction of the tetrazolium salt Nitro BT, the absorbances of the final reaction products deposited in single cells in unfixed sections of various types of tissue increase nonlinearly during the first minute of incubation and then linearly for at least 2 min thereafter (
The purpose of the present study was to obtain evidence to support our hypothesis that the parameter a is a constant related to the diffusion of LDH out of tissue sections and is a measure of the interactions of the enzyme with intracellular components. We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on the constants a for the LDH reactions in situ in hepatocytes and skeletal muscle fibers. Both these protectants have been explicitly assumed in the past to prevent or limit the diffusion of soluble proteins out of tissue sections (
Mouse liver and skeletal muscle both contain predominantly muscle-type (M) isozymes (e.g.,
The initial nonlinearity of the LDH reaction in situ may also be due to inhibition of LDH by pyruvate, a product of lactate oxidation (
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Materials and Methods |
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Tissues
Five adult male ddY mice were sacrificed by cervical dislocation at approximately the same time of day (0945 hr) in January and February. Their gastrocnemius muscles and livers were removed. The livers were cut into pieces no larger than 5 mm3. These pieces and whole gastrocnemius muscles were embedded in Tissue-Tek II OCT compound (Miles Laboratories; Naperville, IL) and immediately quenched in liquid nitrogen. Serial sections of the tissues 4-14 µm thick were cut at -20C at a constant speed in a Bright cryostat (Cambridge, UK) to ensure constancy of intersection thickness (
Model Gel Sections Containing Purified LDH
Purified LDH isolated from rabbit skeletal muscle (Boehringer Mannheim; Mannheim, Germany) was mixed with the ingredients for preparing polyacrylamide gels. The final composition of the mixture was 0.855% LDH, 12% (w/v) acrylamide monomer (Nacalai Tesque) recrystallized from ethanol, 0.6% (w/v) N,N'-methylenebisacrylamide (Nacalai Tesque), 0.188% ammonium peroxodisulfate (Nacalai Tesque) (dissolved just before use), and 75 mM Tris-HCl buffer, pH 7.5. A drop of N,N,N',N'-tetramethylethylenediamine (Nacalai Tesque) was added to the mixture to polymerize it. The mixture was kept at 4C in glass tubes of 5-mm diameter for 5 hr until polymerization was complete. The gel columns were cut in 5-mm-long pieces. The pieces were rinsed at 4C in three changes of 80 mM Tris-HCl buffer, pH 7.5, over 30 min. The buffer solution was removed from the gel surface with filter paper and the gel pieces were embedded in Tissue-Tek II OCT compound and quenched in liquid nitrogen. Serial sections 4-10 µm thick were cut at constant speed and mounted on coverslips in the same way as for tissue sections.
Assay Media for LDH
Three kinds of incubation media were used for the histochemical assays: aqueous substrate media with and without 18% (w/v) PVA (hot-water soluble, weight-average molecular weight 70,000-100,000; Sigma) (
Continuous Monitoring of LDH Activity
The time courses of the LDH reaction in tissue and polyacrylamide model sections were monitored with a real-time ARGUS-100 image analysis system (Hamamatsu Photonics; Hamamatsu, Japan) calibrated as described previously (1/2 = 2.5 nm; Vacuum Optics; Tokyo, Japan) was placed in the light path of the microscope. This wavelength is the isobestic point of the principal final reaction products produced by the reduction of Nitro BT, a red monoformazan and a blue diformazan (
The enzyme reaction was started on the stage of the microscope either by placing a gel substrate film attached to a coverslip on a tissue or model section mounted on a coverslip, or by pouring aqueous substrate medium, with or without 18% PVA, into the space between two coverslips separated by 0.7-0.9 mm, one of which had a section mounted on the under surface. Previously the temperature of the microscope stage and the media had been warmed to and stabilized at 37 ± 0.2C. The images of the section, viewed with a x 40 objective, were captured at 10-sec intervals in real time continuously from the start of incubation for 4.5 min. The 28 images obtained were integrated 16 times before storage. Each frame required 1/30 sec for capture. The absorbances of the final reaction products deposited in a circular or elliptical delimited area (17-2835 pixels) of the sarcoplasm of single skeletal muscle fibers in cross-sections of the lateral head of gastrocnemius, in a circular delimited area (45 pixels) of the cytoplasm of single periportal hepatocytes located 40-80 µm from interlobular veins, and in a circular delimited area (2617 pixels) of the model sections containing 0.855% LDH were determined from the stored images as a function of incubation time. A pixel corresponded to 0.25 µm2. The background absorbances of the tissue and model sections mounted on coverslips (background controls) were determined on gel films containing 0.8% agarose and 80 mM Tris-HCl buffer, pH 7.5 (37C). The absorbances of test samples were corrected for the background control and the background image of each incubation medium (shading correction). The background absorbance controls for mouse periportal hepatocytes in 4-, 7-, and 14-µm-thick sections and gastrocnemius muscle fibers in 4-µm-thick sections were taken as 0.0238, 0.0334, 0.0440, and 0.0149 respectively as determined previously (
The data obtained are plotted in Figure 5 Figure 6 Figure 7. Each experimental point in Figure 5 was the mean of three measurements in the same section, and in Figure 6 and Figure 7, of 10 measurements.
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Initial Reaction Velocity
The observed initial velocities (vi) of LDH reactions in which the activity (absorbance) increase was linear during the first 3 min (Figure 2a and Figure 2b) were determined from the gradients of absorbance-incubation time plots over this time. The observed vi of LDH reactions in which the activity increased nonlinearly during the first minute of incubation (Figure 2c, Figure 3, and Figure 4) were calculated from the absorbance increases between 0 and 10 sec (
Kinetic Parameter a
The parameter a (see Introduction) was calculated from plots of (vi - v) against °A as described elsewhere (
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Results |
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Localization of the LDH Activity
Formazan final reaction products were deposited in the cytoplasm of hepatocytes and skeletal muscle fibers in sections incubated by any of the three histochemical methods described (Figure 1). Three types of fibers, small (Type I), intermediate (Type IIA), and large (Type IIB) fibers (
Time Course of the LDH Reaction
The absorbances of the formazan final reaction products deposited in model acrylamide gel sections containing 0.855% LDH increased linearly during at least the first 3 min of incubation both on the agarose gel films and in the PVA media (Figure 2a and Figure 2b). In contrast, the LDH reaction in model gel sections incubated in aqueous media was nonlinear during the first minute of incubation but was linear for at least 2 min thereafter (the correlation coefficient r = 0.99-1.00, p<0.001, n = 13) (Figure 2c). The activity of pure LDH in the model sections without lactate ("nothing dehydrogenase" activity) was almost zero (Figure 2a-c). Figure 3 shows the comparative time courses of the LDH reaction in mouse periportal hepatocytes in the sections incubated with the three assay media. They were all nonlinear during the first minute of incubation but linear in the next 2 min of incubation. Similar results were obtained for the LDH reaction in the three types of muscle fibers in the gastrocnemius (shown for small fibers in Figure 4). Even the intrinsic or corrected activity curves for LDH, obtained by subtracting the "nothing dehydrogenase" activity at each incubation time point from the corresponding LDH activity, showed similar nonlinear and linear phases (Figure 2 Figure 3 Figure 4). The LDH reaction in periportal hepatocytes and skeletal muscle fibers in the sections incubated with aqueous assay media containing 70 mM L-lactate and 1-100 mM pyruvate showed similar activity curves, i.e., nonlinear and subsequent linear phases, to those in the absence of pyruvate (Figure 3c and Figure 4c), although the levels of the LDH activities were considerably reduced by pyruvate.
Plots of (vi - v) on °A for Determining a
The steady-state velocities v and intercepts °A for the nonlinear LDH reaction were determined from, respectively, the gradients and intercepts of the linear regression best-fits (r = 0.99-1.00, p<0.001, n = 13) for the plots of A584 of the final reaction products on incubation time for times between 1 and 3 min. A typical plot is shown in Figure 2c. Plots of (vi - v) on °A for all nonlinear uncorrected LDH and "nothing dehydrogenase" reactions were approximately linear. Their linear regression lines, of gradients a, passed through or near the origin and their correlation coefficients were highly significant (Figure 5 Figure 6 Figure 7). The plots of (vi - v) on °A for the LDH reaction in periportal hepatocytes in the sections incubated in aqueous substrate media in the presence and absence of pyruvate approximated to the same straight line (Figure 6). A similar result was obtained for three types of skeletal muscle fibers in gastrocnemius muscle. The a value for the linear reaction was zero because the initial velocity was equal to v. Table 1 shows the a values determined by the three assay methods for LDH in model and tissue sections. They were in the ranges 0-1.29, 1.38-2.04, and 1.16-1.50, respectively, for purified LDH in model acrylamide gel sections, LDH in periportal hepatocytes, and LDH in gastrocnemius muscle fibers.
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vi Calculated from v, °A, and a
The initial velocities vi of the nonlinear LDH and "nothing dehydrogenase" reactions were calculated from the equation vi = v + a°A using the a values shown in Table 1 and v and °A values measured more precisely than the observed vi values, which included some errors in starting time of incubation and out-focus of the image at zero incubation time. Previously we found that this gave a more consistent measurement of vi than either curve fitting or tangent fitting at t = 0 of the absorbance-time plot (
The intrinsic LDH velocities (Table 1) were calculated by subtracting the initial velocities of "nothing dehydrogenase" activity from those of uncorrected LDH activity determined at a saturated lactate concentration (70 mM). For all the specimens investigated, the intrinsic LDH velocities determined on the substrate gel films and in the aqueous assay media were similar but were 1.8-3.1 times higher than those determined with PVA media.
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Discussion |
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The constants a in the equation vi - v = a°A that have been determined previously from the linear regression plots of observed vi minus v on °A for the LDH reaction in skeletal muscle fibers, hepatocytes, and cardiac muscle fibers in the sections incubated on gel substrate films were respectively 1.2, 2.2, and 1.5 (
The highest a value (2.0; Table 1) for LDH in periportal hepatocytes in sections incubated on substrate gel films or in aqueous assay media suggests that most of the enzyme in hepatocytes is present in a soluble form in the cytoplasm. However, diffusion of the enzyme out of liver sections can be limited by including 18% PVA in the incubation medium, causing the a value to be lower (1.38). In contrast, the reaction catalyzed by LDH incorporated into polyacrylamide gel sections incubated either on agarose gel films or in PVA media was linear for at least first 3 min of incubation and the a value was zero. This indicates that little or no diffusion of the enzyme out of the sections occurs during that time. In aqueous assay media, on the other hand, pure LDH does diffuse out of polyacrylamide sections (a = 1.29). The a values for the enzyme in three types of skeletal muscle fibers showed similar variations to those for pure LDH incorporated into polyacrylamide gel sections, although the muscle values are higher as a whole than those for the latter (Table 1). Moreover, the a values for skeletal muscle fibers determined with three assay media were lower than those for periportal hepatocytes. These data suggest that PVA is a more effective tissue protectant than agarose gel for LDH in hepatocytes but that both are equally effective for LDH in skeletal muscle fibers. They also indicate that more LDH diffuses from hepatocytes than from muscle fibers and that LDH in skeletal muscle fibers may be restricted or shielded by interactions with structural components or macromolecules in the sarcoplasm.
Several groups (e.g.,
The initial velocities calculated from the equation vi = v + a°A for 0.855% purified LDH in the model sections and LDH in periportal hepatocytes and skeletal muscle fibers in the presence of 18% PVA were found in our study to be respectively 33%, 44-54%, and 37-48% of those in the absence of PVA. This may be explained by the reduction in the catalytic center activity kcat of LDH by the high viscosity of the PVA media. A less probable explanation is the inhibition of the activities by PVA itself because the Michaelis constants of LDH (against L-lactate) in the model and tissue sections are scarcely affected by the presence of 18% PVA (
The activities (velocities) of LDH in situ in the presence of PVA have been reported previously as 7.5 µmoles pyruvate formed/cm3/min for mouse skeletal muscle (
In conclusion, we have found that the constant a in the equation vi = v + a°A is a useful indicator for revealing differences in the microenvironments of LDH in various types of cell. Further investigations are necessary, however, to identify which cytoskeletal protein is responsible for the diverse a values of LDH within cells.
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Acknowledgments |
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We thank Professor Dr C.J.F. Van Noorden, University of Amsterdam, for his advice concerning grades of PVA.
Received for publication April 3, 1997; accepted April 22, 1997.
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