ARTICLE |
Correspondence to: Wouter H. Lamers, Dept. of Anatomy and Embryology, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
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Summary |
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We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (>96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 ± 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (14 mM) and intermediate and pericentral zones
13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to
15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of
5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to
8 mM. These glutamate distribution patterns were in agreement with those detected immunohisto-chemically. (J Histochem Cytochem 45:1217-1229, 1997)
Key Words: glutamate, glutamate dehydrogenase, glutamine synthetase, quantitative histochemistry, image analysis, metabolic zonation, liver lobule
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Introduction |
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In the past decades, several methods have been developed to determine distribution patterns of enzymes in tissues and cells and to quantify their local activity (for review see
The aim of the present study was the development of a simple method for the localization and quantification of endogenous substrate concentrations in different zones of the liver lobule in cryostat sections at the light microscopic level. Glutamate was chosen as a model substrate because it is present in the liver in relatively high concentrations (
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Materials and Methods |
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Principle of the Detection Method
The reactions used to measure endogenous glutamate concentrations in unfixed liver cryostat sections are as follows:
GDH (EC 1.4.1.2) converts glutamate into ammonia and -oxoglutarate and simultaneously reduces NAD+ to NADH. NADH reduces nitroblue tetrazolium salt (NBT) to its formazan via the electron carrier phenazine methosulfate (PMS). The histochemical procedure is described in detail by
Glutamate Measurements in Gelatin Model Sections
For development and validation of the method, gelatin model sections were used (
A sandwich was made as described by -oxoglutarate results in the release of two electrons, whereas four electrons are needed for complete reduction of NBT (
Incubations were performed at 37C in a moistened chamber for various time intervals to convert glutamate present in the first section. After incubation, sections were covered with a glycerin-gelatin mounting medium and a coverslip. Formazan production was measured immediately to avoid any nonspecific formazan production (
Measurements with the Vickers M85a cytophotometer were performed at 585 nm with a x6.3 planachromatic objective (NA 0.20), a band-width setting of 65, a mask with an effective diameter of 190 µm, and a scanning spot with an effective diameter of 3.2 µm. The Photometrics cooled CCD camera was used with a x2.5 objective (NA 0.075). Sections were illuminated with white light from a stabilized power supply and filtered with an infrared blocking filter (
For the assay of glutamate in tissue sections, the sandwich technique used for gelatin model sections was replaced by a method based on a polyvinyl alcohol (PVA)-containing incubation medium. The use of this liquid medium allows more rapid and uniform contact between tissue sections and incubation medium. Therefore, the validity of this method was tested by direct comparison with the sandwich technique using the gelatin model sections. Incubation medium for the glutamate concentration test reaction contained 10% PVA (weight average Mr 70,000-100,000; Sigma, St Louis, MO) in 0.1 M Tris-Hepes buffer (pH 8.0), 225 mM sodium chloride, 5 mM sodium azide, 5 mM EDTA, 2 mM ADP, 0.2 mM PMS, 4 mM NAD+, 5 mM NBT, and 150 U GDH per ml medium. The final pH was 8.0. To start the reaction, a metal spacer (0.5 mm) on a coverslip was filled with incubation medium and placed on top of the section. This ensured an even distribution of the medium over the entire section (
Glutamate Measurement in Rat Liver Cryostat Sections
Adult male rats with free access to food and water, adult male rats fasted for 24 hr, rat embryos at 3 days before birth, and rat neonates at 0 or 7 days after birth, all of the Wistar strain, were used for this study. Environmental temperature was kept at 21-22C and the relative humidity at 60%. Rats were exposed to a 12-hr light-dark cycle (light 0700-1900 hr). The liver was removed between 0900 and 1100 hr to avoid chronobiological variations. The rats were lightly anesthetized with CO2/O2 before decapitation, the abdominal cavity was opened, and the liver was removed within 30 sec. Fragments of the liver were placed in screw-capped aluminum vials (Sanbio; Uden, the Netherlands) and immediately frozen in liquid nitrogen. The frozen material was stored at -70C until further use. Serial sections (8 or 16 µm) were cut and sections were picked up individually onto clean glass slides and kept in the cryostat until further use. For each measurement, a set of three serial sections was used to perform the glutamate concentration test and control reaction and the GDH activity assay for topographical reference. PVA-containing media as described above were used for the test reaction to measure glutamate levels in sections. The glutamate-independent background formation of formazan (control reaction) was determined by including 30 mM -oxoglutarate (Sigma), one of the products of the GDH reaction, in the reaction medium to inhibit deamination of glutamate completely (
Localization of GDH activity was demonstrated using the medium described for the glutamate assay, except that GDH was omitted and 100 mM glutamate (pH 8.0) was added as substrate (
Immunohistochemical Localization of Glutamate in Liver Tissue Sections
Local differences in glutamate concentrations, as measured with the histochemical method, were validated using immunohistochemistry on cryostat and paraffin-embedded sections of livers. Serial cryostat sections were picked up individually onto clean glass slides and fixed immediately. Two types of chemical fixation were applied to the sections: glutaraldehyde 2.5% (w/v)/formaldehyde 1% (w/v) or methanol/acetone/water [40:40:20 (v/v/v)]. Fixation lasted for 15 min at room temperature (RT). The first fixative crosslinks glutamate to proteins, whereas the second fixative precipitates glutamate. After fixation, sections were rinsed briefly in PBS, pH 7.4. The immunohistochemical detection of glutamate was performed with either a commercially available polyclonal antibody against glutamate (1:1,000; Sigma) or a polyclonal antibody against glutamate that was kindly provided by Dr. Pow (1:200,000;
Glutamate Measurement in Isolated Hepatocytes
Total hepatocyte populations were isolated from male Wistar rats (200-250 g) fasted for 20-24 hr, as described by
Intracellular amino acid concentrations were measured after separation of the cells from the incubation mixture by centrifugation through silicone oil (AR 200:20=3:2; Wacker Chemie, Munich, Germany) into a layer of sulfosalicylic acid (10%, m/v). To minimize corrections for amino acids present in the extracellular fluid adhering to the cells, hepatocytes were diluted fivefold with ice-cold Krebs-Henseleit bicarbonate medium before their centrifugation through silicone oil. The protein-free extract of the cells was brought to pH 2.2 with 1 M LiOH and was used to measure intracellular concentrations of amino acids. Amino acid concentrations were measured with an LKB Alpha Plus amino acid analyzer, using a lithium citrate buffer system. Carbamoylphosphate synthetase activity was measured according to
Definition of the Liver Architecture
Different architectural subunits have been described in the past (for overview see
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Results |
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Glutamate Concentration Measurements in Model Sections
Absorbance measurements in gelatin model sections showed that the maximal difference between formazan precipitation in test and control reactions was reached after 20 min of incubation and that this difference remained constant thereafter (data not shown). Absorbance measurements in gelatin model sections of various thicknesses containing different concentrations of glutamate that was converted at three different pHs are depicted in Figure 2. The amount of formazan produced at each concentration of glutamate tested increased with increasing pH (Figure 2A), with the highest conversion seen at pH 8.0 (inset in Figure 2A). Incubations at higher pH were not tested because of spontaneous formation of precipitates in the incubation medium (
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Conversion of glutamate, as detected by formazan production at concentrations above 2 mM, was constant (60 ± 5%; Figure 3). On the basis of these results, it was concluded that the sensitivity of the method was 2 mM at pH 8.0 and that absorbance values had to be multiplied by a factor of 100/60 due to incomplete conversion of glutamate into -oxoglutarate and NH4+. Addition of 30 mM
-oxoglutarate, one of the reaction products, to either the gelatin-substrate section or the gelatin-enzyme section completely prevented formazan production (Figure 3). Addition of higher amounts of GDH to the gelatin medium (up to 500 U/ml) did not result in higher conversion rates than at 150 U/ml, the concentration of GDH that was routinely used.
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Data obtained with the sandwich technique and data obtained with incubations using PVA-containing media were very similar. Again, 60% recovery of gluta-mate in gelatin sections was obtained in the range of 2-20 mM glutamate (data not shown).
Measurement of Glutamate Concentrations in Rat Liver Sections
To prevent conversion of endogenous substrates other than glutamate from contributing to formazan production, enzymes in the tissue sections had to be inactivated without simultaneously changing the distribution of glutamate itself. In biochemical assays, enzymes are usually inactivated by acid precipitation or heat inactivation. Because heat inactivation for a period of 10 min at 100C is sufficient for complete inactivation of enzymes in homogenates [see
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Because these results indicated that it was not possible to inactivate endogenous enzymes in tissue sections completely, another approach was chosen to ensure the exclusive measurement of glutamate-dependent formazan production in cryostat tissue sections. Formazan production was strongly dependent on the addition of exogenous co-enzyme (NAD+) (compare Figure 5, bars 1 and 2). Apparently, the level of endogenous NAD+ was too low to drive the reaction. Addition of GDH to the incubation medium did not significantly increase the amount of formazan produced (Figure 5, bar 3). On the basis of these results, it was concluded that endogenous GDH was responsible for conversion of glutamate in cryostat sections of livers. Absorbance values were reduced to background levels when 30 mM -oxoglutarate was added to the incubation medium that contained NAD+ and GDH (Figure 5, bar 4; cf. Figure 3). This finding is in agreement with our previous observations that 30 mM
-oxoglutarate effectively inhibits GDH activity in liver (
-oxoglutarate was chosen as the proper control to estimate background (i.e. glutamate-independent) formazan production.
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Test and control sections were topographically matched by affine transformation (
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Because differences between test and control absorbance values per pixel were small, images of glutamate distribution patterns were too "noisy" to reliably calculate cellular glutamate concentrations. To obtain reliable glutamate concentrations in periportal and pericentral hepatocytes, additional approaches were taken. In the first approach, 10 periportal and 10 pericentral zones were selected in corresponding test and control sections, each zone being 100 pixels (= 670 µm2) in size. Glutamate concentrations in periportal and pericentral zones were calculated by subtracting absorbance values of test and control sections. This approach confirmed that glutamate concentrations were higher periportally than pericentrally in fed adult male rats (Figure 8A). In the second approach, glutamate concentrations were estimated in zones of low, intermediate, and high GDH activity representing periportal, intermediate, and pericentral zones of the liver lobule, respectively (cf. Figure 1). Glutamate concentrations in zones of low, intermediate, and high GDH activity were estimated by subtracting the summed absorbance values of the corresponding zones in test and control reactions and dividing these values by the number of pixels overlying each of these zones. The results of this approach are shown in Figure 8B and Figure 9B, Figure 9D, Figure 9F, and Figure 9H). The findings obtained with both approaches were comparable, except that the second procedure also yielded information on glutamate concentrations in intermediate zones that could not be delineated properly in the first approach.
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In fed adult male rat livers, periportal zones contained highest concentrations of glutamate (14 mM). The values in pericentral zones were 40% lower (9 mM; Figure 8 and Figure 9). In livers of fasted adult male rats, glutamate concentrations in periportal and intermediate zones were in the same range as those found in fed rats, whereas glutamate concentrations in pericentral zones increased to 13 mM (Figure 8 and Figure 9).
We elaborated the second approach further because the immunohistochemical results showed higher gluta-mate concentrations not only in periportal zones but also in a small rim of hepatocytes around central veins (Figure 7B). To measure the concentration of gluta-mate in this small rim (Figure 1 and Figure 7B), the density slice of the NIH image program was used to select the upper 10% of absorbance values in pericentral zones. This selection indeed corresponded to the small rim that expresses the glutamate-consuming enzyme GS. The rim of hepatocytes contained higher glutamate concentrations than the adjacent pericentral hepatocytes (11 and 15 mM in fed and fasted rats, respectively; Figure 8B), confirming the immunohistochemical distribution pattern of glutamate.
In perinatal rats, hepatic glutamate concentrations were homogeneously distributed. In 19-day-old fetal (Figure 9G) and in 0-day-old neonatal rats, GDH activity was also homogeneously distributed in liver. Hepatic glutamate concentrations were 5 mM in these livers (Figure 8A and Figure 9H). In 7-day-old neonatal rats, a clear distinction between pericentral and periportal zones could be made on the basis of the distribution pattern of GDH activity (Figure 9E), but glutamate concentrations in these livers were found to be homogeneously distributed (7 mM; Figure 8 and Figure 9F).
Measurement of Glutamate Concentrations in Isolated Hepatocytes
Isolated periportal and pericentral hepatocytes of fasted adult male rats were incubated in a mixture of amino acids as is present in the portal blood of fed animals and were used to estimate zonal differences in cellular glutamate biochemically. Periportal hepatocytes were enriched in CPS, whereas pericentral hepatocytes were enriched in GS, as expected (Table 1) (
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Discussion |
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Our study shows that cellular substrate concentrations can be determined in cryostat sections with a quantitative enzyme histochemical method using a colorimetric visualization technique. Glutamate was chosen as the test substrate because it is present in fairly high concentrations and shows a heterogeneous distribution pattern in rat liver (
Evaluation of the Enzyme Histochemical Method
An important issue to be considered is that it takes time to freeze tissues in such a way that they can be analyzed histochemically. We have tried to evaluate the effects of this delay by analyzing glutamate concentrations in isolated periportal and pericentral hepatocytes, i.e., by separating these two populations of hepatocytes before freezing rather than the other way around as is done in histochemical assays. The cells were exposed to a mixture of amino acids as is found in the portal vein after feeding (
The major methodological drawback of the histochemical method to measure local substrate concentrations encountered thus far appears to be the failure to inactivate enzymes in cryostat sections without causing redistribution of substrates (Figure 4). Water in such sections evaporates very rapidly, and the resulting desiccated proteins prove to be extremely stable even at high temperatures. Some reported substrate measurement procedures on cryostat sections rely solely on heat inactivation at 100C for 10 min to inactivate cellular enzymes (
In the present study we were able to obviate these problems by using -oxoglutarate and a favorable equilibrium of GDH reactants to assess glutamate-independent NADH production and hence to specifically determine tissue glutamate-dependent formazan production (
10-fold more sensitive (2 mM vs 200 µM) (Mueller-Klieser and Walanta 1993). Therefore, our method is well suited for substrate measurements provided that concentrations in the millimolar range are present.
An unanticipated finding was that GDH added to the incubation medium, even at high concentrations, did not contribute to glutamate-dependent formazan production in the tissue sections (Figure 5). This finding was not expected because glutamate and GDH interacted without apparent diffusion problems in the gelatin model sections (Figure 2 and Figure 3). Unimpeded diffusion was also found for glucose-6-phosphate and glucose-6-phosphate dehydrogenase in gelatin model sections (
The fact that glutamate is firmly bound to tissue components is probably the reason why spatial resolution in tissue sections enables discrimination of the single GS-positive hepatocyte layer around central veins of approximately 40 µm in diameter (Figure 8 and Figure 9). In gelatin model sections, lateral diffusion was rather significant.
The glutamate measurements on gelatin-model sections revealed a constant recovery of glutamate at concentrations higher than 2 mM in 8-µm-thick gelatin sections. The relatively low conversion of approximately 60% is most likely due to the catalysis by GDH of an equilibrium reaction which is strongly in favour of glutamate synthesis (
Variations of the concentrations of exogenous NAD+, PMS, and NBT did not result in a higher recovery. This indicates that the ratio NADH/NAD+ was constant at different glutamate concentrations, most likely because the reducing equivalents of NADH were effectively captured by the PMS-NBT system (-OG2- or NH4+. This conclusion implies that the degree of conversion depends largely on the equilibrium of the reaction under investigation. In conventional assays, glutamate is always determined at high pH (pH 9) to decrease the [NH4+]/[NH3] ratio and in the presence of hydrazine to trap
-oxoglutarate (
The strong pH dependency of the reaction (Figure 2) is based on at least two factors. Activation of the dehydrogenase reaction by the allosteric effector ADP occurs at alkaline pH (
The advantage of the enzyme histochemical method in combination with image analysis was that interesting regions could be selected in recorded images and that measurements could be performed afterwards. Furthermore, different image processing approaches could be tested on the same tissue sample, allowing comparison of the results of the different methods applied (Figure 8). The method of affine transformation and subsequent subtraction of the control reactions from the test reactions (
Biological Relevance of Histochemical Measurement of Substrate Concentrations
The major advantage of biochemical methods to analyze tissues is that they are based on established and validated techniques, whereas their major drawback is the lack of spatial resolution. Virtually the opposite is true for histochemical methods. From this perspective, biochemical and histochemical methods should therefore be used as complementary rather than as alternative methods. Biochemical measurements of glutamate in purified periportal hepatocytes are in good agreement with the enzyme histochemical and immunohistochemical data of intact liver tissue (cf. Figure 7 and Figure 8A; Table 1). On the other hand, a clear discrepancy between biochemical and enzyme histochemical measurements of glutamate was found in pericentral hepatocytes, particularly in the hepatocytes immediately surrounding the central vein. This rim of cells with a relatively high glutamate concentration was not identified in the isolated pericentral cell population (Table 1) or by the standard histochemical spot assays (Figure 8A) but is clearly identified by the approach presented in Figure 8B and delineated at the cellular level by the immunohistochemical method (Figure 7B). Apparently, the cellular environment of these hepatocytes in vivo differs substantially from that present in the biochemical assay. It has been known for more than 10 years that this rim of cells contains a very high concentration of GS (
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Acknowledgments |
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We are grateful to Drs D.V. Pow and D.K. Crook (Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia) for kindly providing the antibody against glutamate, to Dr W.M. Frederiks for fruitful discussions, to Mr C. Gravemeyer and Mr C. Hersbach for excellent (computer) photographic support, and to Ms T.M.S. Pierik for careful preparation of the manuscript.
Received for publication September 25, 1996; accepted March 19, 1997.
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