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Lobular distribution pattern of lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver
acta histochem. 102, 37±47 (2000) Ó Urban & Fischer Verlag
Lobular distribution pattern of lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver Benjamin Schmidt1, Marco Vogelsang1, Imme Haubitz2, and Reinhard Hildebrand1 1
Institut fuÈr Anatomie, UniversitaÈt MuÈnster, Vesaliusweg 2±4, 48149 MuÈnster, Germany, 2 Rechenzentrum der UniversitaÈt WuÈrzburg, Am Hubland, 97074 WuÈrzburg, Germany
Received 7 May 1999 and in revised form 24 September 1999; accepted 27 September 1999
Summary Lactate dehydrogenase (LDH) and 6-phosphogluconate dehydrogenase (6PGDH) activities were measured in lobular areas expanding between 3 portal tracts and an efferent central vein in the livers of male Wistar rats, using a Lowry technique. The maximum of LDH activity was found in a nearly uniform broad area in the lobular periphery. From that area values decreased along periportal/septal ® perivenous gradients, but only slightly within that area along the periportal ® septal axis of the vascular septum. Maximum values of 6-PGDH activity were present in an intermediate area close to the central vein demonstrating a rather inhomogeneous distribution pattern without a clear definition of zonal limits. Our data on the distribution pattern of LDH are in agreement with the concept of the metabolic lobulus and are supported by a recent evaluation of the vascular architecture in rat liver. The lobular distribution pattern of 6-PGDH cannot be interpreted without doubt in accordance with that concept. Key words: lactate dehydrogenase ± 6-phosphogluconate dehydrogenase ± rat liver ± cellular heterogeneity ± metabolic lobulus
Correspondence to: R. Hildebrand, phone: + 49 25 18 35 52 00, fax: + 49 25 18 35 52 41, e-mail: [email protected] http://www.urbanfischer.de/journals/actahist
0065±1281/00/102/1±037 $ 12.00/0
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Introduction According to the widely accepted concept of metabolic zonation, the morphological, biochemical, and functional heterogeneity that is displayed by hepatocytes is related to zonal distribution patterns of many components in liver parenchyma with either predominantly periportal or perivenous localization (Jungermann and Katz, 1989; Gebhardt, 1992; Jungermann, 1995). However, a satisfactory attribution of this zonal heterogeneity to a simple morphofunctional unit of the liver or even the definition of zonal limits (Teutsch et al., 1992) seems to be an elusive goal (Lamers et al., 1989 a, b). Enzymatic studies along the porto-central axis and sinusoidal profiles of enzyme activities starting at the periphery of a classical lobule in the region between two adjoining septal branches and ending at central veins (Matsumoto et al., 1979; Matsumoto and Kawakami, 1982) provided a new startingpoint for the understanding of the specific parenchymal organization of the liver (Teutsch, 1984, 1985; Ebert et al., 1987; GoÈrgens et al., 1988; Teutsch, 1988; Wack and Haubitz, 1989; Teutsch et al., 1992). Matsumoto's concept of primary lobules with their sickle-shaped high potential pool seemed to be more appropriate to explain the data than Rappaport's acinar concept. According to a recent 3-dimensional reconstruction of parenchymal units, the latter concept cannot be applied to rat liver (Teutsch et al., 1999). Except for individual approaches of total pattern analysis in hepatic lobules (Hildebrand and Schleicher, 1986; Teutsch, 1986, 1988; Teutsch and Chilko, 1986) from a functional point of view, evidence was lacking for any interpretation (Lamers et al., 1989 a, b; Gebhardt, 1992), because data are not available of enzymatic distribution patterns in areas between the sinusoidal paths already analysed. Therefore, the aim of the present microquantitative investigation was to provide such data (1) for LDH which is predominantly located in hepatocytes surrounding small portal tracts like key enzymes of gluconeogenesis (Hildebrand and Fuchs, 1984; Ebert et al., 1987) and (2) for 6-PGDH which is predominantly present perivenously (Hildebrand, 1980; Rieder et al., 1978; Rieder, 1981), in intermediate areas (Bhattacharya et al., 1986, 1987), or in pericentral and intermediate areas (Jonges and Van Noorden, 1989; Jonges et al., 1995), respectively.
Material and methods Animals and tissue sampling. Seven male albino rats (84 days post-partum) of the Wistar strain (Wistar TNO; Harlan Winkelmann GmbH, Borchen, Germany) were housed at 27 ± 1 °C and 45% relative humidity in the animal quarters of the Department of Anatomy, University of MuÈnster. The animals were subjected to an artificial light-dark cycle (dark period 19.00 h±7.00 h) and were maintained on Altromin R and tap water ad libitum. The rats were sacrificed between 11.00 and 11.30 h on two subsequent days under ether anesthesia (time of anesthesia 30 s). After opening of the abdominal cavity, a piece of the median lobe of the livers was dissected with scissors and placed on a metal holder. Then,
Lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver
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the material was immediately immersed in liquid nitrogen-cooled n-pentane. After the freezing process, the tissue block was stored in airtight tubes at ±80 °C. Preparation of tissue samples. Twenty lm thick cryostat sections of the livers were cut at a cabinet temp of ±25 °C and an object temp of ±21 °C (Frigocut 2800 E; Reichert-Jung, Nuûloch, Germany). The sections were subsequently lyophilized under vacuum at 0.785 Pa and ±40 °C for approx 48 h (Lyovac GT 2; Leybold-Heraeus, KoÈln, Germany). For the recognition of hepatocellular heterogeneity the activities of glucose-6-phosphatase were demonstrated histochemically (Maly and Sasse, 1983) in 10 lm thick parallel sections which served as guiding maps. From the lyophilized sections of each rat, 3 quadrangular areas of liver tissue each with 3 small portal tracts and 1 central vein at the corners were dissected free-hand using razor blades under a stereomicroscope at a magnification 50 ´. Only lobules with a typical zonal distribution pattern of glucose-6-phosphatase and an arrangement of portal tracts and central vein as demonstrated in Fig. 1 were selected. The dissected areas were subdivided into 4±6 adjacent strips (Fig. 1). The average length and width of the strips were for the LDH assay (1) 423.0 ± 48.0 lm and 73.4 ± 10.1 lm, respectively, and for the 6-PGDH assay (2) 472.0 ± 76.4 lm and 89.1 ± 15.3 lm, respectively. Subsequently, these strips were further subdivided into 5±10 successive samples of 30±100 lm in the axis of the strips for (1) or 40±100 lm for (2), respectively. Measurements were performed using a calibrated eyepiece micrometer. The dissection steps were documented on
Fig. 1. Histochemical demonstration of glucose-6-phosphatase in male rat liver with a schematic drawing of an excised and microdissected lobular area. PT, portal tract; CV, central vein; SEPT, septal area where blood from portal venules of neighbouring portal tracts meet.
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photographs taken from the dissected areas. Standardization of the assays of enzyme activities in quadrangular samples of similar size is necessary. On the one hand, excision of residues of vessels and connective tissue attached to the samples at the corners of the strips causes great variety in size, but on the other hand, vessels and connective tissue cause variation in measurements. Because the general distribution pattern of enzyme activities was of primary interest, samples with segments of portal tracts and central veins were included in our measurements and calculations despite the fact that measurements at the 4 corners of the strips are affected. The tissue samples were weighed using a quartz fiber fishpole balance where one unit equalled for (1) with 17.45 ng and for (2) with 20.63 ng. The recorded weight of the samples was reduced by 2% to allow for the uptake of N2 and O2, and a further 1% to allow for the uptake of H2O for every 10% of the known relative humidity (Teutsch, 1981). After weighing, the tissue samples were immediately dryloaded into the holes of microtest racks (Greiner, NuÈrtingen, Germany), which served as incubation vessels. Microbiochemical processing. The microbiochemical procedure as well as the preparation of tissue samples was based on the method developed by Lowry and collaborators (Lowry et al., no date; Lowry and Passoneau, 1972). Enzymatic assay for LDH. pyruvate + NADH2
LDH
! lactate + NAD+
Reagents: Tris buffer, 0.1 mol/l, pH 7.6; pyruvate, 2 mmol/l; nicotinamide, 20 mmol/l; NADH, 2 mmol/l; BSA, 0.06% (modified after Morrison et al., 1965; Hildebrand, 1983). The assays were performed in waterbaths at 30 °C for 30 min. The incubation volume was 7.043 ll. The enzymatic reaction was stopped by cooling the rack on crushed ice. Excess of NADH was destroyed by 21.337 ll 0.5 N HCl at 60 °C for 15 min. The reaction was stopped on crushed ice. For the determination of enzyme activity, the fluorescence of NAD+ destroyed in strong alkali was measured. 20.258 ll from the incubation vessels were transferred into fluorotubes containing 204.000 ll 6 N NaOH. After mixing, the tubes were incubated at 60 °C for 25 min. Subsequently, the samples were cooled down to room temp and 1 ml of distilled water was added. Fluorometric measurements were performed with a Farrand Ratio Fluorometer-2 (Farrand Optical, New York NY, USA). In all assays, blanks and a duplicate series of NAD+ standards (46.28 ´ 10±6 mol/l ± 265.73 ´ 10±6 mol/l) were incorporated. Tissue blanks were within the same range as reagent blanks. Before measurements were performed, blanks were adjusted to zero. The correct adjustment was repeatedly proven during measurements. Enzyme activity was expressed of moles substrate/kg dry weight/h. The reactions were linear in the range of dry weights 5±80 ng and time up to 60 min (for periportal regions, correlation coefficient, r = 0.99; for perivenous regions, r = 0.998). Materials. Boehringer (Mannheim, Germany): NAD+, grade 1, free acid; NADH, grade 1, disodium salt; pyruvate, monosodium salt; Serva (Heidelberg, Germany): BSA, crystallized research grade, > 99%; nicotinamide, pharm.; Merck (Darmstadt, Germany): Tris (hydroxymethyl)aminomethane, pro analysi. Enzymatic assay for 6-PGDH. 6-phosphogluconate + NADP+
6-PGDH
! ribulose-5-phosphate + CO2 + NADPH2
Reagents: Tris buffer, 0.1 mol/l, pH 8.4; 6-phosphogluconate, 2 mmol/l; NADP+, 1 mmol/l; EDTA, 0.5 mmol/l; BSA, 0.03% (modified after Burch et al., 1963; Hildebrand, 1984). The assays were performed in waterbaths at 30 °C for 60 min. The incubation volume was 7.622 ll. The enzymatic reaction was stopped by cooling the rack on crushed ice. Excess of NADP+ was destroyed by 23.223 ll Na2CO3, pH 12, at 60 °C for 15 min. The reac-
Lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver
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tion was stopped on crushed ice. For the determination of enzyme activity, fluorescence of NADPH destroyed in strong alkali was measured. 13.814 ll from the incubation vessels were transferred into fluorotubes containing 175.000 ll 6 N NaOH and 0.01% H2O2. After mixing, the tubes were incubated at 60 °C for 25 min. Subsequently, the samples were cooled down to room temp and 1 ml of distilled water was added. Fluorometric measurements were performed with a Farrand Ratio Fluorometer-2. In all assays blanks and a duplicate series of NADPH standards (42.09 ´ 10±6 mol/l ± 377.84 ´ 10±6 mol/l) were incorporated. Tissue blanks were within the same range as reagent blanks. Before measurements were performed, blanks were adjusted to zero. The correct adjustment was repeatedly proven during measurements. Enzyme activity was expressed of moles substrate/kg dry weight/h. The reactions were linear in the range of dry weights 30±111 ng and time up to 90 min (for periportal regions, correlation coefficient, r = 0.861; for perivenous regions, r = 0.883). Materials. Boehringer: 6-phosphogluconate, crystallized trisodium salt; NADP+, 99% disodium salt; Merck: H2O2, Perhydrol 30%; Na2CO3; Tris (hydroxymethyl)aminomethane, pro analysi; Serva: BSA, crystallized research grade, > 99%; Sigma (St. Louis MO, USA): EDTA, 99%. Evaluation of the data. Distribution patterns of enzyme activities in the areas studied were presented as 3-dimensional mapping as Postscript-graph by a modified graphic programme according to Haubitz (1977). Due to the great variability in the excised quadrangular areas as well as in the number, length and width of the samples, all excised areas were transformed to standard squares. These squares were divided with a grid of 200 scales at both x-axis and y-axis per edge resulting in 40,000 points of intersection. At each point, enzyme activity was established. First, the mean values of enzyme activity of corresponding samples in 3 areas investigated were calculated for each animal. In this step of the calculation procedure, missing values due to missing samples automatically disappeared. Subsequently, the means of the means of 7 animals were calculated and finally 3-dimensional representations of the activities of both enzymes were computed. The coordinates on the xaxis and y-axis determined each point in the square with its relevant enzyme activity expressed on the z-axis. The values in the images represent mean enzyme activity for all animals. Shading in the graph is generated by a virtual light source from the left.
Results LDH. The gradient of LDH activity decreased along the entire path between portal tract and central vein (Fig. 2). The slight increase of activity outside the portal tracts is partly due to lower LDH activity in samples containing segments of portal vessels and connective tissue. Distribution patterns between joined septal branches and the central vein displayed the same characteristics (sept ® pv) except for generally lower activity and a missing intial increase. Due to the lower activity here 2 furrows directed towards the perivenous area became apparent with a depth that was less distinct in the vicinity of the central vein. As expected, highest activity was found in the lobular periphery and lowest around central veins. However, in the periphery of lobules, an almost uniform wide area with high periportal and septal activity was found with slight decrease of activity along the pp ® sept axis. The residual standard deviation (Armitage, 1971) over all 7 rats was ± 15.7 (moles/kg dry weight/h).
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Fig. 2. Three dimensional demonstration of lactate dehydrogenase activity in a standardized lobular area of rat liver parenchyma expanding between 3 portal tracts and a central vein. PT, portal tract with adjacent periportal area; CV, central vein with adjacent perivenous area; sept, septal area where blood from portal venules of neighbouring portal tracts meet.
6-PGDH. Activity of 6-PGDH showed a relatively inhomogeneous lobular distribution pattern (Fig. 3). Highest levels of activity were found in intermediate zones along the axis between portal tracts and central veins (pp ® pv). Values in areas of adjoining septal branches were slightly lower. The residual standard deviation (Armitage, 1971) over all 7 rats was ± 0.89 (moles/kg dry weight/h).
Discussion LDH. Previous biochemical, microbiochemical and microphotometric investigations of hepatocellular heterogeneity of LDH in rat liver lobules were either limited to hepatocytes positioned at the periportal beginning and the perivenous end of sinusoids (Hildebrand and Fuchs, 1984; GuzmaÂn and Castro, 1998, 1990), to 3 (Shank et al., 1959; Griffini et al., 1994) or 4 areas (Morrison et al., 1965) or to several measurement sites (Wimmer and Pette, 1979) throughout the entire parenchyma between portal tracts and central veins. Ex-
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Fig. 3. Three dimensional demonstration of 6-phosphogluconate dehydrogenase activity in a standardized lobular area of rat liver parenchyma expanding between 3 portal tracts and a central vein. PT, portal tract with adjacent periportal area; CV, central vein with adjacent perivenous area; sept, septal area where blood from portal venules of neighbouring portal tracts meet.
cept for a homogeneous distribution pattern of LDH activity in mouse liver that was presumably due to species or sex differences (Griffini et al., 1994), significantly higher LDH activity has been reported in periportal zones than in perivenous zones (pp/pv ratio 1.3±1.8) (Jungermann and Katz, 1982; Jungermann, 1983; Hildebrand and Fuchs, 1984; GuzmaÂn and Castro, 1998, 1990). Since enzyme activities in liver cells parallel to the entire length of sinusoids may differ in a non-uniform manner (Teutsch, 1985), profiles of LDH activity were established in the present study (1) along sinusoids between small portal tracts and central veins and (2) along those sinusoids originating at adjoining septal branches and central veins (Ebert et al., 1987). According to the study of hepatic sinusoidal beds by Matsumoto and co-workers (Matsumoto et al., 1979; Matsumoto and Kawakami, 1982), these septal branches arise from different portal tracts in the periphery of a classic liver lobule. Except for the unique position of hepatocytes that surround portal tracts like a cuff, major characteristics of activity in sinusoidal profiles were very much the same with a periportal/septal ® perivenous decline in activity. LDH activity along the septal ® perivenous axis, however, was generally lower than along the periportal ® perivenous axis and, as indicated by regression analy-
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sis, the slope of the activity gradient was less distinct than along the periportal ® perivenous axis. Although highest activity was restricted to periportal zones, the lobular periphery showed a rather uniform broad area of high activity (Fig. 2). This continuous area of high LDH activity at the 2-dimensional level resembles the zonation pattern as described for the metabolic lobule provided by Lamers and co-workers (Lamers et al., 1989 a, b; Wagenaar et al., 1994) and is principally supported by the angioarchitectural study of Matsumoto et al. (1979). Such a correlation, however, does not necessarily implicate conceptual conformity in the 3-dimensional design of the metabolic lobulus and Matsumoto's primary lobules. Up to now, Matsumoto's studies on hepatic angioarchitecture with particular attention to the arrangement of portal venules and their septal branches forming a septum-like sinusoidal network were not only relevant for the interpretation of enzyme distribution patterns in the human liver but in rat liver as well. When comparing formation of sinusoidal vascular beds by parenchymal branches of portal veins in the human liver with the 3-dimensional reconstruction of parenchymal units in the rat, Teutsch and co-workers, however, could not find any evidence for the presence of such septal branches (Teutsch et al., 1999). Additional branches from portal venules of small portal tracts were too short and too few in number to be considered equivalent to septal branches as described previously. In the vascular surface of parenchymal units, portal venules of neighbouring portal tracts are connected by vascular septa. ªPortalº sinusoids originate directly and in close vicinity of portal venules and ,ªseptalº sinusoids arise from those regions along vascular septa where blood flow fronts of neighbouring portal venules meet. The variation in LDH activity with clear periportal/septal ® perivenous and rather slight periportal ® septal gradients as shown here are in agreement with a similar decrease of glucose6-phosphatase activity which has been used as marker of the perimeter of parenchymal units (Teutsch et al., 1999). Since typical septal branches are lacking in rat liver, sickle-shaped localization of enzyme activity may not correspond to similarly shaped high potential pools, from which blood is driven into radial sinusoids. The structure of the continuous vascular septum as described by Teutsch et al. (1999) is considered to be more appropriate to explain the broad area of high LDH activity resembling the enzymatic zonation of the metabolic lobulus (Lamers et al., 1989 a, b; Wagenaar et al., 1994). 6-PGDH. Sinusoidal profiles of 6-PGDH activity have not been established yet. Microbiochemical studies thus far were confined to measurements in periportal and perivenous areas (Hildebrand, 1980, 1984) showing a stronger activity perivenously as was demonstrated by qualitative histochemistry as well (Rieder et al., 1978; Rieder, 1981). In a cytophotometric analysis, maximum activity of the enzyme was approx twice higher in intermediate and pericentral (i. e. perivenous) zones than in periportal zones (Jonges and Van Noorden, 1989; Jonges et al., 1995). A time- and age-dependent heterogeneity
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of 6-PGDH activity in the liver was indicated by quantification of enzyme reactions performed by means of the Trident-microphotometric method. Highest levels of 6-PGDH activity were found in an intermediate area along the periportal ® central axis (Bhattacharya et al., 1986, 1987). Similar results have been obtained in the present study on lobular 6-PGDH activity. Although the enzyme is not a typical marker for hepatocytes surrounding central veins, it may be one of the enzymes with a midlobular localization pattern in the metabolic lobulus (Wagenaar et al., 1994). However, our data reveal a relatively inhomogeneous distribution pattern without a clear definition of zonal limits (Fig. 3). These variations in enzyme activity are also demonstrated by the relatively high residual SD over all 7 rats. Therefore, we have some doubts whether lobular distribution of 6-PGDH as demonstrated here may be interpreted in accordance with the metabolic lobulus concept. Acknowledgement. We wish to thank Frau Annelie Ahle for her expert technical assistance in this project.
References Armitage P (1971) Statistical methods in medical research. Blackwell, Oxford Batthacharya RD, Yamamoto A, Araki T, and Yamada M-O (1986) Further extension of Trident quantification in pattern analysis of liver dehydrogenases. Cell Mol Biol 32: 587±591 Batthacharya RD, Araki T, and Yamada M-O (1987) Quantitative topochemistry of rat liver enzymes during postnatal development in relation to activity-rest cycle. Acta Anat 128: 39±44 Burch H, Lowry OH, Kuhlman AM, Skerjance J, Diamant EJ, Lowry SR, and Von Dippe P (1963) Changes in patterns of enzymes of carbohydrate metabolism in the developing rat liver. J Biol Chem 238: 2267±2273 Ebert S, Hildebrand R, and Haubitz I (1987) Sinusoidal profiles of lactate dehydrogenase activity in rat liver. Histochemistry 87: 371±375 Gebhardt R (1992) Metabolic zonation of the liver: Regulation and implications for liver function. Pharmac Ther 53: 275±354 GoÈrgens HW, Hildebrand R, and Haubitz I (1988) Distribution pattern of alanine aminotransferase activity in rat liver. Histochemistry 88: 383±386 Griffini P, Vigorelli E, Bertone V, Freitas I, and Van Noorden CJF (1994) Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenase in mouse liver. Histochem J 26: 480±486 GuzmaÂn M, and Castro J (1989) Zonation of fatty acid metabolism in rat liver. Biochem J 264: 107±113 GuzmaÂn M, and Castro J (1990) Zonal heterogeneity of the effects of chronic ethanol feeding on hepatic fatty acid metabolism. Hepatology 12: 1098±1105 Haubitz I (1977) Programme zum Zeichnen von allgemeinen FlaÈchenstuÈcken. Computing 18: 295±315 Hildebrand R (1980) Nuclear volume and cellular metabolism. Adv Anat Embryol Cell Biol 60: 1±54 Hildebrand R (1983) Microbiochemical approach to liver cell heterogeneity around terminal hepatic venules. Histochemistry 78: 539±544
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Hildebrand R (1984) Quantitative and qualitative histochemical investigation on NADP+-dependent dehydrogenases in the limiting plate and the residual parenchyma surrounding terminal hepatic venules. Histochemistry 80: 91±95 Hildebrand R, and Fuchs C (1984) Microbiochemical investigation on diurnal rhythmic changes of the activities of the lactate dehydrogenase in the periportal and perivenous zones of the acinus of the rat liver. Histochemistry 81: 477±483 Hildebrand R, and Schleicher A (1986) Image analysis of the histochemistral demonstration of glucose-6-phosphatase activity in rat liver. Histochemistry 86: 181±190 Jonges GN, and Van Noorden CJF (1989) In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus. Histochem J 21: 585±594 Jonges GN, Vogels IMC, and Van Noorden CJF (1995) Effects of partial hepatectomy, phenobarbital and 3-methylcholanthrene on kinetic parameters of glucose-6-phosphate and phosphogluconate dehydrogenase in situ in periportal, intermediate and pericentral zones of rat liver lobules. Biochim Biophys Acta 1243: 59±64 Jungermann K (1983) Functional significance of hepatocyte heterogeneity for glycolysis and gluconeogenesis. Pharmacol Biochem Behav 18 (Suppl 1): 409±414 Jungermann K (1995) Zonation of metabolism and gene expression in liver. Histochemistry 103: 81±91 Jungermann K, and Katz N (1982) Functional hepatocellular heterogeneity. Hepatology 2: 385± 395 Jungermann K, and Katz N (1989) Functional specialization of different hepatocyte populations. Physiol Rev 69: 708±764 Lamers WH, Hilberts A, Furt E, Smith J, Jonges GN, Van Noorden CJF, Gaasbeek Janzen JW, Charles R, and Moorman AFM (1989 a) Hepatic enzymic zonation: A reevaluation of the concept of the liver acinus. Hepatology 10: 72±76 Lamers WH, Moorman AFM, and Charles R (1989 b) The metabolic lobulus, a key to the architecture of the liver. In: Gumucio JJ (Ed) Hepatocyte heterogeneity and liver function. RBC Cell Biol Rev 19: 5±26 Lowry OH, and Passoneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New York, London Lowry OH, Schulz D, and Passoneau JV (no date) The St Louis method. Dept of Pharmacology, Washington University School of Medicine, St. Louis Maly IP, and Sasse D (1983) A technical note on the histochemical demonstration of G6Pase activity. Histochemistry 78: 409±411 Matsumoto T, and Kawakami M (1982) The unit-concept of hepatic parenchyma ± a re-examination based on angioarchitectural studies. Acta Pathol Jpn (Suppl 2) 32: 285±314 Matsumoto T, Komori R, Magara T, Ui T, Kawakami M, Tokuda T, Takasaki S, Hayashi H, Jo K, Hano H, Fujino H, and Tanaka H (1979) A study on the normal structure of the human liver, with special reference to its angioarchitecture. Jikeikai Med J 26: 1±40 Morrison GR, Brock EF, Karl IE, and Shank RE (1965) Quantitative analysis of regenerating and degenerating areas within the lobule of the carbon tetrachloride-injured liver. Arch Biochem Biophys 111: 448±460 Rieder H (1981) NADP-dependent dehydrogenases in rat liver parenchyma III. The description of a liponeogenic area on the basis of histochemically demonstrated enzyme activities and the neutral fat content during fasting and refeeding. Histochemistry 72: 579±615 Rieder H, Teutsch HF, and Sasse D (1978) NADP-dependent dehydrogenases in rat liver parenchyma I. Methodological studies on the qualitative histochemistry of G6PDH, 6PGDH, malic enzyme and ICDH. Histochemistry 56: 283±298 Shank RE, Morrison GR, Chang ChH, Carl I, and Schwartz R (1959) Cell heterogeneity within the hepatic lobule (quantitative histochemistry). J Histochem Cytochem 7: 237±239
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Teutsch HF (1981) Chemomorphology of liver parenchyma. Prog Histochem Cytochem 14: 1± 92 Teutsch HF (1984) Sex-specific regionality of liver metabolism during starvation; with special reference to the heterogeneity of the lobular periphery. Histochemistry 81: 87±92 Teutsch HF (1985) Quantitative histochemical assessment of regional differences in hepatic glucose uptake and release. Histochemistry 82: 159±164 Teutsch HF (1986) A new sample isolation procedure for microchemical analysis of functional liver cell heterogeneity. J Histochem Cytochem 34: 263±267 Teutsch HF (1988) Regionality of glucose-6-phosphate hydrolysis in the liver lobule of the rat: metabolic heterogeneity of ªportalº and ªseptalº sinusoids. Hepatology 8: 311±317 Teutsch HF, and Chilko DM (1986) Use of 3-D-computer graphics for imaging of distribution of hepatic metabolites. Histochemistry 84: 396±400 Teutsch HF, Altemus J, Gerlach-Arbeiter S, and Kyander-Teutsch TL (1992) Distribution of 3hydroxybutyrate dehydrogenase in primary lobules of rat liver. J Histochem Cytochem 40: 213±219 Teutsch HF, Schuerfeld D, and Groezinger E (1999) Three-dimensional reconstruction of parenchymal units in the liver of the rat. Hepatology 29: 494±505 Wack S, and Haubitz I (1989) The distribution pattern of NADP+-dependent isocitrate dehydrogenase in rat liver. Cell Mol Biol 35: 305±312 Wagenaar GTM, Moorman AFM, Chamuleau RAFM, Deutz NEP, De Gier C, De Boer PAJ, Verbeek FJ, and Lamers WH (1994) Vascular branching pattern and zonation of gene expression in the mammalian liver. Anat Rec 239: 441±452 Wimmer M, and Pette D (1979) Microphotometric studies on intraacinar enzyme distribution in rat liver. Histochemistry 64: 23±33
Lobular distribution pattern of lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver Benjamin Schmidt1, Marco Vogelsang1, Imme Haubitz2, and Reinhard Hildebrand1 1
Institut fuÈr Anatomie, UniversitaÈt MuÈnster, Vesaliusweg 2±4, 48149 MuÈnster, Germany, 2 Rechenzentrum der UniversitaÈt WuÈrzburg, Am Hubland, 97074 WuÈrzburg, Germany
Received 7 May 1999 and in revised form 24 September 1999; accepted 27 September 1999
Summary Lactate dehydrogenase (LDH) and 6-phosphogluconate dehydrogenase (6PGDH) activities were measured in lobular areas expanding between 3 portal tracts and an efferent central vein in the livers of male Wistar rats, using a Lowry technique. The maximum of LDH activity was found in a nearly uniform broad area in the lobular periphery. From that area values decreased along periportal/septal ® perivenous gradients, but only slightly within that area along the periportal ® septal axis of the vascular septum. Maximum values of 6-PGDH activity were present in an intermediate area close to the central vein demonstrating a rather inhomogeneous distribution pattern without a clear definition of zonal limits. Our data on the distribution pattern of LDH are in agreement with the concept of the metabolic lobulus and are supported by a recent evaluation of the vascular architecture in rat liver. The lobular distribution pattern of 6-PGDH cannot be interpreted without doubt in accordance with that concept. Key words: lactate dehydrogenase ± 6-phosphogluconate dehydrogenase ± rat liver ± cellular heterogeneity ± metabolic lobulus
Correspondence to: R. Hildebrand, phone: + 49 25 18 35 52 00, fax: + 49 25 18 35 52 41, e-mail: [email protected] http://www.urbanfischer.de/journals/actahist
0065±1281/00/102/1±037 $ 12.00/0
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Introduction According to the widely accepted concept of metabolic zonation, the morphological, biochemical, and functional heterogeneity that is displayed by hepatocytes is related to zonal distribution patterns of many components in liver parenchyma with either predominantly periportal or perivenous localization (Jungermann and Katz, 1989; Gebhardt, 1992; Jungermann, 1995). However, a satisfactory attribution of this zonal heterogeneity to a simple morphofunctional unit of the liver or even the definition of zonal limits (Teutsch et al., 1992) seems to be an elusive goal (Lamers et al., 1989 a, b). Enzymatic studies along the porto-central axis and sinusoidal profiles of enzyme activities starting at the periphery of a classical lobule in the region between two adjoining septal branches and ending at central veins (Matsumoto et al., 1979; Matsumoto and Kawakami, 1982) provided a new startingpoint for the understanding of the specific parenchymal organization of the liver (Teutsch, 1984, 1985; Ebert et al., 1987; GoÈrgens et al., 1988; Teutsch, 1988; Wack and Haubitz, 1989; Teutsch et al., 1992). Matsumoto's concept of primary lobules with their sickle-shaped high potential pool seemed to be more appropriate to explain the data than Rappaport's acinar concept. According to a recent 3-dimensional reconstruction of parenchymal units, the latter concept cannot be applied to rat liver (Teutsch et al., 1999). Except for individual approaches of total pattern analysis in hepatic lobules (Hildebrand and Schleicher, 1986; Teutsch, 1986, 1988; Teutsch and Chilko, 1986) from a functional point of view, evidence was lacking for any interpretation (Lamers et al., 1989 a, b; Gebhardt, 1992), because data are not available of enzymatic distribution patterns in areas between the sinusoidal paths already analysed. Therefore, the aim of the present microquantitative investigation was to provide such data (1) for LDH which is predominantly located in hepatocytes surrounding small portal tracts like key enzymes of gluconeogenesis (Hildebrand and Fuchs, 1984; Ebert et al., 1987) and (2) for 6-PGDH which is predominantly present perivenously (Hildebrand, 1980; Rieder et al., 1978; Rieder, 1981), in intermediate areas (Bhattacharya et al., 1986, 1987), or in pericentral and intermediate areas (Jonges and Van Noorden, 1989; Jonges et al., 1995), respectively.
Material and methods Animals and tissue sampling. Seven male albino rats (84 days post-partum) of the Wistar strain (Wistar TNO; Harlan Winkelmann GmbH, Borchen, Germany) were housed at 27 ± 1 °C and 45% relative humidity in the animal quarters of the Department of Anatomy, University of MuÈnster. The animals were subjected to an artificial light-dark cycle (dark period 19.00 h±7.00 h) and were maintained on Altromin R and tap water ad libitum. The rats were sacrificed between 11.00 and 11.30 h on two subsequent days under ether anesthesia (time of anesthesia 30 s). After opening of the abdominal cavity, a piece of the median lobe of the livers was dissected with scissors and placed on a metal holder. Then,
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the material was immediately immersed in liquid nitrogen-cooled n-pentane. After the freezing process, the tissue block was stored in airtight tubes at ±80 °C. Preparation of tissue samples. Twenty lm thick cryostat sections of the livers were cut at a cabinet temp of ±25 °C and an object temp of ±21 °C (Frigocut 2800 E; Reichert-Jung, Nuûloch, Germany). The sections were subsequently lyophilized under vacuum at 0.785 Pa and ±40 °C for approx 48 h (Lyovac GT 2; Leybold-Heraeus, KoÈln, Germany). For the recognition of hepatocellular heterogeneity the activities of glucose-6-phosphatase were demonstrated histochemically (Maly and Sasse, 1983) in 10 lm thick parallel sections which served as guiding maps. From the lyophilized sections of each rat, 3 quadrangular areas of liver tissue each with 3 small portal tracts and 1 central vein at the corners were dissected free-hand using razor blades under a stereomicroscope at a magnification 50 ´. Only lobules with a typical zonal distribution pattern of glucose-6-phosphatase and an arrangement of portal tracts and central vein as demonstrated in Fig. 1 were selected. The dissected areas were subdivided into 4±6 adjacent strips (Fig. 1). The average length and width of the strips were for the LDH assay (1) 423.0 ± 48.0 lm and 73.4 ± 10.1 lm, respectively, and for the 6-PGDH assay (2) 472.0 ± 76.4 lm and 89.1 ± 15.3 lm, respectively. Subsequently, these strips were further subdivided into 5±10 successive samples of 30±100 lm in the axis of the strips for (1) or 40±100 lm for (2), respectively. Measurements were performed using a calibrated eyepiece micrometer. The dissection steps were documented on
Fig. 1. Histochemical demonstration of glucose-6-phosphatase in male rat liver with a schematic drawing of an excised and microdissected lobular area. PT, portal tract; CV, central vein; SEPT, septal area where blood from portal venules of neighbouring portal tracts meet.
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photographs taken from the dissected areas. Standardization of the assays of enzyme activities in quadrangular samples of similar size is necessary. On the one hand, excision of residues of vessels and connective tissue attached to the samples at the corners of the strips causes great variety in size, but on the other hand, vessels and connective tissue cause variation in measurements. Because the general distribution pattern of enzyme activities was of primary interest, samples with segments of portal tracts and central veins were included in our measurements and calculations despite the fact that measurements at the 4 corners of the strips are affected. The tissue samples were weighed using a quartz fiber fishpole balance where one unit equalled for (1) with 17.45 ng and for (2) with 20.63 ng. The recorded weight of the samples was reduced by 2% to allow for the uptake of N2 and O2, and a further 1% to allow for the uptake of H2O for every 10% of the known relative humidity (Teutsch, 1981). After weighing, the tissue samples were immediately dryloaded into the holes of microtest racks (Greiner, NuÈrtingen, Germany), which served as incubation vessels. Microbiochemical processing. The microbiochemical procedure as well as the preparation of tissue samples was based on the method developed by Lowry and collaborators (Lowry et al., no date; Lowry and Passoneau, 1972). Enzymatic assay for LDH. pyruvate + NADH2
LDH
! lactate + NAD+
Reagents: Tris buffer, 0.1 mol/l, pH 7.6; pyruvate, 2 mmol/l; nicotinamide, 20 mmol/l; NADH, 2 mmol/l; BSA, 0.06% (modified after Morrison et al., 1965; Hildebrand, 1983). The assays were performed in waterbaths at 30 °C for 30 min. The incubation volume was 7.043 ll. The enzymatic reaction was stopped by cooling the rack on crushed ice. Excess of NADH was destroyed by 21.337 ll 0.5 N HCl at 60 °C for 15 min. The reaction was stopped on crushed ice. For the determination of enzyme activity, the fluorescence of NAD+ destroyed in strong alkali was measured. 20.258 ll from the incubation vessels were transferred into fluorotubes containing 204.000 ll 6 N NaOH. After mixing, the tubes were incubated at 60 °C for 25 min. Subsequently, the samples were cooled down to room temp and 1 ml of distilled water was added. Fluorometric measurements were performed with a Farrand Ratio Fluorometer-2 (Farrand Optical, New York NY, USA). In all assays, blanks and a duplicate series of NAD+ standards (46.28 ´ 10±6 mol/l ± 265.73 ´ 10±6 mol/l) were incorporated. Tissue blanks were within the same range as reagent blanks. Before measurements were performed, blanks were adjusted to zero. The correct adjustment was repeatedly proven during measurements. Enzyme activity was expressed of moles substrate/kg dry weight/h. The reactions were linear in the range of dry weights 5±80 ng and time up to 60 min (for periportal regions, correlation coefficient, r = 0.99; for perivenous regions, r = 0.998). Materials. Boehringer (Mannheim, Germany): NAD+, grade 1, free acid; NADH, grade 1, disodium salt; pyruvate, monosodium salt; Serva (Heidelberg, Germany): BSA, crystallized research grade, > 99%; nicotinamide, pharm.; Merck (Darmstadt, Germany): Tris (hydroxymethyl)aminomethane, pro analysi. Enzymatic assay for 6-PGDH. 6-phosphogluconate + NADP+
6-PGDH
! ribulose-5-phosphate + CO2 + NADPH2
Reagents: Tris buffer, 0.1 mol/l, pH 8.4; 6-phosphogluconate, 2 mmol/l; NADP+, 1 mmol/l; EDTA, 0.5 mmol/l; BSA, 0.03% (modified after Burch et al., 1963; Hildebrand, 1984). The assays were performed in waterbaths at 30 °C for 60 min. The incubation volume was 7.622 ll. The enzymatic reaction was stopped by cooling the rack on crushed ice. Excess of NADP+ was destroyed by 23.223 ll Na2CO3, pH 12, at 60 °C for 15 min. The reac-
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tion was stopped on crushed ice. For the determination of enzyme activity, fluorescence of NADPH destroyed in strong alkali was measured. 13.814 ll from the incubation vessels were transferred into fluorotubes containing 175.000 ll 6 N NaOH and 0.01% H2O2. After mixing, the tubes were incubated at 60 °C for 25 min. Subsequently, the samples were cooled down to room temp and 1 ml of distilled water was added. Fluorometric measurements were performed with a Farrand Ratio Fluorometer-2. In all assays blanks and a duplicate series of NADPH standards (42.09 ´ 10±6 mol/l ± 377.84 ´ 10±6 mol/l) were incorporated. Tissue blanks were within the same range as reagent blanks. Before measurements were performed, blanks were adjusted to zero. The correct adjustment was repeatedly proven during measurements. Enzyme activity was expressed of moles substrate/kg dry weight/h. The reactions were linear in the range of dry weights 30±111 ng and time up to 90 min (for periportal regions, correlation coefficient, r = 0.861; for perivenous regions, r = 0.883). Materials. Boehringer: 6-phosphogluconate, crystallized trisodium salt; NADP+, 99% disodium salt; Merck: H2O2, Perhydrol 30%; Na2CO3; Tris (hydroxymethyl)aminomethane, pro analysi; Serva: BSA, crystallized research grade, > 99%; Sigma (St. Louis MO, USA): EDTA, 99%. Evaluation of the data. Distribution patterns of enzyme activities in the areas studied were presented as 3-dimensional mapping as Postscript-graph by a modified graphic programme according to Haubitz (1977). Due to the great variability in the excised quadrangular areas as well as in the number, length and width of the samples, all excised areas were transformed to standard squares. These squares were divided with a grid of 200 scales at both x-axis and y-axis per edge resulting in 40,000 points of intersection. At each point, enzyme activity was established. First, the mean values of enzyme activity of corresponding samples in 3 areas investigated were calculated for each animal. In this step of the calculation procedure, missing values due to missing samples automatically disappeared. Subsequently, the means of the means of 7 animals were calculated and finally 3-dimensional representations of the activities of both enzymes were computed. The coordinates on the xaxis and y-axis determined each point in the square with its relevant enzyme activity expressed on the z-axis. The values in the images represent mean enzyme activity for all animals. Shading in the graph is generated by a virtual light source from the left.
Results LDH. The gradient of LDH activity decreased along the entire path between portal tract and central vein (Fig. 2). The slight increase of activity outside the portal tracts is partly due to lower LDH activity in samples containing segments of portal vessels and connective tissue. Distribution patterns between joined septal branches and the central vein displayed the same characteristics (sept ® pv) except for generally lower activity and a missing intial increase. Due to the lower activity here 2 furrows directed towards the perivenous area became apparent with a depth that was less distinct in the vicinity of the central vein. As expected, highest activity was found in the lobular periphery and lowest around central veins. However, in the periphery of lobules, an almost uniform wide area with high periportal and septal activity was found with slight decrease of activity along the pp ® sept axis. The residual standard deviation (Armitage, 1971) over all 7 rats was ± 15.7 (moles/kg dry weight/h).
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Fig. 2. Three dimensional demonstration of lactate dehydrogenase activity in a standardized lobular area of rat liver parenchyma expanding between 3 portal tracts and a central vein. PT, portal tract with adjacent periportal area; CV, central vein with adjacent perivenous area; sept, septal area where blood from portal venules of neighbouring portal tracts meet.
6-PGDH. Activity of 6-PGDH showed a relatively inhomogeneous lobular distribution pattern (Fig. 3). Highest levels of activity were found in intermediate zones along the axis between portal tracts and central veins (pp ® pv). Values in areas of adjoining septal branches were slightly lower. The residual standard deviation (Armitage, 1971) over all 7 rats was ± 0.89 (moles/kg dry weight/h).
Discussion LDH. Previous biochemical, microbiochemical and microphotometric investigations of hepatocellular heterogeneity of LDH in rat liver lobules were either limited to hepatocytes positioned at the periportal beginning and the perivenous end of sinusoids (Hildebrand and Fuchs, 1984; GuzmaÂn and Castro, 1998, 1990), to 3 (Shank et al., 1959; Griffini et al., 1994) or 4 areas (Morrison et al., 1965) or to several measurement sites (Wimmer and Pette, 1979) throughout the entire parenchyma between portal tracts and central veins. Ex-
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Fig. 3. Three dimensional demonstration of 6-phosphogluconate dehydrogenase activity in a standardized lobular area of rat liver parenchyma expanding between 3 portal tracts and a central vein. PT, portal tract with adjacent periportal area; CV, central vein with adjacent perivenous area; sept, septal area where blood from portal venules of neighbouring portal tracts meet.
cept for a homogeneous distribution pattern of LDH activity in mouse liver that was presumably due to species or sex differences (Griffini et al., 1994), significantly higher LDH activity has been reported in periportal zones than in perivenous zones (pp/pv ratio 1.3±1.8) (Jungermann and Katz, 1982; Jungermann, 1983; Hildebrand and Fuchs, 1984; GuzmaÂn and Castro, 1998, 1990). Since enzyme activities in liver cells parallel to the entire length of sinusoids may differ in a non-uniform manner (Teutsch, 1985), profiles of LDH activity were established in the present study (1) along sinusoids between small portal tracts and central veins and (2) along those sinusoids originating at adjoining septal branches and central veins (Ebert et al., 1987). According to the study of hepatic sinusoidal beds by Matsumoto and co-workers (Matsumoto et al., 1979; Matsumoto and Kawakami, 1982), these septal branches arise from different portal tracts in the periphery of a classic liver lobule. Except for the unique position of hepatocytes that surround portal tracts like a cuff, major characteristics of activity in sinusoidal profiles were very much the same with a periportal/septal ® perivenous decline in activity. LDH activity along the septal ® perivenous axis, however, was generally lower than along the periportal ® perivenous axis and, as indicated by regression analy-
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sis, the slope of the activity gradient was less distinct than along the periportal ® perivenous axis. Although highest activity was restricted to periportal zones, the lobular periphery showed a rather uniform broad area of high activity (Fig. 2). This continuous area of high LDH activity at the 2-dimensional level resembles the zonation pattern as described for the metabolic lobule provided by Lamers and co-workers (Lamers et al., 1989 a, b; Wagenaar et al., 1994) and is principally supported by the angioarchitectural study of Matsumoto et al. (1979). Such a correlation, however, does not necessarily implicate conceptual conformity in the 3-dimensional design of the metabolic lobulus and Matsumoto's primary lobules. Up to now, Matsumoto's studies on hepatic angioarchitecture with particular attention to the arrangement of portal venules and their septal branches forming a septum-like sinusoidal network were not only relevant for the interpretation of enzyme distribution patterns in the human liver but in rat liver as well. When comparing formation of sinusoidal vascular beds by parenchymal branches of portal veins in the human liver with the 3-dimensional reconstruction of parenchymal units in the rat, Teutsch and co-workers, however, could not find any evidence for the presence of such septal branches (Teutsch et al., 1999). Additional branches from portal venules of small portal tracts were too short and too few in number to be considered equivalent to septal branches as described previously. In the vascular surface of parenchymal units, portal venules of neighbouring portal tracts are connected by vascular septa. ªPortalº sinusoids originate directly and in close vicinity of portal venules and ,ªseptalº sinusoids arise from those regions along vascular septa where blood flow fronts of neighbouring portal venules meet. The variation in LDH activity with clear periportal/septal ® perivenous and rather slight periportal ® septal gradients as shown here are in agreement with a similar decrease of glucose6-phosphatase activity which has been used as marker of the perimeter of parenchymal units (Teutsch et al., 1999). Since typical septal branches are lacking in rat liver, sickle-shaped localization of enzyme activity may not correspond to similarly shaped high potential pools, from which blood is driven into radial sinusoids. The structure of the continuous vascular septum as described by Teutsch et al. (1999) is considered to be more appropriate to explain the broad area of high LDH activity resembling the enzymatic zonation of the metabolic lobulus (Lamers et al., 1989 a, b; Wagenaar et al., 1994). 6-PGDH. Sinusoidal profiles of 6-PGDH activity have not been established yet. Microbiochemical studies thus far were confined to measurements in periportal and perivenous areas (Hildebrand, 1980, 1984) showing a stronger activity perivenously as was demonstrated by qualitative histochemistry as well (Rieder et al., 1978; Rieder, 1981). In a cytophotometric analysis, maximum activity of the enzyme was approx twice higher in intermediate and pericentral (i. e. perivenous) zones than in periportal zones (Jonges and Van Noorden, 1989; Jonges et al., 1995). A time- and age-dependent heterogeneity
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of 6-PGDH activity in the liver was indicated by quantification of enzyme reactions performed by means of the Trident-microphotometric method. Highest levels of 6-PGDH activity were found in an intermediate area along the periportal ® central axis (Bhattacharya et al., 1986, 1987). Similar results have been obtained in the present study on lobular 6-PGDH activity. Although the enzyme is not a typical marker for hepatocytes surrounding central veins, it may be one of the enzymes with a midlobular localization pattern in the metabolic lobulus (Wagenaar et al., 1994). However, our data reveal a relatively inhomogeneous distribution pattern without a clear definition of zonal limits (Fig. 3). These variations in enzyme activity are also demonstrated by the relatively high residual SD over all 7 rats. Therefore, we have some doubts whether lobular distribution of 6-PGDH as demonstrated here may be interpreted in accordance with the metabolic lobulus concept. Acknowledgement. We wish to thank Frau Annelie Ahle for her expert technical assistance in this project.
References Armitage P (1971) Statistical methods in medical research. Blackwell, Oxford Batthacharya RD, Yamamoto A, Araki T, and Yamada M-O (1986) Further extension of Trident quantification in pattern analysis of liver dehydrogenases. Cell Mol Biol 32: 587±591 Batthacharya RD, Araki T, and Yamada M-O (1987) Quantitative topochemistry of rat liver enzymes during postnatal development in relation to activity-rest cycle. Acta Anat 128: 39±44 Burch H, Lowry OH, Kuhlman AM, Skerjance J, Diamant EJ, Lowry SR, and Von Dippe P (1963) Changes in patterns of enzymes of carbohydrate metabolism in the developing rat liver. J Biol Chem 238: 2267±2273 Ebert S, Hildebrand R, and Haubitz I (1987) Sinusoidal profiles of lactate dehydrogenase activity in rat liver. Histochemistry 87: 371±375 Gebhardt R (1992) Metabolic zonation of the liver: Regulation and implications for liver function. Pharmac Ther 53: 275±354 GoÈrgens HW, Hildebrand R, and Haubitz I (1988) Distribution pattern of alanine aminotransferase activity in rat liver. Histochemistry 88: 383±386 Griffini P, Vigorelli E, Bertone V, Freitas I, and Van Noorden CJF (1994) Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenase in mouse liver. Histochem J 26: 480±486 GuzmaÂn M, and Castro J (1989) Zonation of fatty acid metabolism in rat liver. Biochem J 264: 107±113 GuzmaÂn M, and Castro J (1990) Zonal heterogeneity of the effects of chronic ethanol feeding on hepatic fatty acid metabolism. Hepatology 12: 1098±1105 Haubitz I (1977) Programme zum Zeichnen von allgemeinen FlaÈchenstuÈcken. Computing 18: 295±315 Hildebrand R (1980) Nuclear volume and cellular metabolism. Adv Anat Embryol Cell Biol 60: 1±54 Hildebrand R (1983) Microbiochemical approach to liver cell heterogeneity around terminal hepatic venules. Histochemistry 78: 539±544
46
B. Schmidt et al.
Hildebrand R (1984) Quantitative and qualitative histochemical investigation on NADP+-dependent dehydrogenases in the limiting plate and the residual parenchyma surrounding terminal hepatic venules. Histochemistry 80: 91±95 Hildebrand R, and Fuchs C (1984) Microbiochemical investigation on diurnal rhythmic changes of the activities of the lactate dehydrogenase in the periportal and perivenous zones of the acinus of the rat liver. Histochemistry 81: 477±483 Hildebrand R, and Schleicher A (1986) Image analysis of the histochemistral demonstration of glucose-6-phosphatase activity in rat liver. Histochemistry 86: 181±190 Jonges GN, and Van Noorden CJF (1989) In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus. Histochem J 21: 585±594 Jonges GN, Vogels IMC, and Van Noorden CJF (1995) Effects of partial hepatectomy, phenobarbital and 3-methylcholanthrene on kinetic parameters of glucose-6-phosphate and phosphogluconate dehydrogenase in situ in periportal, intermediate and pericentral zones of rat liver lobules. Biochim Biophys Acta 1243: 59±64 Jungermann K (1983) Functional significance of hepatocyte heterogeneity for glycolysis and gluconeogenesis. Pharmacol Biochem Behav 18 (Suppl 1): 409±414 Jungermann K (1995) Zonation of metabolism and gene expression in liver. Histochemistry 103: 81±91 Jungermann K, and Katz N (1982) Functional hepatocellular heterogeneity. Hepatology 2: 385± 395 Jungermann K, and Katz N (1989) Functional specialization of different hepatocyte populations. Physiol Rev 69: 708±764 Lamers WH, Hilberts A, Furt E, Smith J, Jonges GN, Van Noorden CJF, Gaasbeek Janzen JW, Charles R, and Moorman AFM (1989 a) Hepatic enzymic zonation: A reevaluation of the concept of the liver acinus. Hepatology 10: 72±76 Lamers WH, Moorman AFM, and Charles R (1989 b) The metabolic lobulus, a key to the architecture of the liver. In: Gumucio JJ (Ed) Hepatocyte heterogeneity and liver function. RBC Cell Biol Rev 19: 5±26 Lowry OH, and Passoneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New York, London Lowry OH, Schulz D, and Passoneau JV (no date) The St Louis method. Dept of Pharmacology, Washington University School of Medicine, St. Louis Maly IP, and Sasse D (1983) A technical note on the histochemical demonstration of G6Pase activity. Histochemistry 78: 409±411 Matsumoto T, and Kawakami M (1982) The unit-concept of hepatic parenchyma ± a re-examination based on angioarchitectural studies. Acta Pathol Jpn (Suppl 2) 32: 285±314 Matsumoto T, Komori R, Magara T, Ui T, Kawakami M, Tokuda T, Takasaki S, Hayashi H, Jo K, Hano H, Fujino H, and Tanaka H (1979) A study on the normal structure of the human liver, with special reference to its angioarchitecture. Jikeikai Med J 26: 1±40 Morrison GR, Brock EF, Karl IE, and Shank RE (1965) Quantitative analysis of regenerating and degenerating areas within the lobule of the carbon tetrachloride-injured liver. Arch Biochem Biophys 111: 448±460 Rieder H (1981) NADP-dependent dehydrogenases in rat liver parenchyma III. The description of a liponeogenic area on the basis of histochemically demonstrated enzyme activities and the neutral fat content during fasting and refeeding. Histochemistry 72: 579±615 Rieder H, Teutsch HF, and Sasse D (1978) NADP-dependent dehydrogenases in rat liver parenchyma I. Methodological studies on the qualitative histochemistry of G6PDH, 6PGDH, malic enzyme and ICDH. Histochemistry 56: 283±298 Shank RE, Morrison GR, Chang ChH, Carl I, and Schwartz R (1959) Cell heterogeneity within the hepatic lobule (quantitative histochemistry). J Histochem Cytochem 7: 237±239
Lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver
47
Teutsch HF (1981) Chemomorphology of liver parenchyma. Prog Histochem Cytochem 14: 1± 92 Teutsch HF (1984) Sex-specific regionality of liver metabolism during starvation; with special reference to the heterogeneity of the lobular periphery. Histochemistry 81: 87±92 Teutsch HF (1985) Quantitative histochemical assessment of regional differences in hepatic glucose uptake and release. Histochemistry 82: 159±164 Teutsch HF (1986) A new sample isolation procedure for microchemical analysis of functional liver cell heterogeneity. J Histochem Cytochem 34: 263±267 Teutsch HF (1988) Regionality of glucose-6-phosphate hydrolysis in the liver lobule of the rat: metabolic heterogeneity of ªportalº and ªseptalº sinusoids. Hepatology 8: 311±317 Teutsch HF, and Chilko DM (1986) Use of 3-D-computer graphics for imaging of distribution of hepatic metabolites. Histochemistry 84: 396±400 Teutsch HF, Altemus J, Gerlach-Arbeiter S, and Kyander-Teutsch TL (1992) Distribution of 3hydroxybutyrate dehydrogenase in primary lobules of rat liver. J Histochem Cytochem 40: 213±219 Teutsch HF, Schuerfeld D, and Groezinger E (1999) Three-dimensional reconstruction of parenchymal units in the liver of the rat. Hepatology 29: 494±505 Wack S, and Haubitz I (1989) The distribution pattern of NADP+-dependent isocitrate dehydrogenase in rat liver. Cell Mol Biol 35: 305±312 Wagenaar GTM, Moorman AFM, Chamuleau RAFM, Deutz NEP, De Gier C, De Boer PAJ, Verbeek FJ, and Lamers WH (1994) Vascular branching pattern and zonation of gene expression in the mammalian liver. Anat Rec 239: 441±452 Wimmer M, and Pette D (1979) Microphotometric studies on intraacinar enzyme distribution in rat liver. Histochemistry 64: 23±33