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Am J Physiol Gastrointest Liver Physiol 293: G838-G849, 2007. First published August 9, 2007; doi:10.1152/ajpgi.00120.2007
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LIVER AND BILIARY TRACT

Flexibility of the hepatic zonation of carbon and nitrogen fluxes linked to lactate and pyruvate transformations in the presence of ammonia

Jurandir Fernando Comar, Fumie Suzuki-Kemmelmeier, Écio Alves Nascimento, and Adelar Bracht

Submitted 8 March 2007 ; accepted in final form 8 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been proposed that key enzymes of ureagenesis and the alanine aminotransferase activity predominate in periportal hepatocytes. However, ureagenesis from alanine, when measured in the perfused liver, did not show periportal predominance and even the release of the direct products of alanine transformation, lactate and pyruvate, was higher in perivenous cells. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea production from precursors such as lactate or pyruvate plus ammonia. In the present work these aspects were investigated in the bivascularly perfused rat liver. The results of the present study confirm that gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region. Alanine synthesis from lactate and pyruvate plus ammonia, however, predominated in the perivenous region. Furthermore, no predominance of ureagenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. These observations corroborate the view that data on enzyme activity or expression alone cannot be extrapolated unconditionally to the living cell. The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureagenesis in periportal cells. However, since urea synthesis occurs at high rates in all hepatocytes with the possible exclusion of those cells not possessing carbamoyl-phosphate synthase, it is probable that ureagenesis is equally important as an ammonia-detoxifying mechanism in the perivenous region.

metabolic zonation; liver; carbon fluxes; ureagenesis; gluconeogenesis

THERE IS GENERAL AGREEMENT that in the liver the metabolic activity is heterogeneously distributed along the hepatic acini. Some routes predominate in periportal and others in perivenous cells. Evidence favoring this view has been obtained in enzyme distribution studies (24, 25), flux measurements in isolated perivenous and periportal hepatocytes (36) and in the perfused rat liver (6, 7, 25) and, more recently, in a study of differential gene expression in periportal and perivenous hepatocytes (9). Despite this general agreement and the numerous studies that have been done, there are several aspects still awaiting a more detailed investigation. Evidence indicates that zonation presents some degree of flexibility in that it may suffer considerable alterations when the metabolic conditions are changed (23). Furthermore, conflicting observations are not uncommon, especially when reciprocally regulated fluxes are analyzed, as for example the carbon and nitrogen fluxes involving amino acids and ammonia. The most common discrepancies are those between enzyme activity or gene expression measurements and the actual metabolic fluxes in the living cell. For example, urea production due to alanine transformation in the perfused rat liver is uniformly distributed over the liver parenchyma and for this reason the molar ratio of ureagenesis to gluconeogenesis is smaller in periportal cells compared with perivenous cells (6). The latter observations are unexpected by virtue of the finding that the expressions of several key enzymes of the urea cycle predominate in the periportal region (9, 17). Moreover, several investigators have found that the alanine aminotransferase activity predominates in periportal hepatocytes (1, 5, 11, 32, 36, 40). This leads us to expect a more intense alanine metabolism in the periportal zone. In fact, higher rates of gluconeogenesis from alanine and the associated increases in oxygen uptake were found in the perfused rat liver (6). Parallel observations strongly suggest, however, that this periportal predominance is not primarily caused by the periportal predominance of the alanine aminotransferase activity. It is more likely the consequence of the general periportal predominance of the gluconeogenic pathway, a conclusion derived from the observation that the overflows of pyruvate and lactate, the first products of alanine transformation, were considerably smaller in periportal cells (6).

The results on the hepatic zonation of alanine metabolism discussed above raise the question about the true functional distribution of alanine aminotransferase and the urea cycle along the hepatic acini. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea production from precursors such as lactate or pyruvate plus ammonia. These are precisely the main purposes of the present work in which alanine and urea production from these precursors were measured in the bivascularly perfused rat liver in antegrade and retrograde perfusion. It has been shown by previous work that infusion of substrates via the hepatic artery in retrograde perfusion allows to reach selectively periportal cells in contrast to the same infusion in antegrade perfusion, a procedure in which all cells are reached. This particularity provides a suitable method for investigating metabolic zonation in the intact rat liver without the disturbances inherent to cells subject to an entirely artificial environment (6, 7, 33). Besides alanine and urea production, several additional parameters related to the carbon and the nitrogen fluxes were measured at different ammonia concentrations and under different redox states, to obtain a more complete picture about the zonation of these fluxes under various conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. The liver perfusion apparatus was built in the workshops of the University of Maringá. Enzymes and coenzymes used in the metabolite assays were purchased from Sigma Chemical (St. Louis, MO). All standard chemicals were from the best available grade (>99.5% purity) and were purchased from Merck (Darmstadt, Germany), Carlo Erba (São Paulo, Brazil), and Reagen (Rio de Janeiro, Brazil).

Bivascular liver perfusion. Male albino rats (Wistar), weighing 180–220 g, were fed ad libitum with a standard laboratory diet (Purina). Food was withdrawn 24 h before the liver perfusion experiments. For the surgical procedure, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). All experiments were done in accordance with the American Physiological Society Guiding Principles in the Care and Use of Animals. Dr. Adelar Bracht and his coworkers were duly authorized by the Coordination of the PhD Program in Biological Sciences of the University of Maringá to conduct this animal study.

Hemoglobin-free, nonrecirculating bivascular liver perfusion was performed either in the antegrade (entry via the portal vein plus hepatic artery and exit via the hepatic vein) or in the retrograde mode (entry via the hepatic vein plus hepatic artery and exit via the portal vein). The surgical procedure was described elsewhere (39). In situ perfusion was carried out, the flow being provided by two peristaltic pumps. The perfusion fluid was Krebs-Henseleit-bicarbonate buffer (pH 7.4) containing 25 mg% bovine serum albumin, saturated with a mixture of oxygen and carbon dioxide (95:5) by means of a membrane oxygenator with simultaneous temperature adjustment (37°C). The portal flow was adjusted between 28 and 32 ml/min and the arterial flow between 2 and 3 ml/min. All perfusion experiments were initiated in the antegrade mode. Retrograde perfusion was established by changing the direction of flow at 15–20 min before initiating sampling of the effluent perfusate.

In all perfusion experiments livers from fasted rats were used so that glycogenolysis and glycolysis from endogenous sources was minimal (2). The substrates were lactate (21 µmol·min–1·g–1) plus ammonia (up to 9.5 µmol·min–1·g–1) or pyruvate (8.5 µmol·min–1·g–1) plus ammonia. Ammonia was infused in the form of ammonium chloride.

Analysis. Samples of the effluent perfusion fluid were collected according to the experimental protocol and analyzed for their metabolite contents. The following compounds were measured by means of standard enzymatic procedures: glucose, lactate, pyruvate, alanine, glutamate, glutamine, urea, and ammonia (3). The oxygen concentration in the outflowing perfusate was monitored continuously, employing a Teflon-shielded platinum electrode adequately positioned in a Plexiglas chamber at the exit of the perfusate (8).

Data treatment and interpretation. The changes in metabolic fluxes caused by lactate plus ammonia or pyruvate plus ammonia infusion into the hepatic artery were expressed as micromoles per minute per milliliter accessible cell space in each perfusion mode (14, 15), after subtracting the basal rates. The basal rates are those measured before any substrate infusion. This procedure effectively normalizes the responses of the liver and provides a basis for comparison despite the different cell spaces that are reached via the hepatic artery in antegrade and retrograde perfusion (see Fig. 1). The effective substrate concentrations along the hepatic acinus, however, depend on the distribution of the arterial flow between the pre- and intrasinusoidal confluences, a fact that must also be taken into account when interpreting the results.


Figure 1
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  		Fig. 1. Diagram of some important features of the hepatic microcirculation. The shaded area represents the liver space that is accessible via the hepatic artery in retrograde perfusion, i.e., in the direction hepatic vein -> portal vein.

 
A simple model for the flux responses that can be expected when substrates are infused into the hepatic artery in antegrade or retrograde perfusion has been proposed in a previous work (6). If Jret and Jant are the cell space-normalized metabolic fluxes due to the infusion of saturating substrate concentrations into the hepatic artery in retrograde and antegrade perfusion, respectively, the following simple relations are valid:

Formula 1(1)

Formula 2(2)
V1 is the maximal rate in the region between the pre- and intrasinusoidal confluence and V2 the maximal rate in the region situated downstream to the intrasinusoidal confluence (Fig. 1); r is the fractional cell volume between the pre- and the intrasinusoidal confluences, which has been determined to be equal to 0.38 for the isolated and hemoglobin-free perfused rat liver (14, 15). Since Jret represents the response of periportal cells and Jant the mean response of all cells, three simple rules can be applied: if Jret = Jant, no zonation exists; if Jret > Jant, periportal predominance exists; if Jant > Jret, the predominance is perivenous.

Because of these simple relationships, the gluconeogenic substrates lactate and pyruvate were infused at saturating concentrations. For lactate this corresponds anyway to the physiological conditions. For pyruvate it is an important experimental condition, i.e., a low NADH-to-NAD+ ratio as opposed to the high ratio in the presence of lactate (38). For ammonia, however, the physiological conditions are normally well below saturation and, for this reason, a wide range of infusion rates must be used if the data are to be appropriately interpreted. Uptake of ammonia by hepatocytes follows apparent Michaelis-Menten kinetics (10), probably because the limiting steps of this process, catalyzed by carbamoyl-phosphate synthase (22) and glutamate dehydrogenase (35), also follow Michaelis-Menten kinetics (Fig. 2). In terms of the microcirculatory peculiarities of the liver, responses to ammonia infusion (Sin) in the presence of lactate or pyruvate in retrograde (Jret) and antegrade (Jant) perfusion obeying the Michaelis-Menten equation can be described by the following equations (6):

Formula 3(3)

Formula 4(4)
Sin represents the rate of ammonia infusion (in µmol min–1·g liver–1). JFormula 4 and JFormula 4 represent the rates of the metabolic fluxes in the absence of ammonia due to the infusion of saturating levels of lactate or pyruvate into the hepatic artery in retrograde and antegrade perfusion, respectively. FT is the total flow through the liver (in ml min–1·g liver–1), FA is the arterial flow, and FAIS is the arterial flow that reaches the intrasinusoidal confluence (see Fig. 1). K1 and K2 are, respectively, the half-saturation concentrations for the region between the pre- and intrasinusoidal confluence and for the region situated downstream to the intrasinusoidal confluence. In retrograde perfusion the substrate concentration that reaches the region between the pre- and intrasinusoidal confluences (shaded area in Fig. 1) corresponds to the term Sin/(FTFA/FAIS). In antegrade perfusion this concentration equals Sin/[(FTFA/(FA – FAIS)] in the region between the pre- and intrasinusoidal confluences; finally, downstream to the intrasinusoidal confluence the mean substrate concentration is given by Sin/FT. It is worth remarking that Eq. 4 describes two independent phenomena, transformation in the region between the pre- and intrasinusoidal confluence and transformation in the region situated downstream to the intrasinusoidal confluence, that occur independently but are measured as a single total transformation rate (Jant). This is analogous to the equation for two independent enzymes catalyzing the same reaction (37), but with corrections for the effective substrate concentrations.


Figure 2
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  		Fig. 2. Schematic representation of the main pathways leading to the production of the metabolites quantified in the present work. Cells expressing carbamoyl-phosphate synthase comprise both periportal and perivenous hepatocytes, with the exception of a small group of perivenous hepatocytes immediately surrounding the hepatic venules (17). These cells are able to express glutamine synthetase (18), which is also found in Kupffer and endothelial cells (4, 29). Ornit, ornithine; Citr, citrulline; Arg, arginine; Suc, succinate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GDH, glutamate dehydrogenase.

 
It should be noted that Sin/FT is always superior to Sin/[(FTFA/ (FA – FAIS)] or Sin/(FTFA/FAIS). Consequently, in practical terms, if the experimental Jret is found greater than Jant at subsaturating conditions (i.e., Jret > Jant), this clearly indicates periportal predominance. And also, if Jant persistently exceeds Jret over a wide concentration range without any tendency of convergence at saturating concentrations (i.e., Jant >> Jret), this can be regarded as an indication of perivenous predominance (6). It should be stressed that these two rules are valid also when Jant and Jret do not obey Michaelis-Menten kinetics, provided that these experimental parameters are saturating functions of the substrate infusion rate.

If the determination of K1, K2, V1, and V2 is possible, the metabolic fluxes in the region between the pre- and intrasinusoidal confluence (J1) and in the region that is situated downstream to the intrasinusoidal confluence (J2) can be calculated for any ammonia concentration:

Formula 5(5)

Formula 6(6)
J1 = J2 will mean absence of zonation, J1 > J2 periportal predominance, and J2 > J1 perivenous predominance.

Treatment of data. Statistical analysis of the data was done by means of the Statistica program (Statsoft, 1998). Fitting of Eqs. 3 and 4 to experimental data was performed by means of an iterative nonlinear least-squares procedure, using the Scientist software from MicroMath Scientific Software (Salt Lake City, UT).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Time courses of the responses to ammonia plus lactate or pyruvate. The scheme in Fig. 2 summarizes the main pathways leading to the production of the metabolites that were measured in the present work. It also gives information about the intra- and intercellular distribution of important enzymes (9, 17, 18). Enzyme gradients along the hepatic acinus, however, are not represented. The time course of the responses of the liver in antegrade and retrograde perfusion to arterial ammonia and lactate infusion are illustrated by Fig. 3; Fig. 3A shows the changes in oxygen uptake and in the production of glucose and pyruvate (electron and carbon fluxes) and Fig. 3B the changes in urea, glutamine, glutamate, and alanine production (nitrogen fluxes). After a preperfusion period (10 min), ammonia was initially infused into the hepatic artery for 14 min. In the experiments illustrated by Fig. 3 the rate of ammonia infusion into the hepatic artery was equal to 2.3 µmol·min–1·g–1. Lactate infusion was initiated at 24 min perfusion time and continued until the end of the experiment (70 min). The lactate infusion rate was equal to 21 µmol·min–1·g–1, which should be saturating for lactate metabolism (20, 42). Except for oxygen uptake, the basal rates (before substrate infusion) were all low. The infusion of ammonia produced significant increases in urea and glutamine production and small increments in oxygen uptake in both perfusion modes, antegrade and retrograde. Lactate infusion in addition to ammonia produced much more pronounced changes, with initially accelerated increases, followed by phases of stabilization. All changes were smaller in retrograde perfusion, an expected event because the cell space that is accessible via the hepatic artery in retrograde perfusion corresponds to only 38% of that accessible in antegrade perfusion (14, 15). The extent of the changes caused by lactate in antegrade and retrograde perfusion, however, was not the same for all parameters. For glucose production and oxygen uptake, for example, the final difference between antegrade and retrograde perfusion was relatively small (Fig. 3A). The urea and alanine production, on the other hand, increased to a relatively small extent in retrograde perfusion so that the differences between antegrade and retrograde perfusion were more pronounced (Fig. 3B).


Figure 3
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  		Fig. 3. Action of arterially infused ammonia and lactate on hepatic carbon fluxes (A), oxygen uptake (A), and nitrogen fluxes (B) in antegrade and retrograde perfusion. Livers from fasted rats were perfused as described in MATERIALS AND METHODS. Lactate and ammonia were infused into the hepatic artery as indicated. Solid symbols represent the results obtained in antegrade perfusion; open symbols represent those obtained in retrograde perfusion. Data are from 9 (antegrade) or 6 (retrograde) liver perfusion experiments. Error bars represent mean standard errors.

 
The experimental protocol and the time courses of the experiments in which pyruvate was used as the carbon source are illustrated by Fig. 4; Fig. 4A shows the changes in oxygen uptake and in the production of glucose and lactate (electron and carbon fluxes) and Fig. 4B shows the changes in urea, glutamine, glutamate, and alanine production (nitrogen fluxes). After a preperfusion period of 10 min, ammonia was initially infused into the hepatic artery for 14 min. Pyruvate infusion was initiated at 24 min perfusion time and continued until the end of the experiment (50 min). The pyruvate infusion rate of 8.5 µmol·min–1·g–1 should be saturating for pyruvate metabolism (20, 42). The responses to pyruvate were similar to the responses to lactate. The main differences to be remarked are that the increments in glucose production and oxygen uptake were smaller, but the increments in alanine and glutamate production were considerably higher, especially in the antegrade mode of perfusion.


Figure 4
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  		Fig. 4. Action of arterially infused ammonia and pyruvate on hepatic carbon fluxes (A), oxygen uptake (A), and nitrogen fluxes (B) in antegrade and retrograde perfusion. Livers from fasted rats were perfused as described in MATERIALS AND METHODS. Lactate and ammonia were infused into the hepatic artery as indicated. Solid symbols represent the results obtained in antegrade perfusion; open symbols represent those obtained in retrograde perfusion. Data are from 9 (antegrade and retrograde) liver perfusion experiments. Error bars represent mean standard errors.

 
Metabolic fluxes in antegrade and retrograde perfusion corrected for the accessible cell spaces. The experiments shown in Figs. 3 and 4 were obtained with an ammonia infusion rate of 2.3 µmol·min–1·g–1. They were repeated without previous ammonia infusion (zero ammonia) and with two additional ammonia infusion rates, namely 4.6 and 9.5 µmol·min–1·g–1. Since different cellular spaces are supplied with substrates infused into the hepatic artery in antegrade and retrograde perfusion (see Fig. 1), normalization is essential for conclusions about zonation. The cellular spaces accessible in each perfusion mode have already been determined in previous work (14, 15). When these cellular spaces are used for normalization it is implicitly assumed that the given metabolites are in fact being produced in these spaces. This is a reasonable assumption for glucose and alanine production and also for oxygen uptake. Urea production can also be analyzed in this way because more than 90% of the hepatocytes express the enzymes of the urea cycle (17, 18). Normalization of the metabolic fluxes consists in dividing each metabolic flux in the presence of ammonia and lactate or pyruvate by the corresponding accessible cell space after subtracting the basal rates. Basal rates are those measured before ammonia infusion. It is thus assumed that the increments caused by lactate or pyruvate plus ammonia are due solely to the transformation of these substrates in the corresponding cell space. In all cases the values at the end of the substrate infusion period (50 min for pyruvate and 70 min for lactate) were used. The results are summarized in Figs. 5 and 6 for each metabolic flux and condition. All data were represented against the rate of ammonia infusion.


Figure 5
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  		Fig. 5. Changes in glucose production (A and B) and oxygen uptake (C and D) due to lactate plus ammonia or pyruvate plus ammonia as a function of the arterial ammonia infusion in antegrade (bullet) and retrograde ({circ}) perfusion. Data from experiments such as those illustrated by Figs. 3 and 4 were used. The changes correspond to the final flux in the presence of both lactate and ammonia (70 min perfusion time in Fig. 3) or pyruvate and ammonia (50 min perfusion time in Figs. 4) subtracted from the basal flux (no substrates, 0–10 min perfusion time in Figs. 3 and 4). All values were expressed as µmol per minute per ml cell space that is accessible via the hepatic artery in each perfusion mode. These spaces are 0.684 and 0.266 ml/g in antegrade and retrograde perfusion, respectively (14, 15). Data were analyzed by variance analysis; asterisks indicate significant differences at the 5% level.

 

Figure 6
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  		Fig. 6. Changes in urea (A and B) and alanine (C and D) production due to lactate plus ammonia or pyruvate plus ammonia as a function of the arterial ammonia infusion in antegrade (bullet) and retrograde ({circ}) perfusion. Data from experiments such as those illustrated by Figs. 3 and 4 were used. The changes correspond to the final flux in the presence of both lactate and ammonia (70 min perfusion time in Fig. 3) or pyruvate and ammonia (50 min perfusion time in Fig. 4) subtracted from the basal flux (no substrates, 0–10 min perfusion time in Figs. 3 and 4). All values were expressed as µmol per minute per ml cell space that is accessible via the hepatic artery in each perfusion mode. These spaces are 0.684 and 0.266 ml/g in antegrade and retrograde perfusion, respectively (14, 15). Data were analyzed by variance analysis; asterisks indicate significant differences at the 5% level.

 
As presented in MATERIALS AND METHODS, the normalized increments in retrograde perfusion (Jret) reflect the periportal region, whereas the increments in antegrade perfusion (Jant) correspond to a weighted mean over the whole liver parenchyma. Figure 5A reveals higher rates of glucose production from lactate in retrograde perfusion at low ammonia concentrations. Since the rate of lactate infusion produced saturating concentrations of this substrate, the observation that Jret > Jant also means predominance of gluconeogenesis in the periportal region. This corroborates previous observations (7). Confirming previous notions, ammonia inhibited glucose production (34, 41), but its action was more pronounced in retrograde perfusion, so that the difference in Jret and Jant diminished progressively as the ammonia infusion was increased. At high ammonia infusion rates the difference between Jret and Jant vanished. The inhibitory action of ammonia on gluconeogenesis from lactate is, thus, stronger in the periportal region. For the oxygen uptake increment caused by lactate (Fig. 5C), the situation Jret > Jant was maintained over the whole range of ammonia infusion, meaning thus periportal predominance of the increment in oxygen uptake. Confirming a previous communication (7), glucose production from saturating pyruvate concentrations was similar in periportal and perivenous cells in the absence of exogenously added ammonia, as revealed by Fig. 5B. With the increasing rates of ammonia infusion, glucose production was inhibited, with a stronger effect in antegrade perfusion. It is worth to remark that this is opposite of what occurred with gluconeogenesis from lactate. The result was that at high rates of ammonia infusion, Jret was superior to Jant, meaning a shift toward periportal predominance under these conditions. The oxygen uptake increments due to pyruvate (Fig. 5D) behaved similarly to glucose production: no difference between Jret and Jant at low rates of ammonia infusion, but a clear tendency toward periportal predominance at high infusion rates.

Urea production with lactate plus ammonia (Fig. 6A) was a saturating function of the ammonia infusion rate in both antegrade and retrograde perfusion. For the whole range of ammonia infusion rates, Jant was superior to Jret, meaning thus predominance in cells situated downstream to the periportal region in the physiological direction. Alanine production from lactate plus ammonia (Fig. 6C) in antegrade and retrograde perfusion were saturating functions of the ammonia infusion rate with a kind of "substrate inhibition" phenomenon at the highest infusion rate of ammonia. Perivenous predominance is clear, however, because of the fact that Jant was always superior to Jret for the whole range of ammonia infusion rates. The increments in urea production with pyruvate plus ammonia revealed clear saturating functions of the ammonia infusion rate (Fig. 6B). Jret and Jant were only slightly different, with a clear tendency of equalizing at high rates of ammonia infusion. By simple inspection it is difficult to conclude about zonation, a more precise model analysis being indispensable (see Model analysis). Figure 6D shows alanine production in antegrade and retrograde perfusion as saturating functions of the ammonia infusion rate; Jant was clearly superior to Jret for the whole range of ammonia infusion rates, meaning perivenous predominance.

Glutamate and glutamine production as a function of the ammonia infusion rates. It is not advisable to normalize glutamine production in terms of the whole cell spaces that are accessible via the hepatic artery because this compound is produced by a relatively small number of cells, probably only by hepatocytes immediately surrounding the hepatic venules (18), by Kupffer cells (4) and endothelial cells (29), as illustrated by the scheme in Fig. 2. For this reason the production of these two metabolites was simply expressed as micromoles per minute per gram wet liver weight and represented against the ammonia infusion rates. The results are shown in Fig. 7. The rates of glutamate release were low. There was no defined increment upon ammonia infusion, except when pyruvate was the substrate in antegrade perfusion (Fig. 7B). Glutamate production in antegrade perfusion was always superior to that in retrograde perfusion. Glutamine production from lactate plus ammonia was a saturable function of the ammonia infusion rate in antegrade perfusion and increased linearly with the infusion rate in retrograde perfusion (Fig. 7C). Surprisingly, this difference in behavior produced higher rates of glutamine production in retrograde compared with antegrade perfusion at the highest rate of ammonia infusion. Glutamine production from pyruvate plus ammonia, however, was always less pronounced in retrograde perfusion (Fig. 7D).


Figure 7
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  		Fig. 7. Changes in glutamate (A and B) and glutamine (C and D) production due to lactate plus ammonia or pyruvate plus ammonia as a function of the arterial ammonia infusion in antegrade (bullet) and retrograde ({circ}) perfusion. Data from experiments such as those illustrated by Figs. 3 and 4 were used. The changes correspond to the final flux in the presence of both lactate and ammonia (70 min perfusion time in Fig. 3) or pyruvate and ammonia (50 min perfusion time in Fig. 4) subtracted from the basal flux (no substrates, 0–10 min perfusion time in Figs. 3 and 4). All values were expressed as µmol per minute per gram liver wet weight.

 
Model analysis. Interpretation of the data in Figs. 5 and 6 can be refined by model analysis that takes into account the different substrate concentrations in the two regions of the sinusoidal bed according to the scheme shown in Fig. 1. Equations 3 and 4 can be fitted to the experimental data to determine the various parameters. These equations represent Michaelian saturation functions, and they will produce reliable parameters only if the experimental data also conform to the same function. Visual inspection of the various relationships in Figs. 5 and 6 reveals that the curves of urea and alanine production from pyruvate vs. the ammonia infusion rates (Fig. 6, B and D) are typical saturation curves. Alanine production from lactate (Fig. 6C) is not adequate because it presents substrate inhibition and urea production from lactate is near to saturation with the lowest rate of ammonia infusion. On the basis of these criteria the data on alanine and urea production from ammonia plus pyruvate were chosen for the analysis and Eqs. 3 and 4 were fitted simultaneously to the experimental data. A nonlinear least squares procedure was utilized and the results are illustrated by Fig. 8. The optimized parameters that were obtained from the fitting procedure are listed in the legend to Fig. 8. Actually two sets of equations were used, one for alanine and the other one for urea. In the legend to Fig. 8 the kinetic parameters for alanine and urea can be distinguished by their different subscripts, namely KA1, KA2, VA1, and VA2 for alanine production and KU1, KU2, VU1, and VU2 for urea production. As explained in MATERIALS AND METHODS, the subscript 1 refers to the region between the presinusoidal and intrasinusoidal arterioportal confluences and the subscript 2 to the region situated downstream to the intrasinusoidal confluence in antegrade perfusion (Fig. 1). Figure 8 reveals good agreement between experiment and theory. The computed arterial flow that reaches the intrasinusoidal confluence (FAIS, 0.205 ml·min–1·g–1) is realistic compared with the total experimental arterial flow (0.35 ml·min–1·g–1). The maximal rates for alanine production in both regions are different, the VA2/VA1 ratio of 2.33 fully corroborating the conclusion that alanine production is faster in the perivenous region. The apparent Michaelis constants for alanine production are in the milimolar range with a KA2/KA1 ratio of 0.68. For urea production, the maximal rates are favoring the periportal region with a VU2/VU1 ratio of 0.63. However, the apparent Michaelis constants also differ considerably, with a KU2/KU1 ratio of 0.29. This means that predominance of urea production in one of the two regions along the hepatic acinus that can be differentiated by the present experimental approach is a function of ammonia concentration. This prediction can be tested by the simulations shown in Fig. 9. These graphs present the saturation curves of alanine and urea production as functions of the extracellular ammonia concentration for the regions along the hepatic acinus that can be differentiated by the present experimental approach. The simulations were done by calculating Michaelian functions with the kinetic parameters obtained in the fitting procedure illustrated by Fig. 8. As expected, for alanine production perivenous predominance is clear for the whole range of ammonia concentration. For ureagenesis, however, the distribution over the liver parenchyma is a function of the ammonia concentration: the perivenous predominance at low ammonia concentrations shifts progressively toward periportal predominance at high concentrations.


Figure 8
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  		Fig. 8. Experimental and theoretical curves of alanine (A) and urea (B) production due to pyruvate and ammonia infusion in antegrade (Jant) and retrograde (Jret) perfusion. Equations 3 and 4 were fitted simultaneously to the experimental urea and alanine production in antegrade and retrograde perfusion shown in Fig. 6, B and D, respectively. The points represent the experimental values and the continuous lines were calculated according to Eqs. 3 and 4 by using the parameters that were optimized by means of a nonlinear least-squares procedure (Scientist). The optimized parameters ± the corresponding standard deviations were the following (see text for definitions): FAIS = 0.205 ± 0.046 ml·min–1·g–1; KA1 = 1.897 ± 0.110 mM; KA2 = 1.294 ± 0.290 mM; VA1 = 0.421 ± 0.298 µmol min–1 ml–1; VA2 = 0.979 ± 0.114 µmol min–1 ml–1; KU1 = 2.606 ± 0.142 mM; KU2 = 0.754 ± 0.034 mM; VU1 = 2.154 ± 0.100 µmol min–1 ml–1; VU2 = 1.355 ± 0.263 µmol min–1 ml–1. The experimentally determined parameters were FT = 3.5 ml·min–1·g–1; FA = 0.35 ml·min–1·g–1; r = 0.38. The standard deviation of the estimate was 0.0180 and the coefficient of determination 0.998.

 

Figure 9
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  		Fig. 9. Expected alanine and urea production from pyruvate and ammonia in 2 different zones along the hepatic parenchyma as a function of the extracellular ammonia concentration. JA1 and JU1 are the alanine and urea production in the periportal region situated upstream to the intrasinusoidal confluence, whereas JA2 and JU2 are the corresponding values in the region situated downstream to the same confluence (see Fig. 1). The curves represent Michaelis-Menten functions, calculated using the optimized parameters listed in the legend to Fig. 8.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The method employed in the present work was originally proposed as an advantageous alternative for studying metabolic zonation (33). The main advantage is the maintenance of the microcirculation and the organ integrity, meaning also maintenance of intercellular cooperation. The recent proposition of a simple model integrating the main microcirculatory features and the differential action of agents and substrates along the hepatic acinus has facilitated the interpretation (6). This model, which has been adapted to the particular experimental protocol utilized in the present work, deals basically with saturating phenomena that can be complex functions of the rates of substrate or inhibitor infusion. Simulation frequently provides a useful basis for interpretation (6). When the model can be fitted to the experimental data because they conform to the simple Michaelis-Menten relation, however, the method allows a fine resolution of the differential effects of a given substrate along the hepatic acini. In the present work this was the case of urea and alanine production from pyruvate plus ammonia as a function of the ammonia concentration. Apparent Michaelis-Menten kinetics with respect to the extracellular substrate concentration is a frequent phenomenon even when the final product results from a long and complex pathway as it is the case of gluconeogenesis and ureagenesis (see Fig. 2). Hepatic gluconeogenesis from lactate, for example, obeys Michaelis-Menten kinetics with respect to the extracellular lactate concentration in the presence and absence of oleate (20). In hepatocytes, ammonia uptake and urea production have also been found to obey apparent Michaelis-Menten kinetics with respect to the extracellular ammonia concentrations (10). The reason for this kind of behavior is that the limiting step or steps also follow Michaelis-Menten kinetics and, under steady-state conditions, the intracellular concentration is generally proportional to the extracellular one. For gluconeogenesis from lactate and pyruvate the limiting step is probably catalyzed by pyruvate carboxylase (20); in the case of ammonia uptake and transformation into urea the phenomenon probably reflects the kinetics of the main enzymes responsible for ammonia detoxification, which are carbamoyl-phosphate synthase (13, 22) and glutamate dehydrogenase (35). Alanine aminotransferase also follows Michaelis-Menten kinetics (21), a phenomenon that could be contributing to the apparent Michaelis-Menten kinetics of alanine production from pyruvate found in the present work. Alanine production from lactate, however, presented substrate inhibition at high lactate concentrations, a phenomenon that frequently superimposes on the Michaelis-Menten kinetics (37) and whose reasons are difficult to identify with the available data.

The parameters KA1, KA2, VA1, and VA2 for alanine production and KU1, KU2, VU1, and VU2 for urea production that were obtained by fitting Eqs. 3 and 4 to the experimental data are complex functions of several variables within the cell. Despite this, they also have a simple physiological meaning in that VA1, VA2, VU1, and VU2 correspond to the maximal attainable rates in the specific regions. In a similar way, the parameters KA1, KA2, KU1, and KU2 correspond to the concentrations of ammonia producing these half-maximal rates in the same regions, which can be regarded as indicators for the apparent affinity of each metabolic pathway for the extracellular precursor. Since multisubstrate regulatory enzymes are involved, these parameters are very complex functions (37). The parameters KA1, KA2, KU1, and KU2 are functions of the concentrations of the intracellular substrates participating in the limiting-step reactions as well as the concentrations of the reaction products. They are also functions of the concentrations of modulators if they act by changing the substrate affinity (13, 37). The same occurs with the maximal rates, which are functions of the intracellular substrate concentrations as well as of the enzyme activities (37). The fact that KU1 and KU2 differed substantially (see legend to Fig. 8) can thus be regarded as a consequence of the different concentrations of substrates and modulators in periportal and perivenous cells.

In addition to the apparent affinity constants for ammonia along the hepatic acinus, fitting of Eqs. 3 and 4 to the data also allowed us to obtain an estimate of the arterial flow that reaches the intrasinusoidal confluence (FAIS in Eqs. 3 and 4; see Fig. 1). The actual value that was obtained, 58.5% of the total arterial flow, is quite realistic and confirms previous conclusions from experiments in which the fractional extraction of arterially infused ATP was investigated (31). In practical terms the determination of FAIS allows calculation of the sinusoidal concentrations of the substrates infused into the hepatic artery in the regions before and after the intrasinusoidal arterioportal confluence (Fig. 1), by use of the formulations discussed in MATERIALS AND METHODS. For an infusion rate of 21 µmol·min–1·g–1, the lactate concentration reaching the region situated before the intrasinusoidal confluence can be estimated as Sin/(FTFA/FAIS) = 3.51 mM in retrograde perfusion and Sin/[FTFA/(FA – FAIS)] = 2.49 mM in antegrade perfusion. In the region situated after the intrasinusoidal confluence in antegrade perfusion, the lactate concentration reaching the sinusoidal can be estimated to be Sin/FT = 6 mM. Similar calculations can be done for the pyruvate infusion rate of 8.5 µmol·min–1·g–1. The pyruvate concentrations reaching the sinusoids can be estimated as 1.42 and 1.0 mM in the region before the intrasinusoidal confluence in retrograde and antegrade perfusion, respectively, and 2.43 mM in the region after the intrasinusoidal confluence in antegrade perfusion. All these concentrations are saturating for lactate and pyruvate metabolism (20, 42).

Besides confirming the general notion of an unequal distribution of the metabolic pathways along the hepatic acinus, the results of the present work also reveal that this distribution is under short-term control and it can even be changed by factors acting in the opposing direction of medium- and long-term factors. As predicted by enzyme activity measurements, gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region of liver parenchyma. Periportal hepatocytes are in principle better equipped than perivenous hepatocytes for gluconeogenesis and oxidative processes. Compared with perivenous hepatocytes, periportal cells have higher activities of key enzymes of gluconeogenesis as well as a greater volume of mitochondria and a greater area of cristae (16, 26, 27). However, this periportal predominance of gluconeogenesis and the associated extra oxygen consumption is not unconditional. Besides the absence of zonation when an oxidized state is induced by pyruvate infusion (7), it is also evident from the observations that zonation can be abolished by ammonia when lactate is the substrate or induced by ammonia when pyruvate is the substrate. If one considers that the physiological condition is characterized by high lactate and low pyruvate concentrations and low ammonia concentrations, gluconeogenesis will predominate in the periportal region although the difference will not be as pronounced as if ammonia were almost absent (7).

The absence of predominance of alanine synthesis from lactate and pyruvate plus ammonia in the periportal region is an unexpected phenomenon by virtue of the reported predominance of alanine aminotransferase in the periportal region (1, 5, 11, 32, 36, 40). This observation is a clear indication that data on enzyme activity or expression alone cannot be extrapolated unconditionally to the living cell. In this specific case it is apparent that under real cellular conditions the activity of the alanine aminotransferase does not conform to the activities measured under artificial conditions. Most likely the conditions surrounding the alanine aminotransferase in periportal and perivenous cells are substantially different, producing also different net fluxes. Substrate availability is an important factor influencing both the reaction rate and the direction of the net flux. In the direction of alanine synthesis the substrates are pyruvate and glutamate (see Fig. 2). Pyruvate was constantly infused in the present work so that its availability was not a limiting factor. Glutamate, however, has to be produced by the amination reaction catalyzed by glutamate dehydrogenase, which is an enzyme operating at near-equilibrium conditions. This enzyme is expressed by all hepatocytes and there are observations indicating either a uniform distribution (5) or perivenous predominance (19). Irrespective of the acinar distribution, however, it has been hypothesized that the perivenous glutamate dehydrogenase is highly active in the direction of glutamate production whereas the opposite occurs in periportal hepatocytes (5). The reduced alanine production from lactate or pyruvate plus ammonia in the periportal hepatocytes observed in the present work could thus be reflecting a lower flux through glutamate dehydrogenase in the amination direction, generating also a reduced flux of glutamate in the direction of pyruvate transamination. This interpretation should be regarded as one of several possibilities needing confirmation by additional experimental work.

No predominance of ureagenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. Especially in the presence of lactate and low ammonia concentrations, conditions close to the physiological ones, the mean ureogenic activity of the whole liver parenchyma clearly exceeded that of the periportal cells, indicating that ureagenesis is more active in hepatocytes localized downstream to the periportal region in the antegrade direction. Absence of periportal predominance of ureagenesis was also found in experiments in which alanine was used as the sole carbon and nitrogen source (6). In these experiments no ammonia was infused and there was a net production of this metabolite in consequence of alanine deamination. Ammonia release from alanine was clearly superior in the periportal region, with Jret/Jant ratios between 3.83 and 3.12. These observations are analogous to the findings of the present work in that they also indicate that the periportal ureogenic activity is relatively low and needs to be strongly complemented by urea synthesis in perivenous cells.

Periportal predominance of the activity and expression of key enzymes of the urea cycle have been found in several studies (9, 17). However, the enzymes of the urea cycle are present in more than 90% of the parenchymal cells. The key enzyme carbamoyl-phosphate synthase, for example, seems to be absent only from those hepatocytes immediately surrounding the hepatic venules (17, 18). The hepatocytes not containing carbamoyl-phosphate synthase are precisely those containing glutamine synthetase (see Fig. 2). The latter have been estimated to comprise maximally 7% (18). Most hepatocytes, thus, are perfectly able to synthesize urea and the final ureogenic activity will depend not only on the maximal activity (which is that detected under artificial conditions) but also on the real cellular conditions in terms of the concentrations of substrates and allosteric regulators. Since there are several enzymes involved, there are also several regulatory possibilities, which, in the absence of direct evidence, can only be discussed as more or less likely possibilities. It could be, for example, that the conditions in periportal cells are less favorable than in downstream localized cells for the production of N-acetylglutamate, the key activator of carbamoyl-phosphate synthase (13, 30). This could result from a reduced amination of 2-oxoglutarate by the glutamate dehydrogenase in periportal hepatocytes, as already mentioned above when discussing the lower rates of alanine production in the periportal region. It should also be remembered that the N-acetylglutamate synthase is itself a regulatory enzyme. As such, it is subject to regulatory mechanisms with participation of arginine (28) and ornithine (12), whose concentrations are not necessarily equal along the hepatic acinus. Even for aspartate, which is an essential amine group donor for the urea cycle, there can be no certainty about its availability along the hepatic acinus. The activity of the aspartate transaminases seems to predominate in periportal cells (1, 5), but it is already clear from the example of alanine aminotransferase discussed above that this is not an imperative for enhanced aspartate production. In this particular case, the substrates for aspartate production, glutamate and oxaloacetate, could be limiting, the former for the same reasons already discussed above and the latter because of the high gluconeogenic activity, especially in the presence of lactate.

The production of glutamine in the periportal region from lactate or pyruvate plus ammonia, similar to the reported glutamine production from alanine (6), is unlikely to occur in periportal hepatocytes. This synthesis occurs more likely in periportal Kupffer and endothelial cells, in which the glutamine synthetase has been detected (4, 29), as illustrated by Fig. 2. As already stated, it is not advisable to analyze glutamine production in terms of the space-normalized parameters Jant and Jret because glutamine is certainly coming from very small cellular spaces compared with the whole liver. Furthermore, recycling along the hepatic acini is highly probable so that the normalized rates of glutamine production would be poor indicators of an heterogeneous distribution of its production. The results seem thus be indicating that Kupffer and endothelial cells localized in the periportal region are perfectly able to produce glutamine as indeed expected from the probable presence of glutamine synthetase in these cells (4, 29). Alternatively, a fraction of the glutamine production in retrograde perfusion could be the result of a decreased hydrolysis of the glutamine produced in perivenous hepatocytes from endogenous sources. This is unlikely, however, if one considers that ammonia is well known for its stimulatory effect on glutaminase (24).

The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureagenesis in periportal cells (25). It certainly continues to be a valid assumption that the perivenous cells immediately surrounding the hepatic venules, which contain glutamine synthetase and do not contain carbamoyl-phosphate synthase, are able to remove ammonia solely by glutamine synthesis (17, 18). However, the fact that ureagenesis, under some specific conditions, can be more active in cells situated downstream to the periportal zone strongly suggests that this pathway is also an important ammonia-detoxifying mechanism in the perivenous region excepting only the small fraction of cells deprived from carbamoyl-phosphate synthase.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and from the Fundação Araucária (PRONEX).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Bracht, Laboratory of Liver Metabolism, Dept. of Biochemistry, Univ. of Maringá, 87020900 Maringá, Brazil (e-mail: adebracht{at}uol.com.br)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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