Aminopyrine N-demethylation by rats with liver cirrhosis

GASTROENTEROLOGY

1987;93:719-26

Aminopyrine IV-Demethylation by Rats With Liver Cirrhosis Evidence for the Intact Cell Hypothesis. A Morphometric-Functional Study J. REICHEN, B. ARTS, U. SCHAFROTH, Th. B. ZELTNER, and T. ZYSSET Departments of Clinical Berne, Switzerland

Pharmacology,

Pathology,

The intact cell hypothesis states that a reduced normal hepatocytes, tonumber of intrinsically gether with hemodynamic alterations, explains decreased drug metabolism in cirrhosis. We explored this hypothesis by comparing results of the aminopyrine breath test with in vitro measurements of aminopyrine N-demethylation and morphometrically determined liver cell volume in a rat model of cirrhosis. Aminopyrine N-demethylation in vivo (ABT-k) was 0.98 ? 0.10/h (mean + SD) in controls. The cirrhotic rats were separated into those with normal (NCR) and those with abnormal ABT-k (PCR). Microsomai aminopyrine N-demethylase averaged 2.08 + 0.77 and 2.09 + 0.54 pmollmin in controls and NCRs, respectively; it was reduced to 1.00 ? 0.81 pmol/min (p < 0.02) in PCRs. Morphometrically determined hepatocellular volume was 18.8 + 2.8, 17.1 ? 1.9, and 11.6 + 6.1 ml in controls, NCRs, and PCRs, respectively, PCRs being lower than controls (p < 0.01) and NCRs (p < 0.05). When N-demethylase and cytochrome PJsO were related to hepatoceilular volume (in milliliters), no significant difference between the three groups was apparent. We conclude that reduced aminopyrine N-demethyReceived December 12, 1986. Accepted April 21, 1987. Address requests for reprints to: Jiirg Reichen, M.D., Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland. B. Arts was supported by a grant from the Stipendiafund K.N.M.P., The Hague, The Netherlands; from the University of Utrecht, The Netherlands; and from the Berne Liver Foundation. J. Reichen was supported by grant 3.823.9.84 and was the recipient of a Research Career Development Award (3.731.0.82) from the Swiss National Foundation for Scientific Research. The authors thank Mrs. Bruderer and H. Saegesser for technical assistance, M. Kappeler for preparation of the artwork, and R. Steiner for secretarial assistance. 0 1987 by the American Gastroenterological Association 0016-5085/87/$3.50

A. ZIMMERMANN,

and Anatomy,

University

of Berne,

lation in progressed cirrhosis is mainly due to a loss of liver cell volume. The function per liver cell volume remains constant, however, thus favoring the intact cell hypothesis for the handling of slowly metabolized compounds such as aminopyrine. The intact cell hypothesis has been formulated to explain impaired drug metabolism in liver cirrhosis (1). It states that a reduced number of intrinsically normal (“intact”) hepatocytes together with hemodynamic alterations, such as intrahepatic shunts alone, is sufficient to account for the abnormal function of the cirrhotic liver (1). The influence of hemodynamic alterations on the disposition of high extraction compounds has been investigated both in rats (1) and humans (z-3). The latter study demonstrated that intrinsic clearance becomes rate-limiting in cirrhosis (3). Reduced intrinsic clearance has been demonstrated in isolated hepatocytes for the disposition of high extraction compounds, such as propranolol (4). In contradistinction to the intact cell hypothesis, “the sick cell hypothesis” postulates an abnormal function of the individual hepatocyte. Evidence for the sick cell hypothesis has recently been provided by the demonstration of an uptake defect for taurocholate, an endobiotic whose uptake is also flow-limited (5). Studies in isolated hepatocytes have the advantage of eliminating hemodynamic factors in the assessment of drug metabolism; thus studies showing impaired clearance of xenobiotics and endobiotics in isolated hepatocytes (4,5) would seem to favor the Abbreviations used in this paper: ABT, aminopyrine breath test; ABTk, slope of the decline in “‘CO, radioactivity versus time; NCR, normal cirrhotic rat (normal ABT-k); PCR, pathologic cirrhotic rat (abnormal ABT-k).

720

REICHEN ET AL.

CASTROENTEROLOGY

sick over the intact cell hypothesis. The yield of hepatocytes isolated from cirrhotic livers is dramatically reduced, however (5), and a sampling artifact therefore cannot be ruled out. One of the main factors in the intact cell hypothesis, namely the reduction in liver cell mass, has never been quantified in conjunction with functional studies. We therefore investigated the metabolism of aminopyrine in vivo and in vitro in rats rendered cirrhotic by chronic exposure to phenobarbital and carbon tetrachloride (6). Hepatocellular volume in the cirrhotic rats and appropriate controls was quantitated by morphometric means.

Materials and Methods Induction

of Cirrhosis

Male Sprague-Dawley rats with an initial body weight of between 125 and 150 g were obtained from the Deutsche Versuchstierfarm Hartmutt-Voss, Tuttlingen, F.R.G. They were maintained on a standard rat diet with free access to tap water in a temperatureand humiditycontrolled animal quarters under a 12-h light/dark cycle. Cirrhosis was induced by chronically exposing 12 rats to phenobarbital and carbon tetrachloride according to McLean et al. (6). Treatment was carried out for 10 consecutive weeks and was ceased 2 wk before the study. A control group (n = 9) consisted of untreated or phenobarbitaltreated littermates. Because the two control groups did not differ in any of the biochemical, functional, oi morphologic parameters studied (data not shown), they were treated as one group for statistical purposes. Aminopyrine

Breath

Test

The aminopyrine breath test (ABT) was performed, as previously described from this institution (71, after intraperitoneal administration of a tracer dose (1 &i/kg body wt) of [14C]dimethylaminoantipyrine (Amersham International, Buckinghamshire, U.K.; sp act 120 mCi/ mmol). Exhaled 14C02 was quantitatively collected over lo-min periods for 2 h; the ABT was characterized by calculating the slope of the decline in 14COZ radioactivity versus time (ABT-k) (7-8). The cirrhotic animals were divided into two groups according to their ABT-k as follows: [a) those with an ABT-k similar to controls [within 2 SD) were termed “normal” cirrhotics (NCRs) and (b) those with an ABT-k 2 SD below the mean ABT-k of controls were termed pathologic cirrhotics (PCRs). Microsomal

Assays

One day after performing the ABT, the animals were anesthetized with ether and killed by exsanguination after a blood sample had been drawn from the abdominal aorta. The liver was then flushed with ice-cold heparinized physiologic saline, removed, weighed, and placed in ice-cold phosphate buffer, 0.1 M, pH 7.4. After random sampling for morphometric analysis (see below), about one-half of the liver was further processed for biochemical analysis.

Vol. 93, No. 4

The specimens for biochemical analysis (610 g) were pooled and homogenized in 3 vol of ice-cold sucrose (0.25 M) in phosphate buffer by 12 strokes of a loose-fitting pestle in a Dounce homogenizer. The microsomal fraction was prepared by differential centrifugation according to Mackinnon et al. (9). Cytochrome PJsO content and cytochrome c reductase activity were determined by spectrophotometric methods (10,ll). Glucose-6-phosphatase activity was measured by estimating the free phosphate liberated in the enzymatic reaction (12). Aminopyrine N-demethylase kinetics were assayed in a reaction mixture containing 1 ml microsomal suspension containing 2-3 mg microsomal protein, glucose-6-phosphate (5 pmol), nicotinamide adenine dinucleotide phosphate [l pmol), semicarbazide (5 wmol), magnesium chloride (5 pmol), and glucose-6-phosphate dehydrogenase (2 IU) in a total incubation volume of 3 ml. Aminopyrine concentration varied from 0 [blank) to 16 mM. Flasks were preincubated at 37°C in a shaking water bath; the reaction was started by adding the microsomal suspension. It was stopped after 15 min with 1 ml trichloroacetic acid (15% wtivol). Trapped formaldehyde was assayed by the Nash method (13). Blank values were subtracted for each preparation. Total conjugated bile acid levels in serum were estimated by a commercially available radioimmunoassay (Becton-Dickinson, Orangeburg, SC.). Protein content was estimated according to Lowry et al. (14). Morphometric

Analysis

Morphometric analysis was performed as described earlier from this laboratory (15). Briefly, after vigorous systematic random sampling of five l-cm’ pieces (16) the tissue was embedded in paraffin, cut into 4-pm sections, and stained with both hematoxylin-eosin and Masson trichrome. Morphometric examinations were performed blindly by one of the authors (U.S.) on the sections stained with Masson trichrome at a magnification of x400. A point counting procedure (17) was employed, counting 1.000-1.800 points per liver. Each point was classified as overlying either hepatocyte, bile duct, nonhepatocyte/nonbile duct parenchyma, sinusoid, nonparenchymatous blood vessel, or connective tissue. Volume fractions were calculated as the fraction of points overlying any given compartment over the total number of points counted (17). The respective absolute volumes were calculated as the product of the volume fractions and liver volume.

Statistical

Evaluation

Results are expressed as mean * 1 SD. Group means were compared by analysis of variance, followed by Student’s t-test if the former showed significance (18). Linear regression analysis was performed by the method of least squares (18). Enzyme kinetics were analyzed in terms of the Michaelis-Menten equation using a nonlinear leastsquares fit (19). Intrinsic clearance was calculated as the ratio V,,,iK,. A probability value of co.05 was considered statistically significant.

AMINOPYRINE N-DEMETHYLATION IN CIRRHOTIC RATS

October 1987

Y

721

p
-n.*.-

rp<0.0011

Controle A

1.0 Ml-k

n

(h-‘1

0 Cirrhotlcs:

?? ABT-k ??MT-k

2 4 5

10

15

Hopatocdlular OJ

Figure

PCR

NCR

Controls

1. Aminopyrine N-demethylation (ABT-k) in control rats (0) and rats with maintained function (A, NCR] and impaired function (A, PCR). Mean * SD values are given.

Results The results of the ABT in vivo are shown in Figure 1. ABT-k did not statistically differ between cirrhotic and control animals (0.98 2 0.10 vs. 0.79 f 026/h; p > 0.05). The cirrhotic animals were separated arbitrarily into a group with normal ABT-k (NCRs) that had an ABT-k undistinguishable from controls (0.97 ? 0.09 vs. 0.98 + 0.10/h; p BO.90) and a second group (PCRs) with an ABT-k I mean - 2 SD of controls (0.55 f. 0.22/h); this group differed significantly from both controls (p < 0.002) and from NCRs (p < 0.001). All animals termed cirrhotic had cirrhosis by macroscopic inspection. This was histologically confirmed in all rats. The organ weights of the different groups are given in Table 1. The body weight of control animals did not differ from that of NCRs but was significantly higher than that of the PCRs (Table 1). The liver weights did not differ statistically among the three groups. Spleen weights were increased 20% and 30% in NCRs and PCRs, respectively, but only the increase in PCRs comTable

1.

Body and Organ Weights in Control Animals and Cirrhotic Animals With Normal and Lowered Slope of the Decline in “C02 Radioactivitv Versus Time NCR

Control iody weight (g) Liver weight (g) Spleen weight (g)

619 ?9

66

583

27 37

PCR 487

*5 129”

21.8

2 3.2

21.1

” 2.6

15.9

? 7.1

0.95

f

1.13

f

1.46

? 0.33b

0.18

0.24

NCR, normal cirrhotic rat (normal ABT-k); PCR, pathologic cirrhotic rat (abnormal ABT-k). Values are given as mean 2 1 SD. ‘p < 0.025 versus controls. bp < 0.002 versus controls.

20

normd decroaaed

25

volume bnl)

Figure 2. Morphometrically determined hepatocellular volume in the three groups of rats. Hepatocellular volume was significantly decreased in PCRs compared to both NCRs and controls (see Table 4).

pared to controls was statistically significant (Table 1). Serum levels of total conjugated bile acids averaged 3.5 + 2.2, 7.9 + 3.3, and 16.1 + 10.6 pmol/L in controls, NCRs, and PCRs, respectively. It separated both NCRs (p < 0.01) and PCRs (p < 0.002) from controls, but did not differentiate between the two cirrhotic groups (p > 0.05). Microsomal protein recovery and the relative specific activity of the microsomal marker enzymes are given in Table 2. The relative specific activities were similar in the three groups, documenting that induction of cirrhosis did not invalidate the differential centrifugation procedure to harvest microsomes. Microsomes prepared from PCRs had reduced activities (per milligram of protein) of cytochrome Ph5,,, cytochrome c reductase, and glucose-6-phosphatase (Table 2).

OJ 0

a

4

q”t

(ml/mlnl

Figure 3. Relationship between aminopyrine metabolism in vivo (ABTk) and in vitro (intrinsic clearance, Clint) in control rats (0) and in cirrhotic rats with maintained (A) and impaired (A) aminopyrine N-demethylation in vivo. The regression line for the whole population (-) is described by y = 0.602 + 0.096x (r = 0.632, p < 0.01). The regression line for the cirrhotic animals (. -_I was y = 0.451 + 0.139x (r = 0.752, p < 0.01).

722

GASTROENTEROLOGY Vol. 93, No. 4

REICHEN ET AL.

Table

2.

Activities

of Marker

Enzymes

of the Endoplasmatic

RSA

0.98 & 0.14 1.02 * 0.15 0.71 * 0.21aJJ

Control NCR PCR

in Control

and

Cytochrome-c-reductase (nmoI/min mg)

Cytochrome P450 (nmollmg) SA

Reticulum

SA 63.5 2 10.9 58.9 * 17.1 36.1 * 10.7b,C

5.2 * 1.2 5.0 + 1.4 3.7 2 1.4

Cirrhotic

Livers

Glucose-6-phosphatase (nmol/h mg) RSA

SA

RSA

6.2 2 1.2 5.9 r 2.1 4.2 2 1.4

13.3 ?I 4.0 14.0 ” 2.1 6.7 2 4.2’,’

4.9 2 1.6 6.3 & 2.7 9.0 f 11.3

NCR, normal cirrhotic rat (normal ABTk); PCR, pathologic cirrhotic rat (abnormal ABT-k). SA, specific activity per milligram of microsomal protein; RSA, ratio of specific activity in microsomes to liver homogenate. Values are given as mean + 1 SD. ’ p < 0.02 versus controls. b p < 0.02 versus normal cirrhotic rat (normal ABT-k). ’ p < 0.001versus controls.

The kinetic parameters of microsomal aminopyrine N-demethylase are given in Table 3. The V,, was significantly reduced in PCRs as compared with both controls and NCRs, whereas K, was unaffected. Intrinsic clearance, calculated as the ratio of V,, (recalculated for the total microsomal fraction), and K, averaged 3.24 + 1.50, 3.30 ? 1.02, and 1.26 f 1.05 ml/min. Intrinsic clearance was significantly decreased in PCRs as compared with controls (p < 0.025) and NCRs (p c O.Ol), whereas the difference between controls and NCRs was not statistically significant (p > 0.45). The morphometric characteristics of the cirrhotic livers match with their histologic appearance in that the respective volume fractions of connective tissue and of bile duct structures were significantly increased in all cirrhotic animals at the expense of parenchymal structures (Table 4). Within the cirrhotic animals the NCR and PCR groups could be distinguished on the basis of their volumetric liver composition. Animals with a reduced ABT-k seemed to show a more pronounced degree of cirrhosis as estimated by their significantly reduced fraction of liver parenchyma and their correspondingly increased density of connective tissue.

Table

3.

Aminopyrine

N-Demethylase

Activity

A considerably different picture emerged, however, when the absolute volumes (volume fraction x liver volume) were considered. Again, the compartments of connective tissue and of bile ducts were significantly increased in cirrhotics when compared with controls. These increases were, however, associated with a reduction of hepatocellular mass only in PCRs, whereas NCRs had preserved a hepatocellular mass in the dimension of the control animals. Thus, the only significant difference in total liver composition between NCRs and PCRs was the volume of hepatocytes (Table 4, Figure 2). The relationship between ABT-k and intrinsic clearance is shown in Figure 3. Neither controls (r = 0.290) nor NCRs (r = 0.280) showed a significant correlation when calculated alone, whereas intrinsic clearance and ABT-k were significantly correlated in PCRs, the slope being much steeper than that of the overall curve (y = 0.316 + 0.182x; r = 0.847, p < 0.05). The same held true when the two cirrhotic groups were pooled (y = 0.451 + 0.139x; r = 0.752, p c 0.01). Both ABT-k (Figure 4) and intrinsic clearance (Figure 5) exhibited a significant overall correlation with hepatocellular volume. Neither aspect of

in Hepatic Microsomes

Vma?.

Control NCR PCR Significance (p) NCR vs. control PCR vs. control PCR vs. NCR

Intrinsic clearance” (mUmin)

(nmolimin mg protein)”

(/*mol/min)b

6.1 ” 1.6 5.3 + 1.1 3.6 5 1.3

2.08 + 0.77 2.09 2 0.54 1.00 2 0.81

0.72 + 0.27 0.64 2 0.05 0.85 f 0.11

3.24 2 1.50 3.30 t 1.02 1.26 ? 1.05

NS co.02 co.05

NS 10.02 co.02

NS NS NS

NS CO.025 co.005

(5)

NCR, normal cirrhotic rat (normal ABT-k); NS, not significant; PCR, pathologic cirrhotic rat (abnormal ABT-k); V,,, maximal velocity. Values are mean ? 1 SD. “Specific activity expressed per milligram microsomal protein. bActivity recalculated for total microsomal fraction. “V,,/K, per total microsomal fraction.

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REICHEN ET AL.

Discussion Our results report the first structural- functional correlation in a rat model of liver cirrhosis induced by chronic administration of phenobarbital and carbon tetrachloride. Morphometric analysis as well as microsomal function testing with the ABT was used to arbitrarily separate the cirrhotic animals into two groups, one with maintained (NCR) and one with impaired (PCR) microsomal function. Further analysis of the data demonstrated the impaired aminopyrine demethylation in the latter group to be due to loss of hepatocellular volume. Our results also suggest the intact cell hypothesis to be true at least as far as cytochrome PdsO and aminopyrine N-demethylation are concerned. Assessment of microsomal function by means of the ABT has been used extensively both in humans (20-23) and rats (7,8,23). It has proved its value as a diagnostic (20,21,24) and prognostic (25,26) tool in clinical medicine. Interpretation of its result could be confounded by extrahepatic metabolism (27) and by the fact that different isoenzymes of cytochrome Pas,, seem to be responsible for aminopyrine Ndemethylation (28). The potential problem of product inhibition (29) has been circumvented by use of a tracer dose in the in vivo studies. The agreement between ABT-k and intrinsic clearance (Figure 3) suggests that neither of those concerns was a major factor in our results and is in agreement with the literature (7,30). A reduction in intrinsic clearance has been identified as a major factor in explaining decreased drug metabolism in liver cirrhosis (2,3). This has been substantiated by studies in isolated hepatocytes, where confounding hemodynamic factors are excluded (22). Measurement of cytochrome P450-related enzyme activities in liver biopsy specimens seemed to indicate that this was due to a specific loss of microsomal enzymes (31,321, thus favoring the sick over the intact cell hypothesis. Our results, in contrast, suggest that the activity of aminopyrine N-demethylase activity per unit of hepatocyte volume is maintained, thereby providing support for the intact cell hypothesis. Our results are applicable, however, to aminopyrine metabolism only. Thus, Farrell et al. (32) have shown different cytochrome PhsO functions to be affected to different extents in liver biopsy specimens from patients with cirrhosis. Moreover, different models of cirrhosis could affect cytochrome Pas,, content to a different extent (33). Thus, Babany et al. (33) described a normal cytochrome PJsO content in a carbon tetrachloride model similar to ours, whereas cytochrome Pas0 content was decreased in cirrhosis induced by bile duct ligation. In our exper-

GASTROENTEROLOGY

Vol. 93, No. 4

iments, cytochrome PbsO content (per milliliter of hepatocyte volume) was clearly maintained, whereas aminopyrine N-demethylase showed a trend to decrease in the PCR group. Therefore, we cannot exclude the existence of a further subgroup where the sick cell hypothesis holds true. Alternatively, a specific alteration of cytochrome PasO isoenzymes, as demonstrated in a model of nutritional cirrhosis (34), could explain the trend toward differential effects on cytochrome PJ5a and the aminopyrine N-demethylase. Statistically, however, the intact cell hypothesis has to be accepted for the present experiment. In contrast to chronic administration of carbon tetrachloride, acute intoxication with this solvent leads to a decrease in both in vivo and in vitro metabolism of aminopyrine (35). The heterogeneity in the cirrhosis model used in the present study is analogous to the heterogeneity of function observed in humans (36). At present, it is unknown whether genetic differences or other factors are responsible for the different hepatic response in this model. Our data demonstrate that the heterogeneity of functional impairment (Figure 1) was related to differences in hepatocellular volume (Figures 2, 4, and 5). Animals with reduced liver cell volume made up for this loss by an increase in connective tissue, which left organ weight unaltered (Tables 1 and 4). This is in agreement with the only other published report on morphometric analysis of the liver cirrhosis induced by carbon tetrachloride (37). This finding emphasizes that some specific estimate of hepatocellular volume should be obtained when analyzing the function of the cirrhotic liver. The correlation between in vivo and in vitro metabolism of aminopyrine and hepatocyte volume (Figures 4 and 5) suggests that the ABT could serve as such a measure at least in the phenobarbital/ carbon tetrachloride model of liver cirrhosis. This correlation, however, was not found in bile ductligated animals (I 5). The correlation between in vivo and in vitro aminopyrine metabolism (Figure 3) is in agreement with the literature (7,30,35). It must be pointed out, however, that the regression calculated for the whole population differed from that calculated for the cirrhotic animals only. This could reflect the described alteration of substrate binding to cytochrome PasO in cirrhotic animals (38). An alternate explanation could be that aminopyrine N-demethylation in vivo depends on intrinsic clearance (Figure 3) or hepatocellular volume (Figure 4) up to a certain critical value; above this value it depends on some other factor, e.g., liver blood flow (39). In evaluating the morphometric data it has to be pointed out that total liver cell volume was assessed; therefore, the different aspects were related to cell

October 1987

volumes rather than cell number as originally proposed in the intact cell hypothesis (1). Furthermore, the different hemodynamic alterations such as intrahepatic shunting (1) and sinusoidal capillarization (40) were not evaluated in the present investigation. Therefore, it appears likely that our results will apply to compounds with relatively low extraction only. This contention is supported by the lack of correlation between bile acid levels and hepatocellular volume (r = 0.270; data not shown). In conclusion, our studies provide evidence for the intact cell hypothesis with respect to aminopyrine metabolism in carbon tetrachloride-induced cirrhosis in the rat. Heterogeneity of aminopyrine metabolism in cirrhotic rats is due to differences in hepatocellular volume, with the enzymatic content of the unit volume of hepatocytes being maintained. The ABT reflects hepatocyte volume in this animal model of liver cirrhosis.

References 1.

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