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Orotic acid overproduction in experimental cirrhosis of rats
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
Orotic Acid Overproduction J.D.
50,
371-384 (1989)
in Experimental
SHOEMAKER
AND W.J.
Cirrhosis
of Rats
VISEK
University of Illinois, College of Medicine at Urbana-Champaign,
Illinois 61801
Received July 5, 1988, and in revised form October 25, 1988 Ammonia clearance, portal blood ammonia, and amino acid concentrations were studied during induction of cirrhosis by carbon tetrachloride in rats. Exposure to Ccl, vapors twice weekly for 7-16 weeks doubled erotic acid excretion. If exposure was discontinued for 7 days, the erotic acid excretion decreased despite the presence of cirrhosis proven histologically. Replacement of dietary casein with soybean protein eliminated the CC&-induced erotic aciduria in growing rats but not in adults. Supplementation of casein with 1.5% arginine did not prevent CClJnduced erotic aciduria. [i4C]Orotate uptake into RNA and DNA of liver was not impaired. Perfusion of livers of cirrhotic animals with ammonia concentrations between 0.2 and 3.0 mM revealed no significant decreases in urea synthesis rates due to cirrhosis and no increase in the tendency to make erotic acid at a given ammonia concentration. However, ammonia uptake by cirrhotic livers was significantly reduced, resulting in higher ammonia concentrations in the effluent when there was moderateto-severe cirrhosis. Portal blood samples taken from rats exposed to Ccl, had higher ammonia concentrations as cirrhosis worsened. The results lend support to the “intact hepatocyte” hypothesis of cirrhosis which attributes metabolic abnormalities to intrahepatic shunts. 0 19S9 Academic Press, Inc.
INTRODUCTION The impaired capacity of cirrhotic liver to metabolize protein is well recognized (Zieve, 1966; Conn et al., 1977; Fraser and Arieff, 1985) but not well understood. Our previously reported studies show increased urinary erotic acid (OA)’ excretion following three forms of chemical hepatic injury (Visek and Shoemaker, 1986). The present report contains data on urinary OA of rats exposed to CC& according to a modification of the McLean ef al. (1969) “instant cirrhosis” protocol. We have used erotic acid as a marker of urea cycle capacity because OA synthesis is enhanced in the liver when ammonia (NH,+, NH4+) concentrations are elevated (Tremblay et al., 1977; Jones, 1980; Milner and Visek, 1973). The present experiments were conducted to determine if the rise in OA excretion by cirrhotic rats was attributable to decreased utilization of OA in nucleic acid synthesis, portal amino acid imbalances, increased portal ammonia, reduced urea synthesis at near-physiological ammonia concentrations, or a change in the relationship between ammonia and OA production due to other actions of CCL,. The biochemical pathways involved in ammonia and erotic acid metabolism are illustrated in Fig. 1. Carbamyl phosphate (CP) of either intramitochondrial or cytoplasmic origin can be used in OA synthesis (Tremblay et al., 1977; Jones, 1980). The first step in urea synthesis combines ammonia with CO2 to form CP within mitochondria. CP is then combined with omithine to form citrulline. Previous studies have shown that when ammonia production is elevated or urea cycle ’ Abbreviations used: AIN-76A, diet formulated according to the American Institute of Nutrition (1977); BW, body weight; CC14, carbon tetrachloride; CP, carbamyl phosphate; HPLC, highperformance liquid chromatography; OA, erotic acid. 371 0014-4800/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHOEMAKER
372
AND VISEK
MlTOCHONDRlON CYTOPLASM
CYTOPL-
NH4+ 4
HC03-
GLUTAMINE
ATP Y
UREA
CARBAMYL-P ORNlTHlNE
-
---a.
*.
1 CARBAMYL-P
--•,ORNlTHlNE
L ARGININE 1 ARGININOSUCCINATE ASPARTATE t CITRULLINE
UMP
FIG. 1. Mitochondrial and cytoplasmic sources of carbamyl phosphate (CP) for the synthesis of erotic acid. CPS I and II are distinct forms of carbamyl phosphate synthetase located in the liver mitochondria and cytosol, respectively. Ammonia excess or lack of urea cycle intermediates may cause CP production in excess of urea cycle capacity. The result is elevated erotic acid synthesis in the cytosol, which is indicated by enhanced urinary erotic acid. Adapted from Tremblay et al. (1977).
capacity is depressed, CP leaves the mitochondria and enhances orotate synthesis in the cytoplasm (Tremblay et al., 1977; Jones, 1980; Milner and Visek, 1973). Arginine supplementation decreases ammonia toxicity (Najarian and Harper, 1956) and OA excretion (Kesner, 1965). Previously, we reported that carbon tetrachloride, ethanol, and galactosamine elevated urinary erotic acid of rats fed purified diets (Visek and Shoemaker, 1986). A feedback relationship between liver damage and erotic acid synthesis, as hypothesized by Faust0 et al. (1975), was strongly supported because supplying extra dietary arginine reduced OA excretion and the extent of hepatic regeneration after partial hepatectomy (Shoemaker and Visek, 1985). MATERIALS
AND METHODS
Animals
Sixty male albino Sprague-Dawley (Harlan-Sprague-Dawley, Madison, WI) rats were randomly assigned either to a group of 45 to be exposed to Ccl, or to a group of 15 controls. Mean starting weights were 192 * 10 and 201 + 4.3 g, respectively. All animals were housed individually in stainless-steel wire-bottom cages in temperature-controlled rooms with a 12-hr light-dark cycle. Glass distilled water was provided ad fibitum. Rats were fed a basal semipurified diet prepared according to AIN-76A (American Institute of Nutrition Report, 1977) guidelines, Table I. Ccl, Exposure
The procedure varied from the McLean et al. (1969) protocol in two respects. First, the animals were exposed to Ccl, vapor only to the point of light anesthesia rather than for a fixed time period, and second, phenobarbital was mixed into the
373
OROTIC ACID AND CIRRHOSIS Diet Composition, Ingredient
TABLE I Based on the Recommendations of the American Institute of Nutrition
(42)
Diet 1: control (g)
Diet 2: 1.5% arginine (8)
Diet 3: soybean protein (g)
200 0 432.4 217 50 50 50 0.6 0 1000
175 0 442.4 217 50 50 50 0.6 15 1000
0 200 432.4 217 50 50 50 0.6 0 1000
Casein” Soybean proteinb Cornstarch’ Sucrose Celluloseb Corn oild Micronutrients’ Phenobarbid Arginine8
G High Protein (95%) Casein, Teklad, ARS-Sprague-Dawley, Madison, WI. * 95% Soybean Protein Isolate Teklad, ARSSprague-Dawley, Madison, WI. ’ A. E. Staley Mfg. Co., Decatur, IL. d Mrs. Tucker’s Corn Oil, Anderson Clayton Foods, Dallas, TX. ’ Micronutrients were premixed as follows: (ah ingredients were from Dyets, Inc., Bethlehem, PA): choline bitartrate, 12 g; methionine, 18 g; AIN-76A vitamin mix, 60 g; AINmineral mix, 210 g. f Sigma Chemical, St. Louis, MO, added in 50 ml of ethanol to corn oil. H Ajinomoto USA, Rahway, NJ.
diet instead of being supplied in the drinking water. Twice weekly, four rats at a time, contained in individual cages, were exposed within a chamber to Ccl, vapors produced by compressed air flowing at 2 liter/min through two successive bottles of CC& held at 20°C. As soon as animals became unable to right themselves, they were removed from the exposure chamber. The animals were fed ad libitum and exhibited significantly shortened pentobarbital sleeping times after 7 days of feeding (Shoemaker, 1984). In a preliminary study this protocol significantly reduced sulfobromophthalein clearance in rats after 10 weeks (Shoemaker, 1984). Phenobarbital feeding alone in previous studies caused no changes in erotic acid excretion in experiments employing a crossover design (Shoemaker, 1984). Urine Collection During the Ccl, exposure period, animals were transferred into stainless-steel wire-bottom metabolism cages at approximately 2-week intervals 24 hr after exposure. They remained in the metabolism cages for 24 hr while their urine was collected, separately from feces, in 50-ml polycarbonate centrifuge tubes containing 5 drops of phosphoric acid. The collection was immediately diluted to 30-40 ml with glass-distilled water and centrifuged at 5OOg for 15 min. Aliquots were decanted and frozen at - 20°C until analyzed. Assignment
to Subgroups
For purposes of these studies, erotic aciduria was defined as an excretion rate exceeding the control mean plus two standard deviations. When rats developed erotic aciduria, which required 7-16 weeks, they were withheld from Ccl, exposure for 1 week, urine was collected, and the rats were assigned to one of three diets (Table 1). The control animals were fed the basal AIN-76A diet (Diet 1) with casein as the source of protein. The arginine-supplemented animals (Diet 2) were
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fed the basal diet containing 1.5% arginine and the soybean protein-fed animals received Diet 3 with soybean protein replacing casein. The arginine-supplemented group remained on the regimen for the remainder of 20 weeks of Ccl, exposure. The control and soybean groups consumed their diets for 4 to 7 days, when urine was collected and their diets were switched. They were subjected to three such crossovers before they were killed. Twelve additional rats which developed erotic aciduria after 16 weeks of CCI, exposure were assigned six to Diet 1 and six to Diet 3 for two crossovers at intervals of 4-7 days. Liver Perfusion
Studies
Liver perfusion was carried out after the animals had been exposed to Ccl, for 16 to 20 weeks. Forty-live minutes prior to the procedure, each rat was injected intraperitoneally with 5 l&i of [6-14C]orotic acid (New England Nuclear Corp., Boston, MA) in 500 p1 of bicarbonate buffer (pH 7.4). A laparotomy was performed under ether anesthesia, 200 l.~l of heparin (1000 U/ml) was injected into the portal vein and 2 ml of portal blood was withdrawn into a heparinized syringe for amino acid analyses. The portal and gastric veins were ligated with surgical silk and the portal vein was then nicked proximally to its ligature where a beveled cannula of polyethylene (PE-60) tubing was inserted and secured with surgical silk. The other end of the cannula was connected to the perfusion apparatus. The inferior vena cava was then transected and perfusion fluid flow was established with the liver in situ. When the caval drainage became clear the liver was dissected free and placed inside of the perfusion chamber. The perfusate was KrebsHenseleit buffer II (Dawson, 1978) with 20 mM alanine, 10 mA4 6-azauridine, and 0.5 or 5.0 mM ammonium bicarbonate added before equilibration with 95% O2 and 5% CO, for 1 hr at 37°C. The pH of the perfusing solution was maintained at 7.4 + 0.1 during perfusion with an automatic titrator (Radiometer, The London Co., Cleveland, OH) which delivered 1 M NaOH into the gas-exchange tube of the perfusion apparatus. The volume of base added was 1.8 f 0.21 ml (mean f SEM) for all perfusions and the volume of perfusate was 50 ml circulated at 16 ml/min. After 30 min of perfusion ornithine was added to the perfusate to a final concentration of 2.3 mM. One-milliliter aliquots were taken at 30 and 60 min for analysis. The viability of the livers was verified in three preliminary cases by comparing the rates of urea synthesis during the first and second 30-min periods of perfusion without ornithine added to the medium. Analyses
of Blood and Liver Per-mates
Immediately after collection, portal blood was mixed with 200 p,l of 4% NaF and centrifuged at 5OOg for 5 min. Five hundred microliters of the plasma was added to 1 ml of ice-cold 7.5% sulfosalicylic acid buffer (pH 1.8) and centrifuged at 48OOg for 15 min. The deproteinized extracts were stored at -70°C until analyzed for amino acids by ion-exchange chromatography (amino acid analyzer Model 119CL, Beckman Instruments, Palo Alto, CA, physiological fluid analysis). One-milliliter aliquots of perfusate taken at 0, 30, and 60 min were immediately chilled on ice and 100 ~1 of concentrated HClO, was added before centrifugation at 4800g for 15 min. The samples were assayed for ammonia by a selective ion electrode with a gas-permeable membrane (Ammonia electrode 95-10, Orion Research Inc., Cambridge, MA), in a lo-ml beaker containing 1.5 ml of deproteinized extract and 1.5 ml of 10 M NaOH as directed by the manufacturer. Two standards
OROTIC ACID AND CIRRHOSIS
375
containing 0.01 to 1 mM ammonia were assayed in parallel with each aliquot tested. Orotic acid was measured by injection of 5 ~1 of deproteinized perfusate directly onto an HPLC column as described below. Urea was measured colorimetrically (Foster and Hochholzer, 1971). Histology
and Chemical
Analysis of the Livers
After perfusion the livers were blotted and weighed. Samples for histological study were fixed with 2% glutaraldehyde in 0.1 M sodium phosphate, pH 7.4, and the severity of cirrhosis was assessed by a board-certified pathologist. The criteria for severity were the thickness of fibrous bands, extent of dissection of lobules, and presence of regenerative nodules (More, 1973) as shown in Photographs l-3. A total of 26 animals were killed for the perfusion studies and 21 livers were carried through 60 min of perfusion including 12 starting with 5 mM and 9 with 0.5 mM ammonia (before O.&O2 equilibration). Remaining animals were used in other studies (Shoemaker, 1984). Of the 21 perfused livers, 14 were CC&-treated and of these, 6 were classified histologically as having moderate-to-severe cirrhosis. Weights of spleen and testes were obtained when the animals were killed. Liver tissue was homogenized (Kinematica Polytron, Brinkman Instruments, Westbury, NY) in 1.5 ml of buffer (Pierson and Brien, 1980) per gram of tissue and the homogenates were stored at -70°C until analyzed. The homogenates were analyzed for dry weight, total fat, RNA, DNA, and total protein. All operations were performed at 4°C according to Glazer and Weber (1971) except as noted. The RNA and DNA fractions were assayed for incorporation of [6-14C]orotic acid. Aliquots of 0.5 ml containing approximately 200 mg of tissue were also transferred into glass centrifuge tubes or aluminum weighing pans. After 24 hr at 70°C the pans were weighed for dry matter determinations and 0.8 ml of cold 1 .O N HC104
PHOTOGRAPH 1. Liver from rat treated for 16 weeks with inhaled Ccl, and phenobarbital representing “mild” cirrhosis: fibrous bands evident, but do not dissect lobules, some fat vacuoles. Magnification 3 1.25X .
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AND
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PHOTOGRAPH 2. Liver from rat treated as in Photograph 1, representing “moderate” fibrous bands dissect lobules and circumscribe some lobules. Magnification 31.25~.
cirrhosis:
was added to the centrifuge tubes which were centrifuged at 48OOg for 15 min. The pellets, resuspended twice in 6 ml of 0.2 N perchloric acid after each addition, were centrifuged for 10 min at 4800g. Lipid was extracted by treating the pellets once with 2.0 ml of 1.O N potassium acetate in absolute ethanol, twice with 4.0 ml chloroform:methanol (l:l), and once with 2 ml absolute ethanol. Each extraction included rehomogenization followed by centrifugation at 4800g for 10 min. The
PHOTOGRAPH 3. Liver from rat treated as in Photograph 1, representing “severe” cirrhosis: disruption of sinusoidal architecture, numerous fat vacuoles, regenerative nodule. Magnification 31.25~.
OROTIC
ACID
AND
CIRRHOSIS
377
solvent extracts were pooled and evaporated overnight in tared vials at 70°C. The fat-free precipitates were dispersed and allowed to stand overnight in 5 .O ml of 1.5 N HClO,. RNA was extracted by resuspending the pellet twice in 2.5 ml of 0.5 N perchloric acid. DNA was extracted by two treatments with 5.0 ml of 0.5 N perchloric acid at 70°C for 20 min, and the protein remaining was dissolved by treatment with 4.0 ml of 1 N KOH for 15 min at 70°C. Radioactivity was assayed on 2.5-ml aliquots of the RNA and DNA extracts each mixed with 7.5 ml Aquasol II (New England Nuclear Corp., Boston, MA) and counted in a Beckman LK 9000 scintillation counter. Calorimetric determinations of RNA, DNA, and protein followed Cerotti (1955), Hubbard et al. (1970), and Lowry et al. (1951), respectively. Assay of Orotic Acid Urinary erotic acid was assayed in the effluent from a high-pressure liquid chromatography column of Partisil-IO SAX (Whatman Corp., Clifton, NJ) eluted in a gradient from distilled water to 10 mM KH,PO,, pH 2.76, according to a procedure adapted from Mills et al. (1979). A stainless-steel 4 x 25-mm column with a flow rate of 2.5 ml/min at 40°C and a solvent program requiring 10 min were used with a Waters Solvent Programmer Curve No. 5 and a convex gradient (Waters Corp., Milford, MA). When a Radial-Pak (Waters Corp.) column was used in the Waters RCM-100, the flow rate was 3 ml/min and the program took 15 min at room temperature. Absorbance was measured at both 280 and 254 nm. The erotic acid peak was identified by its retention time compared to an erotic acid standard (Sigma Chemical Co., St. Louis, MO) and by its ratio of absorbance at 280 and 254 nm which is characteristically high for erotic acid among the other peaks in the chromatogram. The height of the erotic acid peak at 280 nm was compared to that of a standard peak injected with each series of samples. For each lo-ml vol of diluted urine, 1 ~1 of sample was injected. The lower limit of detection was 5-10 ng. RESULTS Two of the 45 animals died from acute CCL, toxicity and 7 died from pneumonia as shown by histopathological examination. There were no consistent effects of treatment on feed intake but there was a statistically significant depression in weight gain, which averaged 8% in the CC&-treated animals compared to that in controls until the 18th week. Compared to Diet 1, no consistent differences were found in the intake of either the arginine-supplemented casein diet (Diet 2) or the soybean protein diet (Diet 3). Cessation of Ccl4 exposure for 1 week at the onset of erotic aciduria lowered erotic acid excretion from 179 2 0.5 to 102 k 6 kg/day (1 = 7.43, P < 0.001) within 7 days. Orotic acid excretion tended to decline as the animals reached adult weight in both the treated and untreated groups (Fig. 2). Arginine supplementation tended to lower erotic acid excretion, but not by a statistically significant difference (Table II). Likewise, 1.5% arginine supplementation did not prevent the elevation of erotic acid excretion when Ccl, treatment was resumed during the following 4 weeks. The ratio of liver protein to DNA decreased with Ccl, exposure (Table III) while liver lipid content and the rate of [14C]orotic acid incorporation into RNA did not differ between the two groups. Spleen weights were
378
SHOEMAKER
AND
VISEK
160-
FIG. 2. Orotic acid excretion by rats exposed to carbon tetrachloride (Ccl,; Week, 1 n = 45; Week 10, n = 37; Week 20, n = 16) compared to that by unexposed controls (Week 1, n = 15; Week 10, n = 13; Week 20, n = 5). Both groups were fed AIN-76A diets containing phenobarbital (Table 1). The means indicated differ significantly (P < 0.0s). Error bars indicate standard errors of the mean. Arrow at 20 weeks indicates cessation of Ccl, exposure.
significantly greater after CCL, exposure, but only one rat, with severe cirrhosis and ascites, had atrophic testicles at necropsy. The data from perfusion of cirrhotic livers are shown by Figs. 3 and 4 and Table IV. There were no significant differences in urea synthesis rates between the first 30 min without added ornithine and the subsequent 30 min of perfusion, when additional ornithine was present in the perfusing medium. Urea synthesis was constant with ammonia concentrations ranging from 0.2 to 3.0 mM. Addition of ornithine caused no significant increase in urea synthesis or ammonia clearance in either controls or cirrhotics, and the histological severity of cirrhosis was not
TABLE II Effect of Casein Soybean and Arginine on Orotic Acid Excretion in CC&-Treated Rats, Means f SEM Group Control (20 weeks, mean) All CC1 rats at onset of erotic aciduria Casein (7-16 weeks) Diet 1 1.5% Arginine (Diet 2) Soybean (7-16 weeks) Diet 3 Casein (16-20 weeks) Diet 1 Soybean (16-20 weeks) Diet 3
Orotic acid excreted Way)
Number of rats
83 f 9”
15
179 +- 9” 213 * H6 162 2 14 139 * 11* 111 f 7 11429
30 4 10 4 6 6
a,* Means with same superscript differ (P < 0.001).
379
OROTIC ACID AND CIRRHOSIS TABLE III Liver, Spleen, and Testis Weights and Liver RNA, DNA Protein, and Lipids in Rats Made Cirrhotic by Inhalation of CCI, for 16-20 Weeks, Means 2 SEM Control (n = 9)
Organ/component
CCLtreated (n = 17)
Body weight (9) Liver (g) Liver (g/kg BW)
355 + 18 14.3 f 1.1 40.1 +- 1.8” 40.9 f 2.5 49.6 f 4.5’ 5.64 + 0.6
365 14.6 44.0 34.1 38.8 35.4 4.82
f f f f k + f
18 1.0 1.96 2.5”,b 1.6 1.7’ 0.4
Liver lipid (% dry wt) Liver (g protein/g DNA) Liver [‘4C]orotate, uptake into RNA (dpm/pg RNA) Liver RNA (m&g FFDW)* RNA (mg/liver) DNA (mg/liver) Protein (g/liver) Spleen (&g BW) Testis @/kg BW)
33.4 72.3 37.9 1.88 1.49 9.26
35.2 72.0 43.2 1.53 2.85 8.91
k + f ” + 2
1.7 2.8 2.7 0.1 0.34” 0.5
f ” + f f +
3.2 4.1 4.2 0.2 0.24d 0.4
(Sacrificed at weeks 16-20) (Sacrificed after 20 weeks)
* t = 2.02, P < 0.05. b t = 3.23, P < 0.01. = t = 3.36, P < 0.01. d t = 3.48, P < 0.01. ’ FFDW, fat free dry weight.
associated with a decrease in urea synthesis rates. For example, the three rats with the most severe cirrhosis synthesized an average of 30.6 + 4.9 kmole of urea/g liver protein/hr before the addition of ornithine. Table III shows changes in organ weights and liver DNA, RNA, lipid, and protein consistent with cirrhosis. Portal serum ammonia and amino acid concentrations (Table V) were also consistent with impairment of liver function by CCL exposure. The difference in the ammonia concentration of portal blood between control and Ccl,-treated rats was not statistically significant, but the subset of animals with the most severe histological cirrhosis, whether fed soybean or casein protein, showed a statisticaIly significant increase in portal ammonia which averaged 0.30 + .Ol compared to 0.20 -+ .08 n&f for controls (t = 2.65, P < 0.05). .F $ 100 \\ F *\ $ b ‘$
BO60-
f
40-
2 4
20-
I --\..
Mod-Severe Disease lllZ6)
I------+ ‘+---,--
t =2.85
-s
PCO.02
-+
t = 5.56
Control + Mild (n=6)
PCO.001 I 30
I 60
Minutes FIG. 3. Percentage of sting ammonia concentration, 2.95 f 0.12 mM (mean -t SEM), remaining in the perfosate as a function of time during perfusion of rat livers with mild or no cirrhosis versus those with moderate-to-severe cirrhosis.
380
SHOEMAKER
AND VISEK
Ammonia
(mM)
FIG. 4. Release of erotic acid into the perfusate by livers of control and CCL-treated rats plotted against ammonia concentration. The correlation for both groups was significant (control, r = 0.76; Ccl,, r = 0.83; both P < 0.01). There were no differences in slope or intercept.
The livers with the most severe histological changes had less capacity to remove ammonia from the per&ate than controls or livers with only mild pathology (Fig. 3). The starting mean ammonia concentration in the perfusate averaged 2.95 2 0.12 mM. There was no difference in the tendency of control or CC&-treated livers to make erotic acid at a given level of ammonia (Fig. 4). Adding 2.3 mik! ornithine to the perfusate raised the calculated threshold ammonia concentration (abscissa-intercept) for causing orotate spillage from 0 to 1.52 mM (kg orotate/hr/g liver protein = 95.2 x NH,[w - 145, r = 0.546, curve not shown). DISCUSSION As control or Ccl,-treated rats achieved adult weight, erotic acid excretion tended to decline, suggesting that competition for arginine between growth and urea synthesis was lessened as the animals matured (Milner and Visek, 1973). Soybean protein, containing more than twice the arginine content of casein (Orr and Watt, 1957), dramatically decreased erotic acid excretion during the growth phase but caused no changes between Weeks 17 and 20 even though the cumulative effects of Ccl, were presumably further advanced at that time. After 7-10 weeks of Ccl, treatment when erotic aciduria first became evident, or after 20 weeks, acute CC& exposure was required to maintain elevated erotic acid excretion. Thus continuous “acute-on-chronic” exposure was essential to maintain elevated erotic acid excretion in this animal model. TABLE IV Urea Synthesis during Perfusion With/Without Omithine of Rat Livers Made Cirrhotic by CCL (kmole/hr/g liver protein), Means k SEM No omithine Controls CC&-treated Mean
32.8 (n 24.7 (n 26.5
2 7.4 = 7) 2 2.4 = 14) -+ 2.5
2.3 mM omithine 30.6 (n 28.7 (n 29.3
f 5.0 = 7) f 4.2 = 14) 2 3.2
Mean 31.7 + 4.3 26.7 f 2.4
OROTIC
ACID
AND
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CIRRHOSIS
TABLE V Portal Plasma Amino Acids in Rats with Carbon Tetrachloride-Induced Cirrhosis, the Effect of Dietary Soybean Protein vs Casein, Means f SEM Treatment Amino acid (mh4) Ammonia Arginine Omithine Citrulline Glutamine Proline Lysine Histidine Tyr + Phe Val + Ile + Leu Ratio Val + Be + Leu Tyr + Phe Tryptophan Met + Cys
Diet 1: control (n = 9)
Diet 1: CCl.,-casein (n = 8)
0.198 f 0.026 0.101 f 0.012 0.047 2 0.003 0.047 2 0.006 0.425 2 0.027 0.178 + 0.027 0.360 + 0.025 0.084 -1-0.008 0.185 f 0.02 0.415 f 0.041 2.24
0.236 0.088 0.055 0.079 0.548 0.416 0.339 0.100 0.255 0.416
+ 0.028 2 0.006 f 0.011 k 0.010 f 0.079 -+ 0.108 * 0.041 * 0.013 k 0.045 2 0.038 1.63
0.060 2 0.010 0.093 k 0.011
0.064 +- 0.006 0.100 2 0.009
Diet 3: CCl,soybean (n = 4) 0.216 0.068 0.073 0.066 0.497 0.229 0.291 0.088 0.177 0.294
f 0.028 2 0.013 2 0.012 f 0.006 f 0.025 f 0.057 2 0.021 + 0.008 + 0.020 f 0.068 1.66
0.073 2 0.008 0.067 2 0.008
The difference in incorporation of labeled erotic acid into RNA of CC&-treated animals compared to that of controls was not statistically significant (Table III) and there was no difference in DNA specific activity. The CC&-treated animals had 5% more RNA per gram of fat-free dry liver and significantly more DNA per unit of protein. Thus, differences in [‘4C]orotate incorporation into RNA did not account for the two- to threefold rise in orotate excretion caused by Ccl4 toxicity. Urea synthesis rates by perfused rat livers in our studies (Table IV) were comparable to those of other investigators (Henley et al., 1975; Perez et al., 1978, 1979), although exact comparisons are precluded because expression of the rates differed between studies. Both Henley et al. (1975) and Perez et al. (1978) employed albumin in their perfusate which introduced another source of nitrogen not used in our studies. Perez et al. (1979) and Henley et al. (1975) also reported differences in urea synthesis rates between cirrhotic and control livers, but the ammonia concentrations they used greatly exceeded the physiological range. Our data (Fig. 3) confirm that cirrhotic livers removed ammonia from the perfusate more slowly than normal liver (Perez et al., 1979). There was no significant difference in the mean urea production between controls and cirrhotic animals (Table IV), and no trend toward lower urea production for the livers with the severest histopathology. The differences between these results and those of Perez et al. (1979) and Henley et al. (1975) are probably due to the concentrations of ammonia used in the perfusing solution. Protein loading studies on intact animals have confirmed that urea synthesis capacity in cirrhosis is near normal (Brewer et al., 1984). The concentration of portal ammonia increased due to Ccl4 exposure as shown in Table V. The ratio of branch chain to aromatic amino acids followed the trend described by Fischer et al. (1975) as indicative of hepatic injury. The “amine pool” amino acids, glutamine and proline, tended to be highest in casein-fed rats with cirrhosis. On the basis of the data in Table V the mean portal ammonia
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concentration in all cirrhotic animals was 0.24 mM compared to 0.20 mA4 for controls. This difference of 0.04 mM if maintained over 24 hr would induce the synthesis of an additional 828 pg of erotic acid per liver (estimated from the regression line in Fig. 4). According to previous findings (Hecht and Potter, 1956), 40% of orotate reaching the liver after intraperitoneal injection is excreted in the urine. If this percentage of the additional erotic acid was excreted daily, the amount would approximate what we observed. Orotic acid production has not previously been measured in perfused cirrhotic livers. Unlike techniques using liver slices or isolated hepatocytes, perfused livers can show differences due to microvascular derangement and intrahepatic shunts described by Wood ef al. (1979), Villeneuve et al. (1978), and Reichen et al. (1987). Our observation of higher portal ammonia with decreased ammonia clearance during perfusion, despite normal urea synthesis capacity concurs with the “intact-hepatocyte” hypothesis put forth by Villeneuve et al. (1978). Intrahepatic shunts may comprise the chronic aspect of the acute-on-chronic toxicity of Ccl, while transient hyperammonemia due to reduced hepatocyte number or effectiveness may result from acute exposure. Arginine supplementation did not prevent the erotic aciduria of Ccl4 toxicity. A previous report showed no effect of arginine in rats with surgical portacaval shunts (Shoemaker, 1984). During the growth phase, soybean protein had a dramatic effect compared to casein in lowering orotate excretion, but not when animals reached adult weight. Lysine is an arginine antagonist and the higher arginineilysine ratio in soybean protein (Orr and Watt, 1957), or differences in digestibility, could have lowered portal ammonia or decreased orotate synthesis after acute Ccl, exposure. Protein of soybeans and other plants has been recommended for diets of patients with hepatic encephalopathy (Greenberger et al., 1977). The present data agree with this recommendation. Enteral or parenteral administration of amino acid mixtures has also been advocated to decrease the incidence of portal systemic encephalopathy (Fischer et al., 1976). Najarian and Harper (1956) reported that arginine lowered blood ammonia in some patients, but a correlation between the degree of encephalopathy and the blood ammonia concentration was not consistently demonstrated. Thomson and Visek (1963) reduced mental obtundation in rats by immunization against jack bean urease which inhibits ammonia formation in the intestine. However, repeated injections of the enzyme are required to initiate and maintain this immunity. More recently, improved mental status from the administration of omithine with or without supplemental branch chain amino acids has been reported (Herlong et al., 1980). Zieve (1986) has suggested that increased urinary erotic acid in a patient with hepatic failure may indicate that sufficient liver function remains to warrant supplementation with omithine or arginine. The present animal study supports this concept with the reservation that intrahepatic shunting may lessen the expected ammonia-lowering effect. Elevated plasma ammonia, reduced hepatic ammonia clearance, and the response of growing rats to soybean protein supported the hypothesis that the erotic aciduria of carbon tetrachloride toxicity was due to acutely elevated ammonia concentrations and the existence of intrahepatic shunts. Continuous exposure to CC& was required to maintain the elevation of urinary erotic acid, probably due to the regenerative activity of the liver.
OROTIC ACID AND CIRRHOSIS
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REFERENCES American Institute of Nutrition Report of the AIN Ad Hoc Committee on Standards for Nutritional Studies (1977). .Z.Nutr. 187, 1340-1348; Second Report of the Ad Hoc Committee on Standards for Nutritional Studies. (1980). .Z.Nutr. 110, 1726. BREWER, T. G., BERRY, W. R., HARMON, J. W., WALKER, S. H., and DUNN, M. A. (1984). Urea synthesis after protein feeding reflects hepatic mass in rats. Hepatology 4, 905-911. CEROTTI, G. (1955). Determination of nucleic acids in animal tissues. J. Biol. Chem. 214, 59-70. CONN, H. O., LEEVY, C. hi., and VLAHCEVIC, Z. R. (1977). Comparison of lactulose and neomycin in the treatment of chronic portal-systemic encephalopathy: A double blind controlled trial. Gnstroenterology 72, 573-583. DAWSON, R. M. C. (1978). Buffers and physiological media. In “Data for Biochemical Reactions” (R. M. C. Dawson, D. C. Elliot, W. H. Elliot, and K. H. Jones, Eds.), 2nd Ed., pp. 507-512. Oxford Univ. Press, Oxford. FAUSTO, N., BRANDT, J. T., and KESNER, L. (1975). interrelationships between the urea cycle, pyrimidine, and polyamine synthesis during liver regeneration. Zn Liver Regeneration after Experimental Injury (R. Lesch, and W. Reutter, Eds.), pp. 215-229. Stratton Intercontinental Medical Book Corp., New York. FISCHER,J. E., FUNOVICS, J. M., AGUIRE, A., et al. (1975). The role of plasma amino acids in hepatic encephalopathy. Surgery 78, 276-290. FISCHER, J. E., RASEN, H. M., EBEID, E. M., JAMES, J. H., KEANE, J. M., and SOETEUS, P. B. (1976). The effect of normalization of plasma amino acids on hepatic encephalopathy in man. Surgery 80, 77-91. FOSTER, L. B., and HOCHHOLZER, J. M. (1971). A single reagent manual method for directly determining urea nitrogen in serum. Clin. Chem. 17, 921-924. FRASER, C. L., and ARIEFF, A. I. (1985). Hepatic encephalopathy. N. Engl. J. Med. 313, 865-873. GLAZER, P. I., and WEBER, G. (1971). Incorporation of [6-‘HIglucose into lipid, protein RNA, and DNA of slices of differentiating rat cerebral cortex. J. Neurochem. 18, 1569-1576. GREENBERGER, N. J., CARLEY, J., SCHENKER, S., BETTINGER, I., STAMNES, C., and BEYER, P. (1977). Effect of vegetable and animal protein diets in chronic hepatic encephalopathy. Amer. J. Dig. Dis. 22, 845-855. HECHT, L. I., and POTTER, V. R. (1956). Nucleic acid metabolism in regenerating rat liver. III. Intermediates in the synthesis of DNA pyrimidine nucleotides. Cancer Res. 16, 999-1004. HENLEY, K. S., CLANCY, P. E., LAUGHREY, E. G., and LYRA, L. G. C. (1975). Nitrogen metabolism in the perfused cirrhotic liver of the rat. .Z. Lab. Clin. Med. 85. 273-280. HERLONG, H. F., MADDREY, W. C., and WALSER, M. (1980). The use of omithine salts of branchedchain ketoacids in portal-systemic encephalopathy. Ann. Intern. Med. 95, 545-550. HUBBARD, R. W., MATTHEW, W. T., and DUBORWIK, D. A. (1970). Factors influencing the determination of DNA with indole. Anal. Biochem. 38, 19&201. JONES, M. E. (1980). Pyrimidine nucleotide biosynthesis in animals: Genes, enzymes and regulation of UMP biosynthesis. Annu. Rev. Biochem. 49, 253-79. KESNER, L. (1%5). The effect of ammonia administration on erotic acid excretion in rats, J. Biol. Chem. 240, 1722-1724. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. MCLEAN, E. K., MCLEAN, A. E. M., and SUTTON, P. M. (1969). Instant cirrhosis. An improved method for producing cirrhosis of the liver in rats by simultaneous administration of carbon tetrachloride and phenobarbitone. Brit. J. Exp. Pathol. 50, 502. MILLS, G. C., SCHMALSTIEG, F. C., NEWKIRK, K. E., and GOLDBLUM, R. M. (1979). Cytosine and erotic acid in urine of immunodeficient children. Clin. Chem. 25, 419-423. MILNER, J. A., and VISEK, W. J. (1973). Orotic aciduria and arginine deficiency. Nature (London) 245, 211-213. MORE, I. A. R. (1973). Biochemical and histological correlations of regeneration after experimental liver damage: significance of cirrhosis. Brit. J. Exp. Pathol. 54, 404-408. NAJARIAN, J. S., and HARPER, H. A. (1956). A clinical study of the effect of arginine on blood ammonia. Amer. J. Med. 21, 832-842. ORR, M. L., and WATT, B. K. (1957). “Amino Acid Content of Foods. Home Economic Research Report No. 4, U.S.D.A.” U.S. Government Printing Oflice, Washington, DC.
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PEREZ, G. O., RIETBERG, B., and SCHIFF, E. R. (1978). Amino acid release by isolated perfused cirrhotic livers. Life Sci. 23, 2533-2538. PEREZ, G. O., RIETBERG, B., OWENS, B., PARKER, T., OBAYA, H., and SCHIFF, E. R. (1979). Urea synthesis by perfused rat liver. Studies of Ccl,-induced cirrhosis. Biochem. Pharmacol. 28, 48% 489.
PIERSON, D. L., and BRIEN, J. M. (1980). Human carbamylphosphate synthetase. J. Biol.
Chem.
255,
7891-7895.
REICHEN, J., ARTS, B., SCHAFROTH, U., ZIMMERMAN, T., ZELTNER, B., and ZYSSETT, T. (1987). Aminopyrine N-demethylation by rats with liver cirrhosis. Gastroenterology 93, 719-722. SHOEMAKER, J. D. (1984). “The Orotic Aciduria of Chemical Hepatotoxicity.” Doctoral thesis, University of Illinois, Urbana. SHOEMAKER, J. D., and VISEK, W. J. (1985). Arginine limits hepatic regeneration. Fed. Proc. 44, 1522. [Abstract] THOMSON, A., and VISEK, W. J. (1963). Some effects of induction of urease immunity in patients with hepatic insufticiency. Amer. J. Med. 35, 804412. TREMBLAY, G. C., CRANDALL, D. E., KNOTT, C. E., and ALFANT, M. (1977). Orotic acid biosynthesis in rat liver: Studies on the source of carbamoyl phosphate. Arch. Biochem. Biophys. 178, 264-277.
VILLENEUVE, J. P., WOOD, A. J. J., SHAND, D. G., ROGERS,L., and BRANCH, R. A. (1978). Impaired drug metabolism in experimental cirrhosis in the rat. Biochem. Pharmacol. 27, 2577-2581. VISEK, W. J., and SHOEMAKER, J. D. (1986). Orotic acid, arginine, and hepatotoxicity. J. Amer. Golf. Nutr. 5, 153-166. WOOD, A. J. J., VILLENEUVE, J. P., BRANCH, R. A., ROGERS, L. W., and SHAND, D. G. (1979). Intact hepatocyte theory of impaired drug metabolism in experimental cirrhosis in the rat. Gastroenterology 76, 13584362. ZIEVE, L. (1966). Pathogenesis of hepatic coma. Arch. Int. Med. 118, 211-223. ZIEVE, L. (1986). Conditional deficiencies of ornithine or arginine. J. Amer. Coil. Nutr. 5, 167-176.
AND
MOLECULAR
PATHOLOGY
Orotic Acid Overproduction J.D.
50,
371-384 (1989)
in Experimental
SHOEMAKER
AND W.J.
Cirrhosis
of Rats
VISEK
University of Illinois, College of Medicine at Urbana-Champaign,
Illinois 61801
Received July 5, 1988, and in revised form October 25, 1988 Ammonia clearance, portal blood ammonia, and amino acid concentrations were studied during induction of cirrhosis by carbon tetrachloride in rats. Exposure to Ccl, vapors twice weekly for 7-16 weeks doubled erotic acid excretion. If exposure was discontinued for 7 days, the erotic acid excretion decreased despite the presence of cirrhosis proven histologically. Replacement of dietary casein with soybean protein eliminated the CC&-induced erotic aciduria in growing rats but not in adults. Supplementation of casein with 1.5% arginine did not prevent CClJnduced erotic aciduria. [i4C]Orotate uptake into RNA and DNA of liver was not impaired. Perfusion of livers of cirrhotic animals with ammonia concentrations between 0.2 and 3.0 mM revealed no significant decreases in urea synthesis rates due to cirrhosis and no increase in the tendency to make erotic acid at a given ammonia concentration. However, ammonia uptake by cirrhotic livers was significantly reduced, resulting in higher ammonia concentrations in the effluent when there was moderateto-severe cirrhosis. Portal blood samples taken from rats exposed to Ccl, had higher ammonia concentrations as cirrhosis worsened. The results lend support to the “intact hepatocyte” hypothesis of cirrhosis which attributes metabolic abnormalities to intrahepatic shunts. 0 19S9 Academic Press, Inc.
INTRODUCTION The impaired capacity of cirrhotic liver to metabolize protein is well recognized (Zieve, 1966; Conn et al., 1977; Fraser and Arieff, 1985) but not well understood. Our previously reported studies show increased urinary erotic acid (OA)’ excretion following three forms of chemical hepatic injury (Visek and Shoemaker, 1986). The present report contains data on urinary OA of rats exposed to CC& according to a modification of the McLean ef al. (1969) “instant cirrhosis” protocol. We have used erotic acid as a marker of urea cycle capacity because OA synthesis is enhanced in the liver when ammonia (NH,+, NH4+) concentrations are elevated (Tremblay et al., 1977; Jones, 1980; Milner and Visek, 1973). The present experiments were conducted to determine if the rise in OA excretion by cirrhotic rats was attributable to decreased utilization of OA in nucleic acid synthesis, portal amino acid imbalances, increased portal ammonia, reduced urea synthesis at near-physiological ammonia concentrations, or a change in the relationship between ammonia and OA production due to other actions of CCL,. The biochemical pathways involved in ammonia and erotic acid metabolism are illustrated in Fig. 1. Carbamyl phosphate (CP) of either intramitochondrial or cytoplasmic origin can be used in OA synthesis (Tremblay et al., 1977; Jones, 1980). The first step in urea synthesis combines ammonia with CO2 to form CP within mitochondria. CP is then combined with omithine to form citrulline. Previous studies have shown that when ammonia production is elevated or urea cycle ’ Abbreviations used: AIN-76A, diet formulated according to the American Institute of Nutrition (1977); BW, body weight; CC14, carbon tetrachloride; CP, carbamyl phosphate; HPLC, highperformance liquid chromatography; OA, erotic acid. 371 0014-4800/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHOEMAKER
372
AND VISEK
MlTOCHONDRlON CYTOPLASM
CYTOPL-
NH4+ 4
HC03-
GLUTAMINE
ATP Y
UREA
CARBAMYL-P ORNlTHlNE
-
---a.
*.
1 CARBAMYL-P
--•,ORNlTHlNE
L ARGININE 1 ARGININOSUCCINATE ASPARTATE t CITRULLINE
UMP
FIG. 1. Mitochondrial and cytoplasmic sources of carbamyl phosphate (CP) for the synthesis of erotic acid. CPS I and II are distinct forms of carbamyl phosphate synthetase located in the liver mitochondria and cytosol, respectively. Ammonia excess or lack of urea cycle intermediates may cause CP production in excess of urea cycle capacity. The result is elevated erotic acid synthesis in the cytosol, which is indicated by enhanced urinary erotic acid. Adapted from Tremblay et al. (1977).
capacity is depressed, CP leaves the mitochondria and enhances orotate synthesis in the cytoplasm (Tremblay et al., 1977; Jones, 1980; Milner and Visek, 1973). Arginine supplementation decreases ammonia toxicity (Najarian and Harper, 1956) and OA excretion (Kesner, 1965). Previously, we reported that carbon tetrachloride, ethanol, and galactosamine elevated urinary erotic acid of rats fed purified diets (Visek and Shoemaker, 1986). A feedback relationship between liver damage and erotic acid synthesis, as hypothesized by Faust0 et al. (1975), was strongly supported because supplying extra dietary arginine reduced OA excretion and the extent of hepatic regeneration after partial hepatectomy (Shoemaker and Visek, 1985). MATERIALS
AND METHODS
Animals
Sixty male albino Sprague-Dawley (Harlan-Sprague-Dawley, Madison, WI) rats were randomly assigned either to a group of 45 to be exposed to Ccl, or to a group of 15 controls. Mean starting weights were 192 * 10 and 201 + 4.3 g, respectively. All animals were housed individually in stainless-steel wire-bottom cages in temperature-controlled rooms with a 12-hr light-dark cycle. Glass distilled water was provided ad fibitum. Rats were fed a basal semipurified diet prepared according to AIN-76A (American Institute of Nutrition Report, 1977) guidelines, Table I. Ccl, Exposure
The procedure varied from the McLean et al. (1969) protocol in two respects. First, the animals were exposed to Ccl, vapor only to the point of light anesthesia rather than for a fixed time period, and second, phenobarbital was mixed into the
373
OROTIC ACID AND CIRRHOSIS Diet Composition, Ingredient
TABLE I Based on the Recommendations of the American Institute of Nutrition
(42)
Diet 1: control (g)
Diet 2: 1.5% arginine (8)
Diet 3: soybean protein (g)
200 0 432.4 217 50 50 50 0.6 0 1000
175 0 442.4 217 50 50 50 0.6 15 1000
0 200 432.4 217 50 50 50 0.6 0 1000
Casein” Soybean proteinb Cornstarch’ Sucrose Celluloseb Corn oild Micronutrients’ Phenobarbid Arginine8
G High Protein (95%) Casein, Teklad, ARS-Sprague-Dawley, Madison, WI. * 95% Soybean Protein Isolate Teklad, ARSSprague-Dawley, Madison, WI. ’ A. E. Staley Mfg. Co., Decatur, IL. d Mrs. Tucker’s Corn Oil, Anderson Clayton Foods, Dallas, TX. ’ Micronutrients were premixed as follows: (ah ingredients were from Dyets, Inc., Bethlehem, PA): choline bitartrate, 12 g; methionine, 18 g; AIN-76A vitamin mix, 60 g; AINmineral mix, 210 g. f Sigma Chemical, St. Louis, MO, added in 50 ml of ethanol to corn oil. H Ajinomoto USA, Rahway, NJ.
diet instead of being supplied in the drinking water. Twice weekly, four rats at a time, contained in individual cages, were exposed within a chamber to Ccl, vapors produced by compressed air flowing at 2 liter/min through two successive bottles of CC& held at 20°C. As soon as animals became unable to right themselves, they were removed from the exposure chamber. The animals were fed ad libitum and exhibited significantly shortened pentobarbital sleeping times after 7 days of feeding (Shoemaker, 1984). In a preliminary study this protocol significantly reduced sulfobromophthalein clearance in rats after 10 weeks (Shoemaker, 1984). Phenobarbital feeding alone in previous studies caused no changes in erotic acid excretion in experiments employing a crossover design (Shoemaker, 1984). Urine Collection During the Ccl, exposure period, animals were transferred into stainless-steel wire-bottom metabolism cages at approximately 2-week intervals 24 hr after exposure. They remained in the metabolism cages for 24 hr while their urine was collected, separately from feces, in 50-ml polycarbonate centrifuge tubes containing 5 drops of phosphoric acid. The collection was immediately diluted to 30-40 ml with glass-distilled water and centrifuged at 5OOg for 15 min. Aliquots were decanted and frozen at - 20°C until analyzed. Assignment
to Subgroups
For purposes of these studies, erotic aciduria was defined as an excretion rate exceeding the control mean plus two standard deviations. When rats developed erotic aciduria, which required 7-16 weeks, they were withheld from Ccl, exposure for 1 week, urine was collected, and the rats were assigned to one of three diets (Table 1). The control animals were fed the basal AIN-76A diet (Diet 1) with casein as the source of protein. The arginine-supplemented animals (Diet 2) were
374
SHOEMAKER
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VISEK
fed the basal diet containing 1.5% arginine and the soybean protein-fed animals received Diet 3 with soybean protein replacing casein. The arginine-supplemented group remained on the regimen for the remainder of 20 weeks of Ccl, exposure. The control and soybean groups consumed their diets for 4 to 7 days, when urine was collected and their diets were switched. They were subjected to three such crossovers before they were killed. Twelve additional rats which developed erotic aciduria after 16 weeks of CCI, exposure were assigned six to Diet 1 and six to Diet 3 for two crossovers at intervals of 4-7 days. Liver Perfusion
Studies
Liver perfusion was carried out after the animals had been exposed to Ccl, for 16 to 20 weeks. Forty-live minutes prior to the procedure, each rat was injected intraperitoneally with 5 l&i of [6-14C]orotic acid (New England Nuclear Corp., Boston, MA) in 500 p1 of bicarbonate buffer (pH 7.4). A laparotomy was performed under ether anesthesia, 200 l.~l of heparin (1000 U/ml) was injected into the portal vein and 2 ml of portal blood was withdrawn into a heparinized syringe for amino acid analyses. The portal and gastric veins were ligated with surgical silk and the portal vein was then nicked proximally to its ligature where a beveled cannula of polyethylene (PE-60) tubing was inserted and secured with surgical silk. The other end of the cannula was connected to the perfusion apparatus. The inferior vena cava was then transected and perfusion fluid flow was established with the liver in situ. When the caval drainage became clear the liver was dissected free and placed inside of the perfusion chamber. The perfusate was KrebsHenseleit buffer II (Dawson, 1978) with 20 mM alanine, 10 mA4 6-azauridine, and 0.5 or 5.0 mM ammonium bicarbonate added before equilibration with 95% O2 and 5% CO, for 1 hr at 37°C. The pH of the perfusing solution was maintained at 7.4 + 0.1 during perfusion with an automatic titrator (Radiometer, The London Co., Cleveland, OH) which delivered 1 M NaOH into the gas-exchange tube of the perfusion apparatus. The volume of base added was 1.8 f 0.21 ml (mean f SEM) for all perfusions and the volume of perfusate was 50 ml circulated at 16 ml/min. After 30 min of perfusion ornithine was added to the perfusate to a final concentration of 2.3 mM. One-milliliter aliquots were taken at 30 and 60 min for analysis. The viability of the livers was verified in three preliminary cases by comparing the rates of urea synthesis during the first and second 30-min periods of perfusion without ornithine added to the medium. Analyses
of Blood and Liver Per-mates
Immediately after collection, portal blood was mixed with 200 p,l of 4% NaF and centrifuged at 5OOg for 5 min. Five hundred microliters of the plasma was added to 1 ml of ice-cold 7.5% sulfosalicylic acid buffer (pH 1.8) and centrifuged at 48OOg for 15 min. The deproteinized extracts were stored at -70°C until analyzed for amino acids by ion-exchange chromatography (amino acid analyzer Model 119CL, Beckman Instruments, Palo Alto, CA, physiological fluid analysis). One-milliliter aliquots of perfusate taken at 0, 30, and 60 min were immediately chilled on ice and 100 ~1 of concentrated HClO, was added before centrifugation at 4800g for 15 min. The samples were assayed for ammonia by a selective ion electrode with a gas-permeable membrane (Ammonia electrode 95-10, Orion Research Inc., Cambridge, MA), in a lo-ml beaker containing 1.5 ml of deproteinized extract and 1.5 ml of 10 M NaOH as directed by the manufacturer. Two standards
OROTIC ACID AND CIRRHOSIS
375
containing 0.01 to 1 mM ammonia were assayed in parallel with each aliquot tested. Orotic acid was measured by injection of 5 ~1 of deproteinized perfusate directly onto an HPLC column as described below. Urea was measured colorimetrically (Foster and Hochholzer, 1971). Histology
and Chemical
Analysis of the Livers
After perfusion the livers were blotted and weighed. Samples for histological study were fixed with 2% glutaraldehyde in 0.1 M sodium phosphate, pH 7.4, and the severity of cirrhosis was assessed by a board-certified pathologist. The criteria for severity were the thickness of fibrous bands, extent of dissection of lobules, and presence of regenerative nodules (More, 1973) as shown in Photographs l-3. A total of 26 animals were killed for the perfusion studies and 21 livers were carried through 60 min of perfusion including 12 starting with 5 mM and 9 with 0.5 mM ammonia (before O.&O2 equilibration). Remaining animals were used in other studies (Shoemaker, 1984). Of the 21 perfused livers, 14 were CC&-treated and of these, 6 were classified histologically as having moderate-to-severe cirrhosis. Weights of spleen and testes were obtained when the animals were killed. Liver tissue was homogenized (Kinematica Polytron, Brinkman Instruments, Westbury, NY) in 1.5 ml of buffer (Pierson and Brien, 1980) per gram of tissue and the homogenates were stored at -70°C until analyzed. The homogenates were analyzed for dry weight, total fat, RNA, DNA, and total protein. All operations were performed at 4°C according to Glazer and Weber (1971) except as noted. The RNA and DNA fractions were assayed for incorporation of [6-14C]orotic acid. Aliquots of 0.5 ml containing approximately 200 mg of tissue were also transferred into glass centrifuge tubes or aluminum weighing pans. After 24 hr at 70°C the pans were weighed for dry matter determinations and 0.8 ml of cold 1 .O N HC104
PHOTOGRAPH 1. Liver from rat treated for 16 weeks with inhaled Ccl, and phenobarbital representing “mild” cirrhosis: fibrous bands evident, but do not dissect lobules, some fat vacuoles. Magnification 3 1.25X .
376
SHOEMAKER
AND
VISEK
PHOTOGRAPH 2. Liver from rat treated as in Photograph 1, representing “moderate” fibrous bands dissect lobules and circumscribe some lobules. Magnification 31.25~.
cirrhosis:
was added to the centrifuge tubes which were centrifuged at 48OOg for 15 min. The pellets, resuspended twice in 6 ml of 0.2 N perchloric acid after each addition, were centrifuged for 10 min at 4800g. Lipid was extracted by treating the pellets once with 2.0 ml of 1.O N potassium acetate in absolute ethanol, twice with 4.0 ml chloroform:methanol (l:l), and once with 2 ml absolute ethanol. Each extraction included rehomogenization followed by centrifugation at 4800g for 10 min. The
PHOTOGRAPH 3. Liver from rat treated as in Photograph 1, representing “severe” cirrhosis: disruption of sinusoidal architecture, numerous fat vacuoles, regenerative nodule. Magnification 31.25~.
OROTIC
ACID
AND
CIRRHOSIS
377
solvent extracts were pooled and evaporated overnight in tared vials at 70°C. The fat-free precipitates were dispersed and allowed to stand overnight in 5 .O ml of 1.5 N HClO,. RNA was extracted by resuspending the pellet twice in 2.5 ml of 0.5 N perchloric acid. DNA was extracted by two treatments with 5.0 ml of 0.5 N perchloric acid at 70°C for 20 min, and the protein remaining was dissolved by treatment with 4.0 ml of 1 N KOH for 15 min at 70°C. Radioactivity was assayed on 2.5-ml aliquots of the RNA and DNA extracts each mixed with 7.5 ml Aquasol II (New England Nuclear Corp., Boston, MA) and counted in a Beckman LK 9000 scintillation counter. Calorimetric determinations of RNA, DNA, and protein followed Cerotti (1955), Hubbard et al. (1970), and Lowry et al. (1951), respectively. Assay of Orotic Acid Urinary erotic acid was assayed in the effluent from a high-pressure liquid chromatography column of Partisil-IO SAX (Whatman Corp., Clifton, NJ) eluted in a gradient from distilled water to 10 mM KH,PO,, pH 2.76, according to a procedure adapted from Mills et al. (1979). A stainless-steel 4 x 25-mm column with a flow rate of 2.5 ml/min at 40°C and a solvent program requiring 10 min were used with a Waters Solvent Programmer Curve No. 5 and a convex gradient (Waters Corp., Milford, MA). When a Radial-Pak (Waters Corp.) column was used in the Waters RCM-100, the flow rate was 3 ml/min and the program took 15 min at room temperature. Absorbance was measured at both 280 and 254 nm. The erotic acid peak was identified by its retention time compared to an erotic acid standard (Sigma Chemical Co., St. Louis, MO) and by its ratio of absorbance at 280 and 254 nm which is characteristically high for erotic acid among the other peaks in the chromatogram. The height of the erotic acid peak at 280 nm was compared to that of a standard peak injected with each series of samples. For each lo-ml vol of diluted urine, 1 ~1 of sample was injected. The lower limit of detection was 5-10 ng. RESULTS Two of the 45 animals died from acute CCL, toxicity and 7 died from pneumonia as shown by histopathological examination. There were no consistent effects of treatment on feed intake but there was a statistically significant depression in weight gain, which averaged 8% in the CC&-treated animals compared to that in controls until the 18th week. Compared to Diet 1, no consistent differences were found in the intake of either the arginine-supplemented casein diet (Diet 2) or the soybean protein diet (Diet 3). Cessation of Ccl4 exposure for 1 week at the onset of erotic aciduria lowered erotic acid excretion from 179 2 0.5 to 102 k 6 kg/day (1 = 7.43, P < 0.001) within 7 days. Orotic acid excretion tended to decline as the animals reached adult weight in both the treated and untreated groups (Fig. 2). Arginine supplementation tended to lower erotic acid excretion, but not by a statistically significant difference (Table II). Likewise, 1.5% arginine supplementation did not prevent the elevation of erotic acid excretion when Ccl, treatment was resumed during the following 4 weeks. The ratio of liver protein to DNA decreased with Ccl, exposure (Table III) while liver lipid content and the rate of [14C]orotic acid incorporation into RNA did not differ between the two groups. Spleen weights were
378
SHOEMAKER
AND
VISEK
160-
FIG. 2. Orotic acid excretion by rats exposed to carbon tetrachloride (Ccl,; Week, 1 n = 45; Week 10, n = 37; Week 20, n = 16) compared to that by unexposed controls (Week 1, n = 15; Week 10, n = 13; Week 20, n = 5). Both groups were fed AIN-76A diets containing phenobarbital (Table 1). The means indicated differ significantly (P < 0.0s). Error bars indicate standard errors of the mean. Arrow at 20 weeks indicates cessation of Ccl, exposure.
significantly greater after CCL, exposure, but only one rat, with severe cirrhosis and ascites, had atrophic testicles at necropsy. The data from perfusion of cirrhotic livers are shown by Figs. 3 and 4 and Table IV. There were no significant differences in urea synthesis rates between the first 30 min without added ornithine and the subsequent 30 min of perfusion, when additional ornithine was present in the perfusing medium. Urea synthesis was constant with ammonia concentrations ranging from 0.2 to 3.0 mM. Addition of ornithine caused no significant increase in urea synthesis or ammonia clearance in either controls or cirrhotics, and the histological severity of cirrhosis was not
TABLE II Effect of Casein Soybean and Arginine on Orotic Acid Excretion in CC&-Treated Rats, Means f SEM Group Control (20 weeks, mean) All CC1 rats at onset of erotic aciduria Casein (7-16 weeks) Diet 1 1.5% Arginine (Diet 2) Soybean (7-16 weeks) Diet 3 Casein (16-20 weeks) Diet 1 Soybean (16-20 weeks) Diet 3
Orotic acid excreted Way)
Number of rats
83 f 9”
15
179 +- 9” 213 * H6 162 2 14 139 * 11* 111 f 7 11429
30 4 10 4 6 6
a,* Means with same superscript differ (P < 0.001).
379
OROTIC ACID AND CIRRHOSIS TABLE III Liver, Spleen, and Testis Weights and Liver RNA, DNA Protein, and Lipids in Rats Made Cirrhotic by Inhalation of CCI, for 16-20 Weeks, Means 2 SEM Control (n = 9)
Organ/component
CCLtreated (n = 17)
Body weight (9) Liver (g) Liver (g/kg BW)
355 + 18 14.3 f 1.1 40.1 +- 1.8” 40.9 f 2.5 49.6 f 4.5’ 5.64 + 0.6
365 14.6 44.0 34.1 38.8 35.4 4.82
f f f f k + f
18 1.0 1.96 2.5”,b 1.6 1.7’ 0.4
Liver lipid (% dry wt) Liver (g protein/g DNA) Liver [‘4C]orotate, uptake into RNA (dpm/pg RNA) Liver RNA (m&g FFDW)* RNA (mg/liver) DNA (mg/liver) Protein (g/liver) Spleen (&g BW) Testis @/kg BW)
33.4 72.3 37.9 1.88 1.49 9.26
35.2 72.0 43.2 1.53 2.85 8.91
k + f ” + 2
1.7 2.8 2.7 0.1 0.34” 0.5
f ” + f f +
3.2 4.1 4.2 0.2 0.24d 0.4
(Sacrificed at weeks 16-20) (Sacrificed after 20 weeks)
* t = 2.02, P < 0.05. b t = 3.23, P < 0.01. = t = 3.36, P < 0.01. d t = 3.48, P < 0.01. ’ FFDW, fat free dry weight.
associated with a decrease in urea synthesis rates. For example, the three rats with the most severe cirrhosis synthesized an average of 30.6 + 4.9 kmole of urea/g liver protein/hr before the addition of ornithine. Table III shows changes in organ weights and liver DNA, RNA, lipid, and protein consistent with cirrhosis. Portal serum ammonia and amino acid concentrations (Table V) were also consistent with impairment of liver function by CCL exposure. The difference in the ammonia concentration of portal blood between control and Ccl,-treated rats was not statistically significant, but the subset of animals with the most severe histological cirrhosis, whether fed soybean or casein protein, showed a statisticaIly significant increase in portal ammonia which averaged 0.30 + .Ol compared to 0.20 -+ .08 n&f for controls (t = 2.65, P < 0.05). .F $ 100 \\ F *\ $ b ‘$
BO60-
f
40-
2 4
20-
I --\..
Mod-Severe Disease lllZ6)
I------+ ‘+---,--
t =2.85
-s
PCO.02
-+
t = 5.56
Control + Mild (n=6)
PCO.001 I 30
I 60
Minutes FIG. 3. Percentage of sting ammonia concentration, 2.95 f 0.12 mM (mean -t SEM), remaining in the perfosate as a function of time during perfusion of rat livers with mild or no cirrhosis versus those with moderate-to-severe cirrhosis.
380
SHOEMAKER
AND VISEK
Ammonia
(mM)
FIG. 4. Release of erotic acid into the perfusate by livers of control and CCL-treated rats plotted against ammonia concentration. The correlation for both groups was significant (control, r = 0.76; Ccl,, r = 0.83; both P < 0.01). There were no differences in slope or intercept.
The livers with the most severe histological changes had less capacity to remove ammonia from the per&ate than controls or livers with only mild pathology (Fig. 3). The starting mean ammonia concentration in the perfusate averaged 2.95 2 0.12 mM. There was no difference in the tendency of control or CC&-treated livers to make erotic acid at a given level of ammonia (Fig. 4). Adding 2.3 mik! ornithine to the perfusate raised the calculated threshold ammonia concentration (abscissa-intercept) for causing orotate spillage from 0 to 1.52 mM (kg orotate/hr/g liver protein = 95.2 x NH,[w - 145, r = 0.546, curve not shown). DISCUSSION As control or Ccl,-treated rats achieved adult weight, erotic acid excretion tended to decline, suggesting that competition for arginine between growth and urea synthesis was lessened as the animals matured (Milner and Visek, 1973). Soybean protein, containing more than twice the arginine content of casein (Orr and Watt, 1957), dramatically decreased erotic acid excretion during the growth phase but caused no changes between Weeks 17 and 20 even though the cumulative effects of Ccl, were presumably further advanced at that time. After 7-10 weeks of Ccl, treatment when erotic aciduria first became evident, or after 20 weeks, acute CC& exposure was required to maintain elevated erotic acid excretion. Thus continuous “acute-on-chronic” exposure was essential to maintain elevated erotic acid excretion in this animal model. TABLE IV Urea Synthesis during Perfusion With/Without Omithine of Rat Livers Made Cirrhotic by CCL (kmole/hr/g liver protein), Means k SEM No omithine Controls CC&-treated Mean
32.8 (n 24.7 (n 26.5
2 7.4 = 7) 2 2.4 = 14) -+ 2.5
2.3 mM omithine 30.6 (n 28.7 (n 29.3
f 5.0 = 7) f 4.2 = 14) 2 3.2
Mean 31.7 + 4.3 26.7 f 2.4
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TABLE V Portal Plasma Amino Acids in Rats with Carbon Tetrachloride-Induced Cirrhosis, the Effect of Dietary Soybean Protein vs Casein, Means f SEM Treatment Amino acid (mh4) Ammonia Arginine Omithine Citrulline Glutamine Proline Lysine Histidine Tyr + Phe Val + Ile + Leu Ratio Val + Be + Leu Tyr + Phe Tryptophan Met + Cys
Diet 1: control (n = 9)
Diet 1: CCl.,-casein (n = 8)
0.198 f 0.026 0.101 f 0.012 0.047 2 0.003 0.047 2 0.006 0.425 2 0.027 0.178 + 0.027 0.360 + 0.025 0.084 -1-0.008 0.185 f 0.02 0.415 f 0.041 2.24
0.236 0.088 0.055 0.079 0.548 0.416 0.339 0.100 0.255 0.416
+ 0.028 2 0.006 f 0.011 k 0.010 f 0.079 -+ 0.108 * 0.041 * 0.013 k 0.045 2 0.038 1.63
0.060 2 0.010 0.093 k 0.011
0.064 +- 0.006 0.100 2 0.009
Diet 3: CCl,soybean (n = 4) 0.216 0.068 0.073 0.066 0.497 0.229 0.291 0.088 0.177 0.294
f 0.028 2 0.013 2 0.012 f 0.006 f 0.025 f 0.057 2 0.021 + 0.008 + 0.020 f 0.068 1.66
0.073 2 0.008 0.067 2 0.008
The difference in incorporation of labeled erotic acid into RNA of CC&-treated animals compared to that of controls was not statistically significant (Table III) and there was no difference in DNA specific activity. The CC&-treated animals had 5% more RNA per gram of fat-free dry liver and significantly more DNA per unit of protein. Thus, differences in [‘4C]orotate incorporation into RNA did not account for the two- to threefold rise in orotate excretion caused by Ccl4 toxicity. Urea synthesis rates by perfused rat livers in our studies (Table IV) were comparable to those of other investigators (Henley et al., 1975; Perez et al., 1978, 1979), although exact comparisons are precluded because expression of the rates differed between studies. Both Henley et al. (1975) and Perez et al. (1978) employed albumin in their perfusate which introduced another source of nitrogen not used in our studies. Perez et al. (1979) and Henley et al. (1975) also reported differences in urea synthesis rates between cirrhotic and control livers, but the ammonia concentrations they used greatly exceeded the physiological range. Our data (Fig. 3) confirm that cirrhotic livers removed ammonia from the perfusate more slowly than normal liver (Perez et al., 1979). There was no significant difference in the mean urea production between controls and cirrhotic animals (Table IV), and no trend toward lower urea production for the livers with the severest histopathology. The differences between these results and those of Perez et al. (1979) and Henley et al. (1975) are probably due to the concentrations of ammonia used in the perfusing solution. Protein loading studies on intact animals have confirmed that urea synthesis capacity in cirrhosis is near normal (Brewer et al., 1984). The concentration of portal ammonia increased due to Ccl4 exposure as shown in Table V. The ratio of branch chain to aromatic amino acids followed the trend described by Fischer et al. (1975) as indicative of hepatic injury. The “amine pool” amino acids, glutamine and proline, tended to be highest in casein-fed rats with cirrhosis. On the basis of the data in Table V the mean portal ammonia
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concentration in all cirrhotic animals was 0.24 mM compared to 0.20 mA4 for controls. This difference of 0.04 mM if maintained over 24 hr would induce the synthesis of an additional 828 pg of erotic acid per liver (estimated from the regression line in Fig. 4). According to previous findings (Hecht and Potter, 1956), 40% of orotate reaching the liver after intraperitoneal injection is excreted in the urine. If this percentage of the additional erotic acid was excreted daily, the amount would approximate what we observed. Orotic acid production has not previously been measured in perfused cirrhotic livers. Unlike techniques using liver slices or isolated hepatocytes, perfused livers can show differences due to microvascular derangement and intrahepatic shunts described by Wood ef al. (1979), Villeneuve et al. (1978), and Reichen et al. (1987). Our observation of higher portal ammonia with decreased ammonia clearance during perfusion, despite normal urea synthesis capacity concurs with the “intact-hepatocyte” hypothesis put forth by Villeneuve et al. (1978). Intrahepatic shunts may comprise the chronic aspect of the acute-on-chronic toxicity of Ccl, while transient hyperammonemia due to reduced hepatocyte number or effectiveness may result from acute exposure. Arginine supplementation did not prevent the erotic aciduria of Ccl4 toxicity. A previous report showed no effect of arginine in rats with surgical portacaval shunts (Shoemaker, 1984). During the growth phase, soybean protein had a dramatic effect compared to casein in lowering orotate excretion, but not when animals reached adult weight. Lysine is an arginine antagonist and the higher arginineilysine ratio in soybean protein (Orr and Watt, 1957), or differences in digestibility, could have lowered portal ammonia or decreased orotate synthesis after acute Ccl, exposure. Protein of soybeans and other plants has been recommended for diets of patients with hepatic encephalopathy (Greenberger et al., 1977). The present data agree with this recommendation. Enteral or parenteral administration of amino acid mixtures has also been advocated to decrease the incidence of portal systemic encephalopathy (Fischer et al., 1976). Najarian and Harper (1956) reported that arginine lowered blood ammonia in some patients, but a correlation between the degree of encephalopathy and the blood ammonia concentration was not consistently demonstrated. Thomson and Visek (1963) reduced mental obtundation in rats by immunization against jack bean urease which inhibits ammonia formation in the intestine. However, repeated injections of the enzyme are required to initiate and maintain this immunity. More recently, improved mental status from the administration of omithine with or without supplemental branch chain amino acids has been reported (Herlong et al., 1980). Zieve (1986) has suggested that increased urinary erotic acid in a patient with hepatic failure may indicate that sufficient liver function remains to warrant supplementation with omithine or arginine. The present animal study supports this concept with the reservation that intrahepatic shunting may lessen the expected ammonia-lowering effect. Elevated plasma ammonia, reduced hepatic ammonia clearance, and the response of growing rats to soybean protein supported the hypothesis that the erotic aciduria of carbon tetrachloride toxicity was due to acutely elevated ammonia concentrations and the existence of intrahepatic shunts. Continuous exposure to CC& was required to maintain the elevation of urinary erotic acid, probably due to the regenerative activity of the liver.
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