Arginine metabolism in rat liver after hepatic damage

BIOCHEMICAL

MEDICINE

27. X6-94

(tY82J

Arginine Metabolism in Rat Liver after Hepatic Damage S. Department

of

VIJAYA

Microbiology. Received

B.

AND Cancer

March

NAGARAJAN Institute,

Madras

600 020, Inditr

13. 1981

L-Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) is known to occur in tissues of both ureotelic and uricotelic animals, the highest activity being found in liver (1). Arginine is a key amino acid involved in a variety of metabolic reactions in the mammalian system. Arginase catalyzes the cleavage of L-arginine to ornithine and urea. In experimentally induced liver damage, serum arginase has been found to be dramatically elevated in mice and rats (2,3) as is serum ornithine carbamyl transferase (OCT) (4,5). Carbon tetrachloride (Ccl,) is a well-known hepatotoxin and hepatocarcinogen. It causes fatty degeneration and necrosis in hepatic cells within a short time after administration, and causes cirrhosis after repeated administration. Liver damage resulting from Ccl, poisoning leads to a number of metabolic changes, one being a profound disturbance in nitrogen metabolism (6-8). Though the changes in urea cycle enzymes in CCL, acute treatment have been studied (9.10). the changes in intermediary metabolites of the urea cycle under this condition have not been studied much. We were interested in studying the time course change in arginine metabolism in both liver and plasma of rats, after Ccl, administration. We also report some lysosomal enzyme changes, 24 hr after treatment with Ccl,. The lysosomal system has been shown to be very sensitive to changes in the intra- and extracellular environment and consequently is involved directly or indirectly in many physiological and pathological processes. It is evidenced that lysosomal integrity is modified during liver injury in the rat. Carbon tetrachloride is a known lysosomal labilizer and releases lysosomal enzymes in vitro and in V~VO, and it is suggested that this action of Ccl, on lysosomes may partly be responsible for its biological effect (11). We believe that lysosomal changes may also be responsible to some extent for the changes in arginase. 86 0006~2944/82/010086-09$02.00/O CopyrIght All rights

0 1982 by Academic Pres. Inc. of reproduction in any form rc\erved

CHANGES INARGININE MATERIALS

METABOLISM

87

AND METHODS

Chemicals. L-Arginine, L-ornithine, dilithium carbamyl phosphate, citrulline, bovine hemoglobin, and ninhydrin were purchased from Sigma Chemical Company; Sephadex G-50, from Pharmacia Chemicals; Dowex AGl x 8 (200-400 mesh), from Bio-Rad. All other reagents used were of analytical grade. Treatment with Ccl,. Young male Wistar rats weighing 120-150 g were used and fed a standard pellet diet and water ad libitum. Rats in both control and treated groups were starved overnight, Ccl, in liquid paraffin (1: 1, v/v) was given by stomach tube (0.5 ml/l00 g body wt) under light ether anesthesia. The control animals were treated with 0.25 ml of liquid paraffin/100 g body wt. The animals were sacrificed at 3, 6, 24, 48, and 72 hr after treatment. The liver tissues were immediately removed and kept in ice at -4°C. Enzyme assays were completed on the same day. Perchloric acid extracts were stored at 4°C for metabolite measurements. Assay of enzymes und metabolites. Liver arginase and other tissue arginases were estimated by the method of Herzfeld ef al. (12). Liver OCT was estimated by the method of Weber et al. (13). Plasma arginase and plasma OCT were estimated as reported earlier (14,15). Arginine and other related metabolites were separated and estimated by the method of Gopalakrishna and Nagarajan (16). In the case of polyamines, after eluting from Dowex AG SOH+ column, the polyamine fractions were evaporated and a portion of the evaporated fraction was subjected to high-voltage electrophoresis (30 V/cm for 45 min) using 0.1 M sodium citrate buffer, pH 4.3; polyamine standards were run side by side. After electrophoresis, the strips were dried and stained with 0.1% ninhydrin in acetone. The polyamine portions were cut and eluted in ethanol: acetic acid: water (4:5:1) containing cadmium acetate 20 mg/ml. The color was read at 505 nm. Urine urea and urine urginase. The animals were kept in metabolic cages and 24-hr urine samples were collected. Urine urea was estimated by the method of Archibald (17) and urine arginase was estimated the same way as plasma arginase after being subjected to centrifuge column dialysis (14). Lysosomal enzymes. For the lysosomal enzyme assays, a 10% tissue homogenate in 0.25 M sucrose was prepared in a Teflon homogenizer by 5-downward movement. To a portion of the homogenate Triton X-100 (final cont. 0.2%) was added and mixed in a Vortex omnimixer for 30 sec. The homogenates were centrifuged at 20,OOOg for 15 min. The supernatant obtained from the homogenate treated with 0.25 M sucrose alone is the free enzyme and the one treated with Triton X-100 is the “total enzyme.” Cathepsin-D and acid phosphatase were assayed by the

method of Barret rt rrl. (IX). Enzyme activities were expressed as units per milligram of protein. Protein was determined according to the method of Lowry et ~1. using bovine serum albumin as standard t 19). Triton X- 100 at this concentration did not interfere with protein determination as well as the enzyme assay. RESULTS Liver Arginase

AND DISCUSSION

und OCT

Table 1 shows liver arginase and OCT activities at various time intervals. At 24 hr liver arginase showed a transient decrease in activity which returned to normal around 72 hr when the regeneration process was completed. OCT showed an increase in the activity in the initial stages of Ccl, poisoning as early as 6 hr. and returned to normal at 72 hr. This was quite in contrast to the profile of the arginase changes seen. Control animals treated with liquid paraffin alone did not show any apparent change. Plusma

Arginase and OCT

Figure I shows the arginase and OCT activities in plasma at various time intervals after CCL, treatment. Both reached the maximum at 24 hr and slowly returned to control values around the sixth day. The increase of these enzymes in plasma is due to necrosis of the liver cell after CCI, administration. We have also seen that plasma arginase, 24 hr after treatment, cross-reacted with antiliver arginase. To see whether any plasma enzyme is excreted in the urine, we have checked urine arginase in 24-hr samples. There was no detectable arginase activity in urine. TABLE EFFECT

OF CARBON

TETRACHLORIDE

LIVER

Time after treatment (hr)

Liver arginase

0 3 6 24 48 72

116.746 106,714 101,739 65,675 97.791 111.610

+ i _f -t 2 i

5112 7994 7172 8611 9477 6296

I

TREATMENT

ON LIVER

AND

SPLEEN

ARGINASE

AND

OCT

Spleen arginase 76.1 -t 6.4 199 -+- 9.5 542 + 132 242 2 24.1 153 ? 18.3

Liver OCT 11,717 18,835 22,499 9.514 8040 11.377

2 f 2 -t 2 +

1004 2378 2226 629 317 473

Note. Values are expressed as mean f SD of four rats in each group. One unit of enzyme is expressed as pmole of product formed/hr/g tissue under the conditions of assay. Animals treated with liquid paraffin alone did not show any change.

CHANGES

IN ARGININE

METABOLISM

89

I--

FIG. 1. Plasma arginase and OCT at different time intervals after Ccl, treatment. 0. Plasma arginase; 0, plasma OCT. Enzyme unit represents *mole of productimllmin. Plasma arginase was 0.005 unit at zero time.

Metabolities

in Liver and Plasmu

Tables 2 and 3 show the content of arginine and related metabolites of liver and plasma of treated animals at 24 and 48 hr. Both the liver and plasma showed a considerable increase in the levels of ornithine, proline, and citrulline. Among the polyamines putrescine and spermidine were elevated in the liver with a fall in the spermine level. The plasma arginine level was low, whereas the liver arginine level showed a moderate increase. The urea level was not altered in both liver and plasma. We have also checked the urea levels in 24-hr urine samples of the CC],treated animals. Urea excretion was not altered much in the treated group. This tends to show that kidney is not affected though the liver function is altered. The decrease in liver arginase 24 hr after Ccl, treatment may be due to either (a) a leakage of considerable amount of liver cell enzyme into the plasma at this time interval or (b) a certain amount of increase in

TABLE

2

0.058 + 0.001

0.544 c 0.049

-

0.040 2 0.088

Omithine

ARGININE

AND

Arginine

METABOLITES

TABLE IN PLASMA

3 OF CARBON

0.132 + 0.029

0.846 f 0.085

0.765 + 0.057

Spermidine

0.547 -c 0.039

0.622 t 0.058

Spermine

0.237 7 0.018 0.243 i 0.013

0.101 i 0.003

Proline

0.080 “_ 0.007

RATS

0.100 t 0.008

0.052 2 0.008

Citrulline

TETRACHLORIDE-TREATED

Urea _-... ..~~~-.--~~~~~.. ~~-0.104 & 0.002 3.700 t 0.503

RELATED

0.021 + 0.006 4.187 + 0.581 24 0.254 2 0.042 48 0.173 -c 0.019 0.041 i- 0.007 3.990 ? 0.601 ._,.. ~~~~~-.~__ _-.~ .-.. ---~~~ Note. Values are expressed as mean t SD in ~moleiml of four rats in each group.

0

Time after treatment (hr)

change.

3.74 + 0.135

4.08 t 0.007

0.024 k 0.005

0.282 2 0.024

RATS

Putrescine

TETRACHLORIDE-TREATED

Proline

OF CARBON ----

Urea Citrulline ~~~~~ ~____ ~-.-2.89 k 0.315 0.042 f 0.004

IN LIVER

0.067 r 0.006 0.473 2 0.037 0.158 2 0.029 1.01 2 0.085 0.544 k 0.047 -~. Note. Values are expressed as mean k SD in p,mole/g of four rats in each group. Animals treated with liquid paraffin alone did not show any

0.035 * 0.007

0.047 t 0.008

0.846 ‘- 0.103

0.733 -t 0.02

24

48

Arginine

RELATED METABOLITES _... -.--..--...

.._~ ..-. 0.237 k 0.021 0.030 t 0.002

Omithine

AND

Time after treatment (hr) -~-. 0

ARGININE

2

c: 2

8

CHANGES

IN ARGININE

91

METABOLISM

liver weight due to liver regeneration. But the increase in plasma arginase does not parallel the decrease in liver arginase. Taking into consideration the increase in liver weight, the total activity of arginase is still less when compared to the control. This agrees with the finding of Flores et al. (IO), who have shown a decrease in the total activities of both arginine synthetase and arginase 24 hr after treatment with Ccl,. So we are of the opinion that some other factors such as release of lysosomal enzymes may play a role in decreasing the liver arginase. In CC&-induced fatty livers the lysosomal enzymes have been reported to be released into the soluble fraction of the cell, and the specific activities of some enzymes such as cathepsin and ribonuclease also increase, when the necrosis is well established (20-22). Release of hydrolytic enzymes would clearly result in extensive damage to proteins, nucleic acids, and other macromolecules in the cell. Our results (Fig. 2) show a moderate increase in the specific activities of both free and total cathepsin at 24 hr, though the increase in acid phosphatase is not much reflected. The increased

Control

Treated

Cathopsin D

Control

Acid

Treated

Phosphatase

FIG. 2. Cathepsin D and acid phosphatase levels in control and CCLtreated rats (24 hr). Each bar represents the mean from four rats. Cathepsin, nmole of Tyr released/min/ mg protein; Acid phosphatase, nmole of P, liberated/min/mg protein. m. Free activity: 0, total activity.

9’

VIJAYA

,AND

NAGAKAJAN

proteolytic enzyme cathepsin may act on the portion of the liver arginase and this will explain in part, the decrease in liver arginase that we see at 24 hr after treatment. The intracellular pH of liver cells also seems to decrease when there is cell damage (23). and this decrease may facilitate the action of proteolytic enzymes on macromolecules (24-27). The mechanism by which an increase in liver OCT occurs in the earlier stages of Ccl, poisoning is not clear when there is leakage of this enzyme into the plasma. McLean et nl. have also reported an increase in two mitochondrial-associated enzymes, ornithine carbamyl transferase and carbamyl phosphate synthase, in early stages of Ccl, poisoning (9). As these authors suggest this increase may indicate some modifications of mitochondrial structure at these very short intervals or the other possibility is that Ccl, action may be mediated by adrenal gland secretion and activation of urea cycle enzymes. The slight fall in OCT at 24 and 48 hr will possibly favor the condition for nucleic acid synthesis, since regeneration processes begin at about 24 hr after CC), administration. The increase in liver and plasma ornithine can be explained as follows. Liver arginase released into the plasma acts on the plasma arginine converting it into ornithine and urea. The increased plasma ornithine may again be taken up by the liver for maintaining the urea cycle activity. The high ornithine content in liver may also act as substrate for both OCT and ornithine transaminase forming more of citrulline and proline. The low level of plasma arginine may be due to the action of elevated plasma arginase on arginine under this condition. The increase in liver arginine 24 hr after Ccl, treatment may be due to the decrease in liver arginase. The unaltered levels of urea in plasma and liver indirectly prove that potential for urea synthesis is maintained, even under these acute toxic conditions. This agrees with the earlier report of McLean et ~11.(9). We feel that the increased putrescine level in the liver is comparable to the situation where the synthesis and accumulation of putrescine in various mammalian tissues are greatly increased after application of certain growth stimuli (28-30). The first change in polyamine metabolism after appropriate growth stimulus is an immense increase in the activity of ornithine decarboxylase with a concomitant accumulation of putrescine. A single injection of Ccl, into rats is known to cause acute liver necrosis followed by a rapid regenerative process. The fall in spermine level during this phase may be due to the increased conversion of spermine to spermidine as suggested by Holta et al. (31). Spleen Arginase

In addition we have also found elevated levels of splenic arginase 24 hr after treatment (Table I). This correlates with the increased plasma

CHANGES

IN ARGININE

METABOLISM

93

arginase activity, where the optimum level is seen at 24 hr. Other tissues do not show any such changes. We have checked the spleen arginase activity in liver regeneration after partial hepatectomy and there also we have seen elevated splenic arginase though not as high as in Ccl, treatment. This is because the release of liver arginase into blood is less in liver regeneration after partial hepatectomy when compared to the release in necrosis after Ccl, administration. It is well known that in animals the spleen serves as a reservoir of blood and releases it by active contraction in response to physiological demand (32). From this we believe that the elevated arginase activity in the spleen may be caused by the blood that has been retained. The possibility that whether the spleen arginase has any specific role under this condition cannot be ruled out and further studies are needed. On the basis of this study on the metabolites and two studies of the urea cycle enzymes, arginase and OCT, in experimental liver injury, it seems that the urea synthesizing capacity of the liver is unaffected in spite of the changes in two of the enzyme profiles. However, studies on other enzymes of the urea cycle such as carbamyl phosphate synthase may substantiate these findings. SUMMARY

The changes in arginine metabolism in liver and plasma were studied through short-term treatment with carbon tetrachloride. There was a considerable decrease in liver arginase at 24 hr and this may be partly related to the rupture of lysosomes and the release of proteolytic enzymes. Both arginase and ornithine carbamyl transferase were released into plasma from the necrosed liver cell. Elevated plasma arginase seems to act on plasma arginine, bringing about changes in arginine related metabolites in liver and plasma. Nonetheless, the net urea production in liver was not affected. ACKNOWLEDGMENT This work was supported by a research grant from the Department of Atomic Energy. Government of India.

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