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Effect of age on the activity of arginase of the liver and kidney cortex of rat
Exp. Geront. Vol. 4. pp. 57-60. Perllmon Press 1969. Printed in Gre~ Britain
Short C o m m - n | r . a t i o n EFFECT OF AGE ON THE ACTIVITY OF ARGINASE THE LIVER AND KIDNEY CORTEX OF RAT
OF
S. P. SHUKLA and M. S. K A N U N C O Department of Zoology, Banaras Hindu University,Varanasi S, India
(Received 16 October 1968) INTRODUCTION EFVICmNT production of urea, which depends on the activity of arginase (L-arginine amino-hydrolase, EC 3.5.3.1)may be of significance in the ageing process. Lightbody (1938) reported an oscillating pattern of arginase activity in the early growth period and a decrease in old age in both the sexes of the rat. While this study was in progress, Charbonneau, Roberge and Berlinguet (1967) reported that the activities of all the enzymes of the urea cycle including the arginase increase with body weight of the rat. Our studies show the alterations in the activity of arginase as a function of age, organ and body weights in the male rats. MATERIALS AND METHODS Male albino rats of Wistar strain were used. They were maintained at 23 + 2°C and were fed standard Anidiet "A" of the Chelsea Chemical Co., Poona, and gram (Cicer arietinum) ad libitum. The ages of the rats were 6, 30 and 96 weeks which covered the entire life span of the animal. They were killed by dislocation of the neck, and the right lobe of the liver and right kidney were removed and weighed. A 10 per cent homogenate of each tissue was made in 0.25 M sucrose using a Potter-Elvehjem homogenizer and a teflon pestle. Homogenization was done at 0 4- 2°C and was completed by 25 up and down strokes of the pestle. The homogenates were then suitably diluted with distilled water for the measurement of the activity of arginase. The assay mixture consisted of 0.2 ml of 0.2 M arginine (pH, 9.5), 0.2 ml water and 0.1 ml of homogenate in a total volume of 0.5 ml. The incubation was done at 37°C for a period of 30 rain as the reaction was linear for this period. The amount of urea liberated was estimated using 0.075 per cent 2,3 butanedione as the colour reagent. The activity of arginase was calculated as units/g wet weight from a standard curve using urea. Statistical analyses of the data were made according to Garret (1956) from the results obtained from 5 or 6 animals of each age. RESULTS AND DISCUSSION Figure 1 shows that the liver of 6-week-old rat has the highest activity of arginase (units/g wet wt.). T h i s indicates that efficient urea production may occur at this age. The activity of arginase of the liver of 30-week-old rat(adult)was only 54 per cent of that of 6 weeks. Thereafter no change was noticed tiU 96 weeks (old age). When the data were analysed on the basis of enzyme units/total organ wt., maximum activity was found at 30 57
S. P. S H U K L A
58
AND
M. S. K A N U N G O
2000
1500
I00C
50C
0
i
i
6
30
i
96 ~(w~ks)
FIG. 1. Activity of arginase (units/g wet wt.) of the liver (©) and kidney cortex (A) of male rats of different ages. weeks, after which no significant change was noticed (Fig. 2). The weight of the liver at 30 weeks was, however, 2.5-fold higher than that at 6 weeks (Fig. 2). So the rate of enzyme synthesis apparently does not keep pace with the increase in the weight of the organ during growth. The weight of the liver at 30 and 96 weeks was almost the same. It is of interest to note that though the weight of the animal increases significantly between 30 and 96 weeks (Fig. 3), there appears to be no further change in the organ weight or the enzyme activity. Thus on the basis of the wet weight, the 6-week-old liver is more efficient in producing urea from nitrogenous waste products. Though the weight of the animal and of other organs increased considerably after 30 weeks, the formation of urea did not show corresponding increase. The activity of arginase (units/g wet wt.) in the kidney cortex also declined between 6 and 30 weeks by about 33 per cent (Fig. 1). The weight of the cortex at 30 weeks was 2.3-fold higher than that at 6 weeks (Fig. 2). Thus the efficiency of urea production also decreases in the kidney cortex as in the liver between 6 and 30 weeks. This is followed by a significant increase in the activity of the enzyme up to 96 weeks. The rates of increase in the enzyme activity and of the weight of the kidney cortex were almost parallel throughout the life span of the animal (6-96 weeks). The rate of increase of arginase between 6-30 weeks was, however, slower in the kidney cortex as compared to that of the weight of the animal in this period (Fig. 3). Thereafter, the rates of increase of arginase activity and of the weight of the animal were parallel unlike that of the liver. The data further show that the activity of arginase of the liver is about 70-fold higher than that of the kidney cortex at 6 weeks. The activity of the enzyme of the liver at 30 weeks is about 50-fold higher than that of the kidney cortex at this age. The enzyme activity was not observed in the medulla of the kidney with the present technique. The total production of urea in the liver due to arginase is less in the adult and the old than in the young on the basis of wet weight. The production of urea in the kidney cortex, however, does not decrease in old age. Rather there is a tendency for an increase in urea production in this tissue in the old.
EFFECT OF AGE ON THE A C T M T Y OF ARGINASE IN RATS
59
/
7500
6000 ~
4500
iI
~
x5
~
3~ ~3000
ill tl
1500
'
'o Agg(wg¢ks)
FIG. 2. Activity of arginase (traits/organ) of the liver (O) and kidney cortex (A) of male rats of different ages. O, weight of whole liver; A, weight of whole kidney cortex.
7500
250
~
6000
200
4500
150 "E '
E
t/ ,
3OO0
20(
I
"6 O0 ~
I
50
1500
o
I
o Age(w~gks)
FIG. 3. Activity of arginase (units/organ) of the liver (O) and kidney cortex (A) of male rats of different ages. @, weight of the rat.
Acknowledgement,v--Theauthors are thankful to Professor S. P. RAY-CHAUDHURIfor encouragement. This research was supported by grants from the Nuffield Foundation, England, and the Council of Scientific and Industrial Research, New Delhi, to M.S.K.S.P.S. thanks the C.S.I.R. for a Junior Research Fellowship.
60
S . P . SHUKLA AND M. S. KANUNGO REFERENCES
CmmaONN~U, R., ROBERGE, A. and BERLINGUET, L. (1967) Canad. ft. Biochem. 45, 1427. Gxamrr, H. E. (1956) In Elementary Statistics. Longmans, Green, New York. LIOHTBODY, H. D. (1938).7. biol. Chem. 124, 169.
S u m m a r y - - T h e activity of arginase in the homogenates of the liver and the kidney cortex of 6-, 30- and 96-week-old rats was determined to evaluate the efficiency of urea production during ageing. T h e activity of the liver enzyme was higher at 6 weeks when expressed as units/g wet wt. However, on the basis of both the organ and body weights, highest activity was observed at 30 weeks after which there was no change. T h e activity of the enzyme of the kidney cortex showed a similar pattern of change as that of the liver. It is suggested that the 6-week-old rat is more efficient in eliminating its ammonia as urea than the old rat. This may be of significance in the ageing process of the rat.
Short C o m m - n | r . a t i o n EFFECT OF AGE ON THE ACTIVITY OF ARGINASE THE LIVER AND KIDNEY CORTEX OF RAT
OF
S. P. SHUKLA and M. S. K A N U N C O Department of Zoology, Banaras Hindu University,Varanasi S, India
(Received 16 October 1968) INTRODUCTION EFVICmNT production of urea, which depends on the activity of arginase (L-arginine amino-hydrolase, EC 3.5.3.1)may be of significance in the ageing process. Lightbody (1938) reported an oscillating pattern of arginase activity in the early growth period and a decrease in old age in both the sexes of the rat. While this study was in progress, Charbonneau, Roberge and Berlinguet (1967) reported that the activities of all the enzymes of the urea cycle including the arginase increase with body weight of the rat. Our studies show the alterations in the activity of arginase as a function of age, organ and body weights in the male rats. MATERIALS AND METHODS Male albino rats of Wistar strain were used. They were maintained at 23 + 2°C and were fed standard Anidiet "A" of the Chelsea Chemical Co., Poona, and gram (Cicer arietinum) ad libitum. The ages of the rats were 6, 30 and 96 weeks which covered the entire life span of the animal. They were killed by dislocation of the neck, and the right lobe of the liver and right kidney were removed and weighed. A 10 per cent homogenate of each tissue was made in 0.25 M sucrose using a Potter-Elvehjem homogenizer and a teflon pestle. Homogenization was done at 0 4- 2°C and was completed by 25 up and down strokes of the pestle. The homogenates were then suitably diluted with distilled water for the measurement of the activity of arginase. The assay mixture consisted of 0.2 ml of 0.2 M arginine (pH, 9.5), 0.2 ml water and 0.1 ml of homogenate in a total volume of 0.5 ml. The incubation was done at 37°C for a period of 30 rain as the reaction was linear for this period. The amount of urea liberated was estimated using 0.075 per cent 2,3 butanedione as the colour reagent. The activity of arginase was calculated as units/g wet weight from a standard curve using urea. Statistical analyses of the data were made according to Garret (1956) from the results obtained from 5 or 6 animals of each age. RESULTS AND DISCUSSION Figure 1 shows that the liver of 6-week-old rat has the highest activity of arginase (units/g wet wt.). T h i s indicates that efficient urea production may occur at this age. The activity of arginase of the liver of 30-week-old rat(adult)was only 54 per cent of that of 6 weeks. Thereafter no change was noticed tiU 96 weeks (old age). When the data were analysed on the basis of enzyme units/total organ wt., maximum activity was found at 30 57
S. P. S H U K L A
58
AND
M. S. K A N U N G O
2000
1500
I00C
50C
0
i
i
6
30
i
96 ~(w~ks)
FIG. 1. Activity of arginase (units/g wet wt.) of the liver (©) and kidney cortex (A) of male rats of different ages. weeks, after which no significant change was noticed (Fig. 2). The weight of the liver at 30 weeks was, however, 2.5-fold higher than that at 6 weeks (Fig. 2). So the rate of enzyme synthesis apparently does not keep pace with the increase in the weight of the organ during growth. The weight of the liver at 30 and 96 weeks was almost the same. It is of interest to note that though the weight of the animal increases significantly between 30 and 96 weeks (Fig. 3), there appears to be no further change in the organ weight or the enzyme activity. Thus on the basis of the wet weight, the 6-week-old liver is more efficient in producing urea from nitrogenous waste products. Though the weight of the animal and of other organs increased considerably after 30 weeks, the formation of urea did not show corresponding increase. The activity of arginase (units/g wet wt.) in the kidney cortex also declined between 6 and 30 weeks by about 33 per cent (Fig. 1). The weight of the cortex at 30 weeks was 2.3-fold higher than that at 6 weeks (Fig. 2). Thus the efficiency of urea production also decreases in the kidney cortex as in the liver between 6 and 30 weeks. This is followed by a significant increase in the activity of the enzyme up to 96 weeks. The rates of increase in the enzyme activity and of the weight of the kidney cortex were almost parallel throughout the life span of the animal (6-96 weeks). The rate of increase of arginase between 6-30 weeks was, however, slower in the kidney cortex as compared to that of the weight of the animal in this period (Fig. 3). Thereafter, the rates of increase of arginase activity and of the weight of the animal were parallel unlike that of the liver. The data further show that the activity of arginase of the liver is about 70-fold higher than that of the kidney cortex at 6 weeks. The activity of the enzyme of the liver at 30 weeks is about 50-fold higher than that of the kidney cortex at this age. The enzyme activity was not observed in the medulla of the kidney with the present technique. The total production of urea in the liver due to arginase is less in the adult and the old than in the young on the basis of wet weight. The production of urea in the kidney cortex, however, does not decrease in old age. Rather there is a tendency for an increase in urea production in this tissue in the old.
EFFECT OF AGE ON THE A C T M T Y OF ARGINASE IN RATS
59
/
7500
6000 ~
4500
iI
~
x5
~
3~ ~3000
ill tl
1500
'
'o Agg(wg¢ks)
FIG. 2. Activity of arginase (traits/organ) of the liver (O) and kidney cortex (A) of male rats of different ages. O, weight of whole liver; A, weight of whole kidney cortex.
7500
250
~
6000
200
4500
150 "E '
E
t/ ,
3OO0
20(
I
"6 O0 ~
I
50
1500
o
I
o Age(w~gks)
FIG. 3. Activity of arginase (units/organ) of the liver (O) and kidney cortex (A) of male rats of different ages. @, weight of the rat.
Acknowledgement,v--Theauthors are thankful to Professor S. P. RAY-CHAUDHURIfor encouragement. This research was supported by grants from the Nuffield Foundation, England, and the Council of Scientific and Industrial Research, New Delhi, to M.S.K.S.P.S. thanks the C.S.I.R. for a Junior Research Fellowship.
60
S . P . SHUKLA AND M. S. KANUNGO REFERENCES
CmmaONN~U, R., ROBERGE, A. and BERLINGUET, L. (1967) Canad. ft. Biochem. 45, 1427. Gxamrr, H. E. (1956) In Elementary Statistics. Longmans, Green, New York. LIOHTBODY, H. D. (1938).7. biol. Chem. 124, 169.
S u m m a r y - - T h e activity of arginase in the homogenates of the liver and the kidney cortex of 6-, 30- and 96-week-old rats was determined to evaluate the efficiency of urea production during ageing. T h e activity of the liver enzyme was higher at 6 weeks when expressed as units/g wet wt. However, on the basis of both the organ and body weights, highest activity was observed at 30 weeks after which there was no change. T h e activity of the enzyme of the kidney cortex showed a similar pattern of change as that of the liver. It is suggested that the 6-week-old rat is more efficient in eliminating its ammonia as urea than the old rat. This may be of significance in the ageing process of the rat.