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Biochimica et Biophysica A cta, 402 (1975) 113--123 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98358 ALBUMIN SYNTHESIS AND CATABOLISM FOLLOWING PARTIAL HEPATECTOMY IN THE RAT. THE EFFECTS OF AMINO ACIDS AND ADRENOCORTICAL STEROIDS ON ALBUMIN SYNTHESIS A F T E R PARTIAL HEPATECTOMY
ELWYN A. LLOYD, STUART J. SAUNDERS, LESLEY O'C. FRITH and JOAN E. WRIGHT Department of Medicine, Medical School, Observatory 7900, Cape Town (South Africa)
(Received February 7th, 1975)
Summary Plasma albumin levels were measured in partially hepatectomized, sham operated and control rats. The levels fell in both the partially hepatectomized and sham operated groups; while the latter group returned to normal within a few days, the low plasma albumin in the partially hepatectomized animals was sustained. Albumin synthesis rates in the isolated perfused rat liver were measured in the three groups of animals at varying intervals after partial hepatectomy. There was a significant depression of albumin synthesis rate in terms of both liver and whole animal weights when compared to the sham operated and control animals. This depression was almost completely reversed by the addition of arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, t r y p t o p h a n and vaiine added together to 10 times their normal plasma concentrations. The addition of hydrocortisone had no effect on the albumin synthesis rate after partial hepatectomy. Studies in vivo in the three groups of animals (partially hepatectomized, sham operated and control animals) revealed a fall in the albumin catabolic rate after partial h e p a t e c t o m y coinciding with the fall in the albumin synthesis rate. An hypothesis whereby the amino acids may have their stimulatory effect is proposed.
Introduction Changes in albumin metabolism following partial h e p a t e c t o m y have been measured [1--8]. The results of these studies are conflicting, some indicating a fall [4,6,8], some no change [2,3,7] and others an increase in the albumin synthesis rate [5].
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These widely differing results, together with the paucity of literature available concerning albumin catabolism after partial hepatectomy, and the lack of information concerning the influence of amino acids on albumin synthesis in the regenerating liver, prompted the work reported here. Materials and Methods Male Wistar rats, weighing between 250 and 300 g, housed in individual cages under controlled conditions of temperature, h u m i d i t y and lighting were permitted water ad libitum and unless otherwise stated had free access to a balanced diet. Partial hepatectomies were performed under diethylether anaesthesia with the removal of median and left lateral lobes as described by Higgins and Anderson [9]. Sham operations consisted of laparotomy and palpation of the liver only.
Radioactive materials All the radioactive materials used in these experiments were obtained from the Radio Chemical Centre, Amersham, England. 1. Na~ ~4 CO3, specific radioactivity 58.9 Ci/mol. 2. Na ~3 l I, specific radioactivity 40 Ci/1.3. Na ~2 s I, specific radioactivity 100 Ci/1.
1. Measurements of plasma albumin levels (in vivo) following partial hepatectomy Eighteen rats were divided into three groups: control, sham and partiallyhepatectomized, in such a way that each control had a sham operated and a partially hepatectomized partner of similar weight. On day O, partial hepatectomies were performed on the six rats in that group. They were weighed post operatively and returned to their individual cages where they received water ad libitum and in which there was a preweighed a m o u n t of food contained in a vessel designed to minimize spillage. The animals had become accustomed to feeding from these vessels prior to surgery. On day I the sham operations were performed and that group of rats weighed post-operatively. The control animals were weighed and they and the sham operated animals were given exactly that a m o u n t of food which their partially hepatectomized partners had eaten during the preceding 24 h. Each day animals were weighed and each trio (partially hepatectomized, sham operated and control) consumed the same weight of food as indicated above. In the first experiment the partially hepatectomized animals were exsanguinated on day 3, and the sham operated and control rats exsanguinated on day 4. In the second experiment the same procedure was adopted, except that the animals were exsanguinated on days 10 and 11 respectively. Serum albumin was estimated by the m e t h o d of Fernandez et al. [10].
2. Measurement of albumin synthesis rates (in vitro) following partial hepatectomy Perfusion. A perfusion cabinet designed to house isolated liver perfusions
115 at constant temperature (37°C) and h u m i d i t y was built in the workshop of the Department of Medicine, University of Cape Town. The livers were perfused as described by Hems et al. [11]. All perfusions were carried out between 10.00 a.m. and noon to avoid variations due to circadian r h y t h m s and in groups of 3, partially hepatectomized, sham operated and control, each group of three consuming the same weight of food during the pre-perfusion period. At the end of each perfusion the liver was flushed out with cold plasmalyte B, excised from the carcass, dried between two pieces of gauze, and weighed. The liver was then placed in a dry oven (120°C} and weighed repeatedly until no change in weight occurred. Perfusate. The perfusate consisted of: 1 . 5 6 ml whole blood from rats the same weight and kept under the same conditions as those perfused. Blood was obtained by cardiac puncture under diethyl ether anaesthesia. 2 . 4 ml Heparin (1000 units/ml, heparin injection B.P., Evans Medical Ltd., Speke, Liverpool, England). 3. 25 ml Plasmalyte B (Baxter, Saphar Laboratories Ltd., Johannesburg, South Africa) containing g/100 ml: NaC1 0.6, KC1 0.03, MgC12 0.03 and NaHCO3 0 . 2 3 . 4 . 2 ml of 4.2% NaHCO3. The final haematocrit was 25%. Albumin synthesis rate. Na2 J 4 CO3 was added to the reservoir after 30 min of perfusion by means of a constant infusion pump, 250 pCi being infused over a period of 2 h. Albumin synthesis rate was measured by the m e t h o d of McFarlane [12] and Reeve et al. [13]. Urea synthesis rate was determined from the difference between the perfusate urea concentrations before and after perfusion. The radioactivity incorporated into the guanidine carbon of arginine in albumin and into the carbon of urea was obtained from the end perfusion sample. Urea specific radioactivity was measured on deproteinized samples, incubated with urease to produce ~4 CO~. which was then released by the addition of acid. The volume of gas produced was measured manometrically on a high-vacuum gas line, collected in p h e n y l e t h y l a m i n o m e t h a n o l (1 : 1, v/v) and counted for radioactivity in a toluene liquid scintillator (2,5-diphenyl-oxazole and 1,4-bis-(4-methyl-5-phenyloxazol-2-yl) benzene) in a Beckman automatic scintillation spectrometer. Samples were counted to an error of less than 3%. The specific radioactivity of the guanidine carbon of arginine in albumin was obtained by extraction of albumin from the plasma by the acid-ethanol method of Korner and Debro [14]. After acid hydrolysis at 110°C, the sample was passed through a bicarbonate-resin column to separate arginine, which was then incubated with arginase to produce urea. The sample was then treated as for urea. The rate of albumin synthesis was calculated as follows: Albumin synthesis rate
Total ~4 C in the guanidine carbon of albumin in arginine Specific activity of newly formed urea
100 5.95
The factor 100/5.95 is obtained from the a m o u n t of arginine present in albumin, i.e. 100 g of albumin contains 5.95 g of arginine. Addition o f amino acids. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, t r y p t o p h a n and valine were added together to a concentration of 10 times their normal peripheral blood concentration as previously defined [15]. Only L-isomers of amino acids
116 (British Drug Houses, Poole, Dorset, U.K.) were added to the perfusate. Addition of hydrocortisone. 30 min after the start of the perfusion, 2 mg hydrocortisone (hydrocortisone sodium succinate injection B.P. Glaxo Laboratories Ltd., Greenford, U.K.) was added to the reservoir as a bolus and 0.949 mg/h as a constant infusion.
3. Measurement o f albumin catabolic rates (in vivo) after partial hepatectomy Albumin for this purpose was fractionated from rat plasma by the polyethylene glycol m e t h o d of Polson and Parker [16]. Iodination with 13 ~I or 2 s I was achieved by the iodine monochloride m e t h o d of McFarlane [17], residual free iodine being removed by passage through an anion-exchange resin column or by dialysis against distilled water. At least 99% labelling efficiency was obtained at mean substitution levels of 1 atom iodine per molecule albumin. Trichloroacetic acid precipitation invariably showed less than 1% free iodine in the final sample. Samples were sterilised by Seitz filtration with the addition of small amounts of carrier albumin or plasma to reduce losses and to protect against radiation damage. Final preparations were checked by cellulose-acetate electrophoresis to ensure absolute chemical purity and homogeneity of the labelled product. For at least 24 h prior to the injection and throughout the experiment, rats were given drinking water containing 0.008% sodium iodide to block thyroidal uptake of radio-iodine released by breakdown. Eighteen rats were divided into three groups and food intake was carefully controlled as described above. In the first experiment, partial hepatectomies were performed on day 0. Immediately after operation 0.5 ml of iodinated albumin containing 15--20 pCi of ~3~ I was administered intravenously, following which whole body counts were obtained by placing the rats in a well ventilated tin which was counted in a ring of 6 matched Geiger-Muller tubes to represent 100% of the administered dose. 15 min after administration of the ~3 ~I 0.8 ml of venous blood was withdrawn for the estimation of total blood volume. On day I the sham operations were performed and they and the control group were injected with ' 3 ~I and samples taken as above. 0.8 ml venous blood and whole body counts were obtained every 24 h, the partially hepatectomized group being sacrificed on day 10 and the sham operated and control groups on day 11. Since intravenously administered albumin does not reach equilibrium within the extravascular space for approx. 48 h [18], the above experiment does not provide information regarding the immediate changes in albumin catabolic rate following partial hepatectomy. A second series of experiments was designed to try to get this information. Here the partially hepatectomized group were injected intravenously with 0.5 ml of iodinated albumin containing 15--20 pCi of ~ 3 ~I on day 0, the sham operated animals and controls being injected intravenously with the same amounts of ~3~I on day 1. Immediately after intravenous administration of 3 ~I, whole body counts were obtained to represent 100% of the administered dose. On day 2 partial hepatectomies were performed on the partially hepatectomized group and immediately post operatively they were injected intravenously with 0.5 ml of albumin iodinated with ~2 s I, 15--20 pCi. On day 3 the
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sham operations were performed and this group injected intravenously, immediately post-operatively, with the same a m o u n t of J251 as were the control group. 15 min after the intravenous administration of ~2 .~I 0.8 ml of venous blood was withdrawn for estimation of total blood volume. Separation of the contributions of J 2 s I and ~3 ~I were achieved by appropriate voltage discrimination. 0.8 ml of venous blood and whole body counts were obtained every 12 h, the partially hepatectomised group being sacrificed on day 5 and the sham operated and control groups on day 6, that is 72 h after operation. Catabolic rate was calculated from the ratio of daily fall in whole body radioactivity and mean plasma specific activity during the corresponding 12 or 24 h. This ratio defined the fraction of plasma albumin catabolized per day; the product of this fraction and the plasma albumin pool provided an absolute catabolic rate (mg albumin per day). This m e t h o d was specifically designed by Matthews for catabolic measurements in the unsteady state [19]. Results
The results of the investigation of serum albumin levels following partial h e p a t e c t o m y and sham operation together with controls are shown in Table I. On day 3 the difference between the serum albumin levels of the controls and the sham operated animals is significant (P < 0.001) while there is no significant difference between the sham operated and partially hepatectomized animals. On day 10 the difference between the control and sham operated animals is no longer significant, while that between the control and partially hepatectomized animals remains so (P < 0.001). At this time the difference between the partially hepatectomized and sham operated animals has now become significant (0.02 > P > 0.01). The results of the measurements of albumin synthesis rates following partial h e p a t e c t o m y are shown in Table II and the statistical significance of these results is set o u t in Table III. The effect of adding 10 times the normal plasma concentration of certain amino acids and the effect of adding cortisone to the perfusate is seen in Table IV. The difference between the albumin synthesis rate of the partially hepatectomized liver and that after partial h e p a t e c t o m y with added amino acids is significant (P < 0.05) both in terms of liver and animal weight. The increase in
TABLE I P L A S M A A L B U M I N L E V E L S (g%) Six a n i m a l s in e a c h o f t h e t h r e e g r o u p s . See t e x t f o r s t a t i s t i c a l a n a l y s i s o f results. Interval after partial hepatectomy Type of experiment Mean plasma albumin level (g%) -+ S t a n d a r d e r r o r o f m e a n * Partial h e p a t e e t o m y .
3 days Control
2.99 -+0.09
10 d a y s P.H.*
2.29 -+0.03
Sham-operated
2.42 -+0.07
Control
2.77 -+0.11
P.H.*
2.09 -+0.13
Sham-operated
2.58 -+0.12
2.66 -+0.48 1.09 +_0.17
Control (9)
0.36 -+0.13 0.36 +-0.13
PH (6)
C o n t r o l vs s h a m
S h a m vs p a r t i a l hepatectomy
C o n t r o l vs p a r t i a l hepatectomy
Interval after partial hepatectomy E x p r e s s i o n of r e s u l t s
P < 0.001
P < 0.001
Per 300 g rat P < 0.001
P < 0.001
Per g dry liver
0.005 >P> 0.001 0.005 >P> 0.001 n.s.
Per 300 g rat
P < 0.001
n.s.
P < 0,001
Per g dry liver
12 h
2.47 +_0.25 0.97 +-0.11
(7)
Sham
6 h
S t u d e n t s ' t ' test. n.s., n o t s i g n i f i c a n t .
STATISTICAL SIGNIFICANCE: ALBUMIN SYNTHESIS RATES
TABLE III
R e s u l t s e x p r e s s e d p e r g d r y liver
Results expressed per 300 g rat
T y p e of experiment:
6 h
0.01
0.01 n.s.
0.001 p < 0.001
n.s.
0.02 >P>
0.02 >P>
0.005 >p>
rat
Per 300 g
72h
0.71 +_0.10 0.59 +-0.08
PH (8)
liver
Per g dry
1.9 +_0.20 0.85 +_0.10
Sham (6)
22 h
Per 300 g rat
22 h
0.25 -+0.04 0.26 +-0.04
(7)
PH
12 h
I n t e r v a l a f t e r paxtial h e p a t e c t o m y
0.05
0.1 >P>
liver
Per g dry
1.77 +0.23 0.76 +-0.09
(7)
Sham
(4)
PH
72 h
0.005
0.01 >P>
rat
Per 300 g
7 days
1.02 -+0.33 0.58 +-0.16
ALBUMIN SYNTHESIS RATES R e s u l t s ( m e a n a l b u m i n s y n t h e s i s r a t e + S.E.) are e x p r e s s e d as m g p e r h. P H , p a r t i a l h e p a t e c t o m y . N u m b e r o f e x p e r i m e n t s is g i v e n in p a r e n t h e s i s .
T A B L E II
0.001
0.02 >P>
Per g dry liver
1.18 -+0.07 0.6 -+0.06
(5)
PH
7 days
0o
119
TABLE IV ALBUMIN SYNTHESIS
RATES
T h e r a t e s are e x p r e s s e d as m g p e r h ( m e a n a l b u m i n s y n t h e s i s r a t e i S.E.). E x p e r i m e n t s animals 12 h after partial hepatectomy. See text for statistical significance.
No of experiments Results expressed per 300 g rat Results expressed per g dry liver
were performed
on
No addition to p e r f u s a t e
1 0 x 11 a m i n o a c i d s added to perfusate
Cortisone added to p e r f u s a t e
7 0.25 ± 0.04 0 . 2 6 -+ 0 . 0 4
7 0.517 ± 0.12 0.517 ± 0.09
6 0.31 ± 0.03 0.30-+ 0.03
the albumin synthesis rate of the partially hepatectomized liver by adding amino acids to the perfusate is such that when considered in terms of liver weight there is n o w no significant difference between it and the sham operated animal. The increase in albumin synthesis rate of the partially hepatectomized animal, following the addition of cortisone to the perfusate, is not significant and the albumin synthesis rate of the sham operated animal remains significantly different. The albumin catabolic rates following partial hepatectomy are given in Table V and the statistical significance in Table VI. The albumin catabolic rate of the partially hepatectomized animals is significantly lower than catabolic rates of the sham operated and control animals in both sets of experiments.
TABLE V ALBUMIN CATABOLIC
RATES
R a t e s e x p r e s s e d as m g / h p e r 3 0 0 g r a t ( m e a n c a t a b o l i c r a t e ± S . E . ) .
Control Sham operated Partial h e p a t e c t o m y
Measurements taken every 12 h for 72 h
Measurements taken every 24 h for 10 days
11.22 ± 0.97 1 2 . 0 6 -+ 1 . 3 1 7.36 ± 0.76
9.47 ± 0.38 11.04 ± 1.14 7.47 ± 0.42
TABLE VI TABLE OF STATISTICAL
SIGNIFICANCE:
ALBUMIN
CATABOLIC
RATES
S t u d e n t s ' t ' t e s t . n.s., n o t s i g n i f i c a n t .
Control vs p a r t i a l h e p a t e c t o m y C o n t r o l vs s h a m S h a m vs p a r t i a l h e p a t e c t o r n y
12 H o u r l y s a m p l e s in 72 h
24 H o u r l y s a m p l e s in 10 d a y s
0.02 > P > 0.01 n.~. 0 . 0 2 > P :> 0 . 0 1
0.005 > P > 0.001 n.s. 0.02 > P > 0.01
120 Discussion
In addition to those workers [1--8] who have investigated albumin metabolism in the rat, many have observed a fall in plasma albumin in man following partial h e p a t e c t o m y [20--28]. Our results are in agreement with this observation. The fall of serum albumin seen in the partially hepatectomized animals after three days (2.99--2.29 g%) was mirrored to almost the same extent by the sham operated animals (2.99--2.42 g%). By the tenth day, however, the serum albumin of the partially hepatectomized animals had fallen further to 2.09 g%, while that of the sham operated group had been almost completely restored (2.58 g%) to the control levels (2.77 g%). Kirsch et al. [29] demonstrated the immediate fall in the albumin synthesis rate which followed protein deprivation and we have attributed the fall in the serum albumin of our control animals over the 10 day period (2.99--2.77 g%) to protein-calorie deprivation, for during the entire 10 day post-operative period the partially hepatectomized animals are considerably less than normal animals fed ad libitum, and since the groups were "pair-fed" the controls were allowed substantially less than their normal diet to eat. The initial fall in the serum albumin of the sham operated animals is attributed to the sequestration of albumin into non-exchangeable extravascular compartments that has been shown to follow surgery [30--32]. Mourisden and Faber [30] and Mourisden [31] have shown an increased elimination of 13 ~I labelled albumin from plasma of patients undergoing major surgery and have demonstrated a "migration of albumin into the oedema of the operative field". Hoye et al. [32] found a reduction in plasma volume during major surgery disproportionate to the reduction in total red blood cell volume and a fall in the albumin concentration of the lymph. There is a highly significant increase in protein concentration and excretion in the urine in patients following surgery [33], this anomaly being greater in those patients undergoing major surgery. Besides these observations, other less well defined factors are involved in circulating albumin loss and include: loss into the abdominal cavity [34]; increased capillary permeability [35]; intravenous infusion [36,37] and an increase in fractional catabolic rate [38]. The albumin synthesis rate is seen to fall significantly after partial hepat e c t o m y . This fall is greatest at 12 h and least at 22 h after the operation. Indeed by 22 h the recovery of the albumin synthesis rate of the liver is such that when the results are expressed in terms of liver weight, the synthesis rates of the partially hepatectomized and sham operated animals are no longer significantly different. Other workers [6] found that 24 h after partial h e p a t e c t o m y the albumin synthesis rate was significantly depressed when expressed in terms of animal weight, although the rate expressed in terms of liver weight was within normal limits. Further, these workers found that 72 h after partial hepatectomy, the albumin synthesis rates were significantly increased when expressed in terms of both animal weight and liver weight. In contrast, in our studies the albumin synthesis rate was still significantly depressed when compared to control animals both in terms of animal weight and liver weight 72 h after partial hepa-
121 t e c t o m y . At no time during this period did we find an elevation of albumin synthesis rate in the partially hepatectomized animals. Our findings are more in keeping with the results obtained from the investigations into serum albumin levels and albumin catabolic rates in this experimental situation. It is important to stress that all groups of animals were "pair-fed" during the experiments carried out to determine albumin synthesis rates, as well as in those investigating serum albumin levels and albumin catabolic rates after partial hepatectomy. It is thus clear that even when the values are considered in terms of liver weight, the albumin synthesis rates of the partially hepatectomized livers are depressed. Rothschild et al. [39] have postulated an extra-vascular osmotic regulatory system in the liver controlling albumin synthesis and it is known that albumin is sequestered in extra-vascular compartments post-operatively. It is possible that the albumin synthesis rate is depressed due to raised extravascular osmotic pressure caused b y this sequestration of albumin. Although the albumin synthesis rates of the partially hepatectomized and sham operated animals are no longer significantly different 22 h post-operatively (when expressed in terms of liver weight) this hypothesis does not provide for the highly significant differences between these two groups at the earlier intervals postoperatively, even when considered in terms of liver weight. Christensen et al. [40] and Braun et al. [3] showed that the levels of amino acids in the liver are increased at an early stage after partial h e p a t e c t o m y and it has been demonstrated that amino acids are mobilized for plasma protein synthesis in in vivo experiments on nephrotic rats [41]. These findings were confirmed more recently by Ferris and Clark [42] who showed a rise in both hepatic and plasma free amino acid concentrations during the first four hours after partial h e p a t e c t o m y as compared to sham operated animals. Using in vitro techniques Marsh and Drabkin [43] and Peters [44] were unable to demonstrate an accelerating effect of added amino acids on the albumin synthesis rates. Clemens [ 4 5 ] , however, has shown an increase in protein synthetic activity in cell free systems after exposure to high concentrations of amino acids, while Kelman et al. [46] demonstrated the ability of certain amino acids, in high concentrations, to restore the albumin synthesis rates of isolated perfused livers from starved rats to normal levels. We have shown that by adding 10 times the normal plasma concentration of certain amino acids to perfusate, the albumin synthesis rate is doubled, the increase being such that it is significant (P < 0.05) in terms of both liver and animal weight. In addition it is no longer significantly different from the sham operated animal when expressed in terms of liver weight. Braun et al. [3] have claimed that in the early stages of liver regeneration precedence is given to the biosynthesis of cellular proteins and that after the regenerative process is well advanced, the increased construction of plasma albumin is metabolically favoured. One mechanism whereby amino acids stimulate albumin synthesis could be that as a result of the redistribution of cellular amino acids following partial hepatectomy, the supply of primary substrate for albumin synthesis is limiting. Alternatively, if depression of albumin synthesis was due to a limited availability of specific tRNAs or amino acid t R N A synthetases, this restriction could be partly reversed by increasing cellular amino acid concentrations above normal levels.
122 Another factor of importance in relation to albumin synthesis is total hormonal balance. While changes in the availability of specific amino acids released from altered tissue degradation may also play a role in tissue culture, cortisone has been shown to increase albumin synthesis per se [47]. We were unable to demonstrate any alteration in the albumin synthesis rate following the addition of cortisone to the perfusate. This is in keeping with the findings of Kelman et al. [48] who was able to influence ribosomal profiles by adding 10 times the normal concentration of amino acid to the system, but not by adding hormones. Two sets of experiments were carried out to investigate albumin catabolism following partial hepatectomy; a short term one to investigate the immediate effect of partial h e p a t e c t o m y on albumin catabolism and a longer term one to allow these changes to be observed for 10 days. There is excellent correlation between the two sets of experiments which indicate an immediate significant fall in the albumin catabolic rate following partial hepatectomy as the system endeavours to maintain the plasma albumin pool. This finding is in keeping with the findings of Kirsch et al. [29] who observed that a fall in the albumin catabolic rate of malnourished rats occurred following the fall in their albumin synthesis rates and plasma albumin pools as a result of protein calorie deprivation. Acknowledgments We are indebted to the following for their valuable advice and technical assistance: Mr V.M. Wells, Chief Technician, MRC/UCT Liver Research Group; Mr Milton Stofile and Mr M. Parker, Laboratory Attendants, Department of Medicine, University of Cape Town; Dr R. Stead, Ph.D. (Natal), Senior Lecturer, Department of Biochemistry, University of Cape Town. This work is part of the programme of the MRC/UCT Liver Research Group and was supported by the South African Medical Research Council, the S.A. Atomic Energy Board, Cancer Research Trust and Staff Research Fund of the University of Cape Town. References I 2 3 4 5 6 7 8 9 10 II 12
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E x p e r i m e n t a l w o r k is in p r o g r e s s in this l a b o r a t o r y i n v e s t i g a t i n g a l b u m i n s y n t h e s i s r a t e s and t h e e f f e c t s of s u p p l e m e n t a r y a m i n o acids in vivo.
123 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
R e e v e , E.B., P e a r s o n , J . R . a n d M a r t z , D,C. ( 1 9 6 3 ) S c i e n c e 1 3 9 , 9 1 4 - - 9 1 6 K o r n e r , A. a n d D e b r o , J . R . ( 1 9 5 6 ) N a t u r e 1 7 8 , 1 0 6 7 K i r s c h , R . E . , B r o c k , J . F . a n d S a u n d e r s , S.J. ( 1 9 6 8 ) A m . J. Clin. N u t r . 2 1 , 8 2 0 - - 8 2 6 P o i s o n , A. a n d P a r k e r , J . R . ( 1 9 7 3 ) P r e p . B i o c h e m . 3~ 3 1 - - 4 5 M c F a r l a n e , A.S. ( 1 9 5 8 ) N a t u r e 1 8 2 , 5 3 S t e r l i n g , K. ( 1 9 5 1 ) J. Clin. Invest. 3 0 , 1 2 2 8 - - 1 2 3 7 M a t t h e w s , C.M.E. ( 1 9 6 5 ) R a d i o a c t i v e I s o t o p e in K l i n i k u n d F o r s c h u n g (K. F e l l i n g e r a n d R. H S f e r , eds), U r b a n & S c h w a r z e n b e r g , M u n i c h I s l a m i , A., P a c k , G . T . , Miller, T . R . , V a n a m e e , P., R a n d a l l , H . T . a n d R o b e r t s , K . E . ( 1 9 5 6 ) S u r g e r y 39, 551--556 P a c k , G . T . a n d M o l a n d e r , D.W. ( 1 9 6 0 ) A r c h . S u r g . 8 0 , 6 8 5 - - 6 9 2 M c D e r m o t t , W.V., G r e e n b e r g e r , N . J . , I s s e l b a c h e r , K . J . a n d W e b e r , A . L . ( 1 9 6 3 ) S u r g e r y 5 4 , 5 6 - - 6 6 M o n a c o , A.P., H a l l g r i m s s o n , J. a n d M c D e r m o t t , W.V. ( 1 9 6 4 ) A n n . S u r g . 1 5 9 , 5 1 3 - - 5 1 9 M c D e r m o t t , W.V. a n d O t t i n g e r , L.W. ( 1 9 6 6 ) A m . J. S u r g . 1 1 2 , 3 7 6 - - 3 8 1 A l m e r s j S , O. a n d B e n g m a r k , S. ( 1 9 6 9 ) A c t a C h i t . S c a n d . 1 3 5 , 3 1 1 - - 3 1 9 A r o n s e n , K . F . , E r i c s s o n , B. a n d Phil, B. ( 1 9 6 9 ) A n n . S u r g . 1 6 9 , 1 0 2 - - 1 1 0 Dillard, B.M. ( 1 9 6 9 ) S u r g . G y n e c . O b s t e t . 1 2 9 , 2 4 9 - - 2 5 7 F l a t m a r k , A., R 5 , J . S . , J a k o b s e n , A., E n g e , I a n d S k r e d e , S. ( 1 9 7 3 ) S c a n d . J. G a s t r o e n t . 8 , 6 0 9 - - 6 1 3 K i t s c h , R . E . , F r i t h , L., B l a c k , E. a n d H o f f e n b e r g , R. ( 1 9 6 8 ) N a t u r e 2 1 7 , 5 7 8 - - 5 7 9 M o u r i s d e n , H . T . a n d F a b e r , M. ( 1 9 6 6 ) L a n c e t ii, 7 2 3 - - 7 2 4 M o u r i s d e n , H . T . ( 1 9 6 7 ) Clin. Sci. 3 3 , 3 4 5 - - 3 5 4 H o y e , R . C . , B e n n e t t , S . H . , G e e l h o e d , G.W. a n d G o r s c h b o t h , H. ( 1 9 7 2 ) J. A m . Med. A s s o c . 2 2 2 , 1255--1261 M a c B e t h , W . A . A . G . a n d P o p e , G . R . ( 1 9 6 8 ) L a n c e t i, 2 1 5 - - 2 1 7 J a r n u m , S. ( 1 9 6 1 ) G a s t r o e n t e r o l o g y 4 1 , 1 0 7 - - 1 1 8 Birke, G., L i i j e d a h l , S . O . , P l a n t i n , L . O . a n d W e t t e r f o r s , J. ( 1 9 5 9 ) A c t a Chir. S c a n d . 1 1 8 , 3 5 3 - - 3 6 6 W a s s e r m a n , K. a n d M a y e r s o n , H.S. ( 1 9 5 2 ) A m . J. P h y s i o l . 1 7 0 , 1 - - 1 0 K a i i s e r , L. a n d R e i g e r , A. ( 1 9 6 8 ) A c t a C h i t . S c a n d . 1 3 4 , 1 5 - - 2 6 G r o s s m a n , 3., Y a l o w , A . A . a n d W e s t o n , R . E . ( 1 9 6 0 ) M e t a b o l i s m 9, 5 2 8 - - 5 5 0 R o t h s c h i l d , M . A . , O r a t z , M., E v a n s , C. a n d S c h r e i b e r , S. ( 1 9 6 6 ) A m . J. P h y s i o l . 2 1 0 , 5 7 - - 6 2 C h r i s t e n s e n , H . N . , R o t h w e l l , J . T . , S e a r s , R . A . a n d S t r e i c h e r , J . A . ( 1 9 4 8 ) J. Biol. C h e m . 1 7 5 , 1 0 1 - - 1 0 5 D r a b k i n , D . L . , M a r s h , J.B. a n d B r a u n , G . A . ( 1 9 6 2 ) M e t a b o l i s m 1 1 , 9 6 7 - - 9 7 7 F e r r i s , G.M. a n d C l a r k , J.B. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 7 3 , 7 3 - - 7 9 M a r s h , J.B. a n d D r a b k i n , D . L . ( 1 9 5 8 ) J. Biol. C h e m . 2 3 0 , 1 0 7 3 - - 1 0 8 1 P e t e r s , T. ( 1 9 5 9 ) H i s t o c h e m . C y t o c h e m . 7 , 2 2 4 - - 2 3 4 C l e m e n s , M.J. ( 1 9 7 2 ) B i o c h e m . J. 1 2 9 , 3 P - - 5 P K e l m a n , L., S a u n d e r s , S . J . , W i c h t , S., F r i t h , L., C o r r i g a l , A., K i t s c h , R . E . a n d T e r b l a n c h e , J. ( 1 9 7 2 ) B i o c h e m . J. 1 2 9 , 8 0 5 - - 8 0 9 B a n c r o f t , F . C . , L e v i n e , L. a n d T a s h j i a n , A . H . ( 1 9 6 9 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 7 , 1028--1035 K e l m a n , L., S a u n d e r s , S.J., W i c h t , S , a n d F r i t h , L . O ' C . ( 1 9 7 3 ) S. A f r . Meal. J. 4 7 , 1 7 5 1 - . 1 7 5 3
Biochimica et Biophysica A cta, 402 (1975) 113--123 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98358 ALBUMIN SYNTHESIS AND CATABOLISM FOLLOWING PARTIAL HEPATECTOMY IN THE RAT. THE EFFECTS OF AMINO ACIDS AND ADRENOCORTICAL STEROIDS ON ALBUMIN SYNTHESIS A F T E R PARTIAL HEPATECTOMY
ELWYN A. LLOYD, STUART J. SAUNDERS, LESLEY O'C. FRITH and JOAN E. WRIGHT Department of Medicine, Medical School, Observatory 7900, Cape Town (South Africa)
(Received February 7th, 1975)
Summary Plasma albumin levels were measured in partially hepatectomized, sham operated and control rats. The levels fell in both the partially hepatectomized and sham operated groups; while the latter group returned to normal within a few days, the low plasma albumin in the partially hepatectomized animals was sustained. Albumin synthesis rates in the isolated perfused rat liver were measured in the three groups of animals at varying intervals after partial hepatectomy. There was a significant depression of albumin synthesis rate in terms of both liver and whole animal weights when compared to the sham operated and control animals. This depression was almost completely reversed by the addition of arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, t r y p t o p h a n and vaiine added together to 10 times their normal plasma concentrations. The addition of hydrocortisone had no effect on the albumin synthesis rate after partial hepatectomy. Studies in vivo in the three groups of animals (partially hepatectomized, sham operated and control animals) revealed a fall in the albumin catabolic rate after partial h e p a t e c t o m y coinciding with the fall in the albumin synthesis rate. An hypothesis whereby the amino acids may have their stimulatory effect is proposed.
Introduction Changes in albumin metabolism following partial h e p a t e c t o m y have been measured [1--8]. The results of these studies are conflicting, some indicating a fall [4,6,8], some no change [2,3,7] and others an increase in the albumin synthesis rate [5].
114
These widely differing results, together with the paucity of literature available concerning albumin catabolism after partial hepatectomy, and the lack of information concerning the influence of amino acids on albumin synthesis in the regenerating liver, prompted the work reported here. Materials and Methods Male Wistar rats, weighing between 250 and 300 g, housed in individual cages under controlled conditions of temperature, h u m i d i t y and lighting were permitted water ad libitum and unless otherwise stated had free access to a balanced diet. Partial hepatectomies were performed under diethylether anaesthesia with the removal of median and left lateral lobes as described by Higgins and Anderson [9]. Sham operations consisted of laparotomy and palpation of the liver only.
Radioactive materials All the radioactive materials used in these experiments were obtained from the Radio Chemical Centre, Amersham, England. 1. Na~ ~4 CO3, specific radioactivity 58.9 Ci/mol. 2. Na ~3 l I, specific radioactivity 40 Ci/1.3. Na ~2 s I, specific radioactivity 100 Ci/1.
1. Measurements of plasma albumin levels (in vivo) following partial hepatectomy Eighteen rats were divided into three groups: control, sham and partiallyhepatectomized, in such a way that each control had a sham operated and a partially hepatectomized partner of similar weight. On day O, partial hepatectomies were performed on the six rats in that group. They were weighed post operatively and returned to their individual cages where they received water ad libitum and in which there was a preweighed a m o u n t of food contained in a vessel designed to minimize spillage. The animals had become accustomed to feeding from these vessels prior to surgery. On day I the sham operations were performed and that group of rats weighed post-operatively. The control animals were weighed and they and the sham operated animals were given exactly that a m o u n t of food which their partially hepatectomized partners had eaten during the preceding 24 h. Each day animals were weighed and each trio (partially hepatectomized, sham operated and control) consumed the same weight of food as indicated above. In the first experiment the partially hepatectomized animals were exsanguinated on day 3, and the sham operated and control rats exsanguinated on day 4. In the second experiment the same procedure was adopted, except that the animals were exsanguinated on days 10 and 11 respectively. Serum albumin was estimated by the m e t h o d of Fernandez et al. [10].
2. Measurement of albumin synthesis rates (in vitro) following partial hepatectomy Perfusion. A perfusion cabinet designed to house isolated liver perfusions
115 at constant temperature (37°C) and h u m i d i t y was built in the workshop of the Department of Medicine, University of Cape Town. The livers were perfused as described by Hems et al. [11]. All perfusions were carried out between 10.00 a.m. and noon to avoid variations due to circadian r h y t h m s and in groups of 3, partially hepatectomized, sham operated and control, each group of three consuming the same weight of food during the pre-perfusion period. At the end of each perfusion the liver was flushed out with cold plasmalyte B, excised from the carcass, dried between two pieces of gauze, and weighed. The liver was then placed in a dry oven (120°C} and weighed repeatedly until no change in weight occurred. Perfusate. The perfusate consisted of: 1 . 5 6 ml whole blood from rats the same weight and kept under the same conditions as those perfused. Blood was obtained by cardiac puncture under diethyl ether anaesthesia. 2 . 4 ml Heparin (1000 units/ml, heparin injection B.P., Evans Medical Ltd., Speke, Liverpool, England). 3. 25 ml Plasmalyte B (Baxter, Saphar Laboratories Ltd., Johannesburg, South Africa) containing g/100 ml: NaC1 0.6, KC1 0.03, MgC12 0.03 and NaHCO3 0 . 2 3 . 4 . 2 ml of 4.2% NaHCO3. The final haematocrit was 25%. Albumin synthesis rate. Na2 J 4 CO3 was added to the reservoir after 30 min of perfusion by means of a constant infusion pump, 250 pCi being infused over a period of 2 h. Albumin synthesis rate was measured by the m e t h o d of McFarlane [12] and Reeve et al. [13]. Urea synthesis rate was determined from the difference between the perfusate urea concentrations before and after perfusion. The radioactivity incorporated into the guanidine carbon of arginine in albumin and into the carbon of urea was obtained from the end perfusion sample. Urea specific radioactivity was measured on deproteinized samples, incubated with urease to produce ~4 CO~. which was then released by the addition of acid. The volume of gas produced was measured manometrically on a high-vacuum gas line, collected in p h e n y l e t h y l a m i n o m e t h a n o l (1 : 1, v/v) and counted for radioactivity in a toluene liquid scintillator (2,5-diphenyl-oxazole and 1,4-bis-(4-methyl-5-phenyloxazol-2-yl) benzene) in a Beckman automatic scintillation spectrometer. Samples were counted to an error of less than 3%. The specific radioactivity of the guanidine carbon of arginine in albumin was obtained by extraction of albumin from the plasma by the acid-ethanol method of Korner and Debro [14]. After acid hydrolysis at 110°C, the sample was passed through a bicarbonate-resin column to separate arginine, which was then incubated with arginase to produce urea. The sample was then treated as for urea. The rate of albumin synthesis was calculated as follows: Albumin synthesis rate
Total ~4 C in the guanidine carbon of albumin in arginine Specific activity of newly formed urea
100 5.95
The factor 100/5.95 is obtained from the a m o u n t of arginine present in albumin, i.e. 100 g of albumin contains 5.95 g of arginine. Addition o f amino acids. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, t r y p t o p h a n and valine were added together to a concentration of 10 times their normal peripheral blood concentration as previously defined [15]. Only L-isomers of amino acids
116 (British Drug Houses, Poole, Dorset, U.K.) were added to the perfusate. Addition of hydrocortisone. 30 min after the start of the perfusion, 2 mg hydrocortisone (hydrocortisone sodium succinate injection B.P. Glaxo Laboratories Ltd., Greenford, U.K.) was added to the reservoir as a bolus and 0.949 mg/h as a constant infusion.
3. Measurement o f albumin catabolic rates (in vivo) after partial hepatectomy Albumin for this purpose was fractionated from rat plasma by the polyethylene glycol m e t h o d of Polson and Parker [16]. Iodination with 13 ~I or 2 s I was achieved by the iodine monochloride m e t h o d of McFarlane [17], residual free iodine being removed by passage through an anion-exchange resin column or by dialysis against distilled water. At least 99% labelling efficiency was obtained at mean substitution levels of 1 atom iodine per molecule albumin. Trichloroacetic acid precipitation invariably showed less than 1% free iodine in the final sample. Samples were sterilised by Seitz filtration with the addition of small amounts of carrier albumin or plasma to reduce losses and to protect against radiation damage. Final preparations were checked by cellulose-acetate electrophoresis to ensure absolute chemical purity and homogeneity of the labelled product. For at least 24 h prior to the injection and throughout the experiment, rats were given drinking water containing 0.008% sodium iodide to block thyroidal uptake of radio-iodine released by breakdown. Eighteen rats were divided into three groups and food intake was carefully controlled as described above. In the first experiment, partial hepatectomies were performed on day 0. Immediately after operation 0.5 ml of iodinated albumin containing 15--20 pCi of ~3~ I was administered intravenously, following which whole body counts were obtained by placing the rats in a well ventilated tin which was counted in a ring of 6 matched Geiger-Muller tubes to represent 100% of the administered dose. 15 min after administration of the ~3 ~I 0.8 ml of venous blood was withdrawn for the estimation of total blood volume. On day I the sham operations were performed and they and the control group were injected with ' 3 ~I and samples taken as above. 0.8 ml venous blood and whole body counts were obtained every 24 h, the partially hepatectomized group being sacrificed on day 10 and the sham operated and control groups on day 11. Since intravenously administered albumin does not reach equilibrium within the extravascular space for approx. 48 h [18], the above experiment does not provide information regarding the immediate changes in albumin catabolic rate following partial hepatectomy. A second series of experiments was designed to try to get this information. Here the partially hepatectomized group were injected intravenously with 0.5 ml of iodinated albumin containing 15--20 pCi of ~ 3 ~I on day 0, the sham operated animals and controls being injected intravenously with the same amounts of ~3~I on day 1. Immediately after intravenous administration of 3 ~I, whole body counts were obtained to represent 100% of the administered dose. On day 2 partial hepatectomies were performed on the partially hepatectomized group and immediately post operatively they were injected intravenously with 0.5 ml of albumin iodinated with ~2 s I, 15--20 pCi. On day 3 the
117
sham operations were performed and this group injected intravenously, immediately post-operatively, with the same a m o u n t of J251 as were the control group. 15 min after the intravenous administration of ~2 .~I 0.8 ml of venous blood was withdrawn for estimation of total blood volume. Separation of the contributions of J 2 s I and ~3 ~I were achieved by appropriate voltage discrimination. 0.8 ml of venous blood and whole body counts were obtained every 12 h, the partially hepatectomised group being sacrificed on day 5 and the sham operated and control groups on day 6, that is 72 h after operation. Catabolic rate was calculated from the ratio of daily fall in whole body radioactivity and mean plasma specific activity during the corresponding 12 or 24 h. This ratio defined the fraction of plasma albumin catabolized per day; the product of this fraction and the plasma albumin pool provided an absolute catabolic rate (mg albumin per day). This m e t h o d was specifically designed by Matthews for catabolic measurements in the unsteady state [19]. Results
The results of the investigation of serum albumin levels following partial h e p a t e c t o m y and sham operation together with controls are shown in Table I. On day 3 the difference between the serum albumin levels of the controls and the sham operated animals is significant (P < 0.001) while there is no significant difference between the sham operated and partially hepatectomized animals. On day 10 the difference between the control and sham operated animals is no longer significant, while that between the control and partially hepatectomized animals remains so (P < 0.001). At this time the difference between the partially hepatectomized and sham operated animals has now become significant (0.02 > P > 0.01). The results of the measurements of albumin synthesis rates following partial h e p a t e c t o m y are shown in Table II and the statistical significance of these results is set o u t in Table III. The effect of adding 10 times the normal plasma concentration of certain amino acids and the effect of adding cortisone to the perfusate is seen in Table IV. The difference between the albumin synthesis rate of the partially hepatectomized liver and that after partial h e p a t e c t o m y with added amino acids is significant (P < 0.05) both in terms of liver and animal weight. The increase in
TABLE I P L A S M A A L B U M I N L E V E L S (g%) Six a n i m a l s in e a c h o f t h e t h r e e g r o u p s . See t e x t f o r s t a t i s t i c a l a n a l y s i s o f results. Interval after partial hepatectomy Type of experiment Mean plasma albumin level (g%) -+ S t a n d a r d e r r o r o f m e a n * Partial h e p a t e e t o m y .
3 days Control
2.99 -+0.09
10 d a y s P.H.*
2.29 -+0.03
Sham-operated
2.42 -+0.07
Control
2.77 -+0.11
P.H.*
2.09 -+0.13
Sham-operated
2.58 -+0.12
2.66 -+0.48 1.09 +_0.17
Control (9)
0.36 -+0.13 0.36 +-0.13
PH (6)
C o n t r o l vs s h a m
S h a m vs p a r t i a l hepatectomy
C o n t r o l vs p a r t i a l hepatectomy
Interval after partial hepatectomy E x p r e s s i o n of r e s u l t s
P < 0.001
P < 0.001
Per 300 g rat P < 0.001
P < 0.001
Per g dry liver
0.005 >P> 0.001 0.005 >P> 0.001 n.s.
Per 300 g rat
P < 0.001
n.s.
P < 0,001
Per g dry liver
12 h
2.47 +_0.25 0.97 +-0.11
(7)
Sham
6 h
S t u d e n t s ' t ' test. n.s., n o t s i g n i f i c a n t .
STATISTICAL SIGNIFICANCE: ALBUMIN SYNTHESIS RATES
TABLE III
R e s u l t s e x p r e s s e d p e r g d r y liver
Results expressed per 300 g rat
T y p e of experiment:
6 h
0.01
0.01 n.s.
0.001 p < 0.001
n.s.
0.02 >P>
0.02 >P>
0.005 >p>
rat
Per 300 g
72h
0.71 +_0.10 0.59 +-0.08
PH (8)
liver
Per g dry
1.9 +_0.20 0.85 +_0.10
Sham (6)
22 h
Per 300 g rat
22 h
0.25 -+0.04 0.26 +-0.04
(7)
PH
12 h
I n t e r v a l a f t e r paxtial h e p a t e c t o m y
0.05
0.1 >P>
liver
Per g dry
1.77 +0.23 0.76 +-0.09
(7)
Sham
(4)
PH
72 h
0.005
0.01 >P>
rat
Per 300 g
7 days
1.02 -+0.33 0.58 +-0.16
ALBUMIN SYNTHESIS RATES R e s u l t s ( m e a n a l b u m i n s y n t h e s i s r a t e + S.E.) are e x p r e s s e d as m g p e r h. P H , p a r t i a l h e p a t e c t o m y . N u m b e r o f e x p e r i m e n t s is g i v e n in p a r e n t h e s i s .
T A B L E II
0.001
0.02 >P>
Per g dry liver
1.18 -+0.07 0.6 -+0.06
(5)
PH
7 days
0o
119
TABLE IV ALBUMIN SYNTHESIS
RATES
T h e r a t e s are e x p r e s s e d as m g p e r h ( m e a n a l b u m i n s y n t h e s i s r a t e i S.E.). E x p e r i m e n t s animals 12 h after partial hepatectomy. See text for statistical significance.
No of experiments Results expressed per 300 g rat Results expressed per g dry liver
were performed
on
No addition to p e r f u s a t e
1 0 x 11 a m i n o a c i d s added to perfusate
Cortisone added to p e r f u s a t e
7 0.25 ± 0.04 0 . 2 6 -+ 0 . 0 4
7 0.517 ± 0.12 0.517 ± 0.09
6 0.31 ± 0.03 0.30-+ 0.03
the albumin synthesis rate of the partially hepatectomized liver by adding amino acids to the perfusate is such that when considered in terms of liver weight there is n o w no significant difference between it and the sham operated animal. The increase in albumin synthesis rate of the partially hepatectomized animal, following the addition of cortisone to the perfusate, is not significant and the albumin synthesis rate of the sham operated animal remains significantly different. The albumin catabolic rates following partial hepatectomy are given in Table V and the statistical significance in Table VI. The albumin catabolic rate of the partially hepatectomized animals is significantly lower than catabolic rates of the sham operated and control animals in both sets of experiments.
TABLE V ALBUMIN CATABOLIC
RATES
R a t e s e x p r e s s e d as m g / h p e r 3 0 0 g r a t ( m e a n c a t a b o l i c r a t e ± S . E . ) .
Control Sham operated Partial h e p a t e c t o m y
Measurements taken every 12 h for 72 h
Measurements taken every 24 h for 10 days
11.22 ± 0.97 1 2 . 0 6 -+ 1 . 3 1 7.36 ± 0.76
9.47 ± 0.38 11.04 ± 1.14 7.47 ± 0.42
TABLE VI TABLE OF STATISTICAL
SIGNIFICANCE:
ALBUMIN
CATABOLIC
RATES
S t u d e n t s ' t ' t e s t . n.s., n o t s i g n i f i c a n t .
Control vs p a r t i a l h e p a t e c t o m y C o n t r o l vs s h a m S h a m vs p a r t i a l h e p a t e c t o r n y
12 H o u r l y s a m p l e s in 72 h
24 H o u r l y s a m p l e s in 10 d a y s
0.02 > P > 0.01 n.~. 0 . 0 2 > P :> 0 . 0 1
0.005 > P > 0.001 n.s. 0.02 > P > 0.01
120 Discussion
In addition to those workers [1--8] who have investigated albumin metabolism in the rat, many have observed a fall in plasma albumin in man following partial h e p a t e c t o m y [20--28]. Our results are in agreement with this observation. The fall of serum albumin seen in the partially hepatectomized animals after three days (2.99--2.29 g%) was mirrored to almost the same extent by the sham operated animals (2.99--2.42 g%). By the tenth day, however, the serum albumin of the partially hepatectomized animals had fallen further to 2.09 g%, while that of the sham operated group had been almost completely restored (2.58 g%) to the control levels (2.77 g%). Kirsch et al. [29] demonstrated the immediate fall in the albumin synthesis rate which followed protein deprivation and we have attributed the fall in the serum albumin of our control animals over the 10 day period (2.99--2.77 g%) to protein-calorie deprivation, for during the entire 10 day post-operative period the partially hepatectomized animals are considerably less than normal animals fed ad libitum, and since the groups were "pair-fed" the controls were allowed substantially less than their normal diet to eat. The initial fall in the serum albumin of the sham operated animals is attributed to the sequestration of albumin into non-exchangeable extravascular compartments that has been shown to follow surgery [30--32]. Mourisden and Faber [30] and Mourisden [31] have shown an increased elimination of 13 ~I labelled albumin from plasma of patients undergoing major surgery and have demonstrated a "migration of albumin into the oedema of the operative field". Hoye et al. [32] found a reduction in plasma volume during major surgery disproportionate to the reduction in total red blood cell volume and a fall in the albumin concentration of the lymph. There is a highly significant increase in protein concentration and excretion in the urine in patients following surgery [33], this anomaly being greater in those patients undergoing major surgery. Besides these observations, other less well defined factors are involved in circulating albumin loss and include: loss into the abdominal cavity [34]; increased capillary permeability [35]; intravenous infusion [36,37] and an increase in fractional catabolic rate [38]. The albumin synthesis rate is seen to fall significantly after partial hepat e c t o m y . This fall is greatest at 12 h and least at 22 h after the operation. Indeed by 22 h the recovery of the albumin synthesis rate of the liver is such that when the results are expressed in terms of liver weight, the synthesis rates of the partially hepatectomized and sham operated animals are no longer significantly different. Other workers [6] found that 24 h after partial h e p a t e c t o m y the albumin synthesis rate was significantly depressed when expressed in terms of animal weight, although the rate expressed in terms of liver weight was within normal limits. Further, these workers found that 72 h after partial hepatectomy, the albumin synthesis rates were significantly increased when expressed in terms of both animal weight and liver weight. In contrast, in our studies the albumin synthesis rate was still significantly depressed when compared to control animals both in terms of animal weight and liver weight 72 h after partial hepa-
121 t e c t o m y . At no time during this period did we find an elevation of albumin synthesis rate in the partially hepatectomized animals. Our findings are more in keeping with the results obtained from the investigations into serum albumin levels and albumin catabolic rates in this experimental situation. It is important to stress that all groups of animals were "pair-fed" during the experiments carried out to determine albumin synthesis rates, as well as in those investigating serum albumin levels and albumin catabolic rates after partial hepatectomy. It is thus clear that even when the values are considered in terms of liver weight, the albumin synthesis rates of the partially hepatectomized livers are depressed. Rothschild et al. [39] have postulated an extra-vascular osmotic regulatory system in the liver controlling albumin synthesis and it is known that albumin is sequestered in extra-vascular compartments post-operatively. It is possible that the albumin synthesis rate is depressed due to raised extravascular osmotic pressure caused b y this sequestration of albumin. Although the albumin synthesis rates of the partially hepatectomized and sham operated animals are no longer significantly different 22 h post-operatively (when expressed in terms of liver weight) this hypothesis does not provide for the highly significant differences between these two groups at the earlier intervals postoperatively, even when considered in terms of liver weight. Christensen et al. [40] and Braun et al. [3] showed that the levels of amino acids in the liver are increased at an early stage after partial h e p a t e c t o m y and it has been demonstrated that amino acids are mobilized for plasma protein synthesis in in vivo experiments on nephrotic rats [41]. These findings were confirmed more recently by Ferris and Clark [42] who showed a rise in both hepatic and plasma free amino acid concentrations during the first four hours after partial h e p a t e c t o m y as compared to sham operated animals. Using in vitro techniques Marsh and Drabkin [43] and Peters [44] were unable to demonstrate an accelerating effect of added amino acids on the albumin synthesis rates. Clemens [ 4 5 ] , however, has shown an increase in protein synthetic activity in cell free systems after exposure to high concentrations of amino acids, while Kelman et al. [46] demonstrated the ability of certain amino acids, in high concentrations, to restore the albumin synthesis rates of isolated perfused livers from starved rats to normal levels. We have shown that by adding 10 times the normal plasma concentration of certain amino acids to perfusate, the albumin synthesis rate is doubled, the increase being such that it is significant (P < 0.05) in terms of both liver and animal weight. In addition it is no longer significantly different from the sham operated animal when expressed in terms of liver weight. Braun et al. [3] have claimed that in the early stages of liver regeneration precedence is given to the biosynthesis of cellular proteins and that after the regenerative process is well advanced, the increased construction of plasma albumin is metabolically favoured. One mechanism whereby amino acids stimulate albumin synthesis could be that as a result of the redistribution of cellular amino acids following partial hepatectomy, the supply of primary substrate for albumin synthesis is limiting. Alternatively, if depression of albumin synthesis was due to a limited availability of specific tRNAs or amino acid t R N A synthetases, this restriction could be partly reversed by increasing cellular amino acid concentrations above normal levels.
122 Another factor of importance in relation to albumin synthesis is total hormonal balance. While changes in the availability of specific amino acids released from altered tissue degradation may also play a role in tissue culture, cortisone has been shown to increase albumin synthesis per se [47]. We were unable to demonstrate any alteration in the albumin synthesis rate following the addition of cortisone to the perfusate. This is in keeping with the findings of Kelman et al. [48] who was able to influence ribosomal profiles by adding 10 times the normal concentration of amino acid to the system, but not by adding hormones. Two sets of experiments were carried out to investigate albumin catabolism following partial hepatectomy; a short term one to investigate the immediate effect of partial h e p a t e c t o m y on albumin catabolism and a longer term one to allow these changes to be observed for 10 days. There is excellent correlation between the two sets of experiments which indicate an immediate significant fall in the albumin catabolic rate following partial hepatectomy as the system endeavours to maintain the plasma albumin pool. This finding is in keeping with the findings of Kirsch et al. [29] who observed that a fall in the albumin catabolic rate of malnourished rats occurred following the fall in their albumin synthesis rates and plasma albumin pools as a result of protein calorie deprivation. Acknowledgments We are indebted to the following for their valuable advice and technical assistance: Mr V.M. Wells, Chief Technician, MRC/UCT Liver Research Group; Mr Milton Stofile and Mr M. Parker, Laboratory Attendants, Department of Medicine, University of Cape Town; Dr R. Stead, Ph.D. (Natal), Senior Lecturer, Department of Biochemistry, University of Cape Town. This work is part of the programme of the MRC/UCT Liver Research Group and was supported by the South African Medical Research Council, the S.A. Atomic Energy Board, Cancer Research Trust and Staff Research Fund of the University of Cape Town. References I 2 3 4 5 6 7 8 9 10 II 12
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