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The role of the liver in the maintenance of plasma proteins and amino acids in the eel, Anguilla anguilla L., with reference to amino acid deamination
Comp. Biochem. Physiol., 1967, Vol. 22, pp. 169 to 175. Pergamon Press. Printed in Great Britain
T H E ROLE OF T H E LIVER IN T H E M A I N T E N A N C E OF PLASMA PROTEINS AND A M I N O ACIDS IN T H E EEL, A N G U I L L A A N G U I L L A L., W I T H REFERENCE TO A M I N O ACID D E A M I N A T I O N ALWYN J E A N E T T E KENYON Department of Zoology, The University, Sheffield 10, England
(Received 27 ~anuary 1967) A b s t r a c t - - 1 . The effect of hepatectomy on plasma proteins and amino acids
and ammonia excretion in the freshwater eel (Anguilla anguilla L.) has been investigated. 2. Removal of the liver was followed by a 25% fall in the concentration of plasma proteins and a 65% rise in the concentration of plasma amino acids, measured 10 days after operation. 3. Eel liver does not appear to be essential in amino acid deamination, as ammonia excretion continues unchanged in hepatectomized eels. However, excess exogenous amino acids cannot be deaminated in the absence of the liver. 4. The function of the eel liver is discussed and compared with that of the mammal. INTRODUCTION
IN A REVIEWon mammalian plasma proteins, Madden & Whipple (1940) suggested that the liver was the site of plasma protein synthesis. Later, Tarver & Reinhardt (1947), comparing the incorporation of asS-labelled methionine into the tissues of intact and hepatectomized dogs, showed that the liver produces most plasma proteins. Miller et al. (1951) and Miller & Bale (1954), working with perfused liver, and Peters & Anfinsen (1950), using liver slices, have confirmed this in the rat. Removal of the liver brings about a sharp rise in the concentration of all plasma amino acids and the cessation of urea production in dogs (Bollman et al., 1926), rabbits (Svedberg et al., 1938) and monkeys (Maddock & Svedberg, 1938). As there is no appreciable rise in ammonia in the blood or urine of these animals, it appears that deamination ceases in the absence of the liver. Smith (1929) showed that the freshwater goldfish (Carassius auratus) and carp (Cyprinus carpio) excrete nitrogen mainly in the form of ammonia. Marine teleosts also excrete 80 per cent of the total waste nitrogen as ammonia (Wood, 1950). Goldstein et al. (1964), McBean et al. (1966) and Pequin & Serfaty (1963, 1966) consider that the liver is the site of ammonia formation in the teleosts Myoxocephalus scorpius, Anguilla rostrata and Cyprinus carpio respectively. The purpose of the experiments reported here was to investigate the role of the liver of the freshwater 169
170
ALWYN JEANETTE KENYON
eel (Anguilla anguilla L.) in the maintenance of plasma proteins and amino acids. From observations on the effect of hepatectomy on ammonia excretion it was hoped to obtain some information as to the possible site of amino acid deamination in the eel. MATERIALS AND METHODS Freshwater yellow or silver eels, weighing 150-250 g, were obtained from Billingsgate market. They were kept, without food, in tanks of aerated Sheffield tap water at 16°C during the course of the experiments.
Hepatectomy Eels were anaesthetized in MS 222 (Sandoz Ltd., approximately 1 g/l). Twenty minutes was normally sufficient time to ensure that the animal remained anaesthetized during the operation. A ventral incision was made, extending from just behind the heart to about 1 cm behind the posterior end of the liver. The mesentery attaching the liver to the dorsal body wall was slit and ligatures were tied loosely round the hepatic portal vein and the hepatic vein, where they entered the liver. As much blood as possible was squeezed out of the liver before securely tying off the blood vessels. The liver and gall bladder were cut away, making sure that no tissue remained attached to the cut ends of the main blood vessels. The body wall was stitched together, with the edges of the wound closely apposed to prevent any leakage of water into the body. Sham-operated eels were treated in the same way except that the liver and gall bladder were left intact in the animal.
Preparation of plasma Ten days after operation, eels were anaesthetized in MS 222 and the skin on the ventral surface in the region of the heart was removed. The posterior part of the tongue muscle was cut away to expose the heart and a slit made in the pericardium. Blood was withdrawn from the conus arteriosus into a heparinized syringe, centrifuged immediately and the plasma retained.
Plasma proteins Plasma was diluted 1 : 2 0 with water and the total protein concentration estimated by the biuret method (Racusen & Johnstone, 1961).
Ninhydrin-positive material in the plasma One-tenth millilitre of 50% trichloroacetic acid (TCA) was added to 1.0 ml fresh plasma which was then centrifuged. Total ninhydrin-positive material in the supernatant was estimated by the method of Rosen (1957). TCA was removed from pooled samples from sixteen control eels and eighteen hepatectomized eels. The samples were then chromatographed on an automatic amino acid analyser (Evans Electroselenium Ltd.) as follows. (1) Acidic and neutral amino acids; 1-0-ml samples of eel plasma were run on a 150-cm column at 30-50°C at a pH of 3-25-4.25.
MAINTENANCE OF PLASMA PROTEINS AND AMINO ACIDS BY EEL LIVER
171
(2) Basic amino acids; 1.0-ml samples of eel plasma were run on a 15-cm column at 50°C at a pH of 5.28.
Ammonia excretion Sham-operated and hepatectomized eels were kept in 400 ml of Sheffield tap water overnight (16 hr). Ammonia was estimated on 1.0-ml samples of water using either Nessler's reagent or the method of Chaney & Marbach (1962).
RESULTS
Effect of hepatectomy on plasma proteins and amino acids Ten days after operation the concentration of total plasma proteins in ten hepatectomized eels was significantly lower, 53.3 + 6.33 mg/ml, than that of the twelve sham-operated controls, 72.75 + 1.69 mg/ml (Table 1). The concentration of plasma amino acids (total ninhydrin-positive material) had risen by 65 per cent from 9.96 + 1-09/~moles/ml in four control eels, to 16.50 + 3.71/zmoles/ml in four hepatectomized eels (Table 1). Analysis of individual amino acids showed that the concentration of all of them had increased after hepatectomy. There was a very marked rise in the amount of serine, tyrosine and histidine in the plasma (Table 2). Summation of the quantities of individual amino acids measured with the analyser gave figures for total amino acids which were 80 per cent lower in control animals and 60 per cent lower in hepatectomized animals than the values obtained from measuring total ninhydrin-positive material. TABLE 1--EFFECT OF HEPATECTOMY ON PLASMA PROTEINS AND AMINO ACIDS IN THE FRESHWATER YELLOW EEL Plasma proteins (mg/ml)
Sham-operated Hepatectomized * P = 0"01.
72-75 + 1"68 53"3 + 6'33*
Plasma amino acids (/zmoles/ml)
9"96 + 1-09 16"50 + 3"71t
t P = 0"01.
Proteins were estimated by the biuret method as described in the text. Total ninhydrinpositive material was estimated on protein-free samples of eel plasma by the method of Rosen (1957).
Effect of hepatectomy on total ammonia excretion The daily ammonia excretion was measured for up to 7 days after operation. There was no significant difference between ammonia excretion of sham-operated and hepatectomized eels. Intraperitoneal injection of L-c~-alanine [200 mg (2"0 ml) -1 (150 g)-l] into sham-operated eels produced a 43 per cent rise in the rate of ammonia excretion (Table 3). The rise was apparent 2 hr after injection
172
ALWYN JEANETTE KENYON TABLE 2--EFFECT OF HEPATECTOMY ON PLASMA AMINO ACIDS IN THE YELLOW EEL
C o n c e n t r a t i o n of a m i n o acid (mM) C Taurine Lysine Valine Isoleucine Leucine Alanine Phenylalanine Glycine G l u t a m i c acid Threonine Serine Arginine Tyrosine Histidine Methionine A s p a r t i c acid Total
H
0"290 0-281 0"162 0" 155 0-143 0-130 0" 100 0"094 0"080" 0'056 0'053 0"044 0"026 0"024* 0'024 0-017
0"962 0"644 0"298" 0-190" 0"233 * 0"689 * 0"352" 0-423 * 0-285 * 0"232" 0'585 * 0" 173 0"265" 0"262 * 0"128" 0"073 *
1"679
5"794
H/C 3-32 2"29 1"84 1-23 1 "63 5" 30 3"52 4-50 3"56 4"14 11-04
3" 93 10-19 10.92 5-33 4'29
F i g u r e s are m e a n s of t h r e e or two (*) e s t i m a t i o n s . Pooled samples f r o m sixteen c o n t r o l (C) a n d e i g h t e e n h e p a t e c t o m i z e d eels (H) were c h r o m a t o g r a p h e d o n an a u t o m a t i c a m i n o acid analyser ( E v a n s E l e c t r o s e l e n i u m Ltd.). Acidic a n d n e u t r a l a m i n o acids were s e p a r a t e d o n a 1 5 0 - c m c o l u m n at 3 0 - 5 0 ° C a n d p H 3-25-4-25. Basic a m i n o acids were s e p a r a t e d o n a 1 5 - c m c o l u m n at 50°C a n d p H 5"28.
TABLE 3--EFFECT OF HEPATECTOMY ON AMMONIA EXCRETION IN THE
YELLOW EEL Ammonia excretion * ( m g . h r -1 kg - t b o d y wt.)
Sham-operated Hepatectomized
C
A
2"12 _+0-21 1-78 +_0'10
3"73 _+0"22 1.76 _+0"26
* M e a n s + S.E. for n i n e a n i m a l s . A n i m a l s were i n j e c t e d w i t h e i t h e r 0 - 9 % NaC1 (C) or L - a - a l a n i n e in 0"9% N a C I [200 m g (2"0 ml) -1 (150 g ) - l ] (A). Eels were k e p t in jars in 400 m l aerated Sheffield tap water. S a m p l e s of w a t e r were t a k e n for a m m o n i a e s t i m a t i o n u s i n g N e s s l e r ' s r e a g e n t or b y t h e m e t h o d of C h a n e y & M a r b a c h (1962).
MAINTENANCE OF PLASMA PROTEINS AND AMINO ACIDS BY I ~ L LIVER
173
and continued at an almost constant rate for 25 hr (Fig. 1). It was calculated that 24 hr after injection, ammonia equivalent to 16 per cent of the exogenous alanine had been excreted. There was no change in ammonia excreted by hepatectomized eels injected with the same dose of L-a-alanine (Fig. 2). IO0--
g 75 |
~
°~°
} o
0
5I
I I I0 1. 5 Time, hr
I 20
I :>5
FIG. 1, Effect of an intraperitoneal injection of L-o~-alanine on ammonia excretion in the sham-operated freshwater eel. © ~ 0 , control injection of 0"9% NaCI. • • L-c~-alanine (200 mg/150 g body wt). I00
.~
--
75
3
-~ .o o
5
I0 Time,
15
20
25
hr
FIG. 2. Effect of an intraperltoneal injection of L-o~-alanine on ammonia excretion in the hepatectomized freshwater eel. © ©, control injection of 0.9% NaCI. • • , L-~-alanine (200 mg/150 g body wt).
174
ALWYN JEANETTE KENYON
DISCUSSION There appears to be a basic similarity in the part played by the liver of teleosts and mammals in the formation of plasma proteins. Studies on hepatectomized dogs, reviewed by Madden & Whipple (1940), showed that in the absence of the liver, plasma protein levels fall. Essentially the same effect was found after removal of the liver of the eel. The eels used in the present work were not fed and gradually lost weight. Teleosts excrete the majority of their total waste nitrogen in the form of ammonia (Smith, 1929; Wood, 1950) which in eels is equivalent to a loss in body weight of approximately 0.27 g (100 g)-i day-1. A starved eel loses approximately 0.35 per cent of its body weight per day (Keys, 1933) and, as about 15 per cent of the body is protein (McCance, 1944), the ammonia excretion observed in the present work can be accounted for by catabolism of the tissues. In mammals, the sharp rise in the concentration of amino acids in plasma and tissues after hepatectomy indicates that protein catabolism has ceased at the level of deamination (Mann, 1927). However, removal of the liver in eels did not alter the rate of ammonia excretion. Even up to 7 days after operation, the rate of ammonia excretion was not significantly different from that of sham-operated control animals. The experiments of van Slyke & Meyer (1912) and Gerok et al. (1963) suggest that mammalian liver regulates plasma amino acid levels. An increase in plasma amino acids is counteracted by deamination in the liver, while a decrease causes the liver to release amino acids into the blood. There is evidence (Miller, 1962) that extrahepatic tissues of the rat are capable of oxidizing a large group of "nonessential" amino acids. This may also apply to fish, for Pequin (1962) reported high concentrations of ammonia in blood coming from the kidney of the carp (Cyprinus carpio). It therefore seems possible that, in the non-feeding hepatectomized eel, the extrahepatic tissues could maintain the normal rate of deamination at a higher steady-state concentration of plasma amino acids. Amino acids, greatly in excess of the normal net rate of release from tissues, that are being utilized to provide energy (e.g. alanine injection in the present work) would normally be dealt with by the liver.
Acknowledgements--The author wishes to thank Professor I. Chester Jones, in whose department this work was carried out, for the provision of many facilities, Dr. D. Bellamy for his supervision and continual encouragement and Dr. W. Ferdinand of the Department of Biochemistry, the University of Sheffield, for carrying out the amino acid analyses. This work was carried out during the tenure of an S.R.C. Research Studentship. REFERENCES BOLLMANJ. L., MANNF. C. & MAGATHT. B. (1926) Studies on the physiology of the liver-15. Effect of total removal of the liver on deaminisation. Am. J. Physiol. 78, 258-269. CHANEYA. L. & MARBACHE. P. (1962) Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130-132. GrROK W., MITZ~T H. J. & SCHIMASSEKH. (1963) Untersuchungen zum Aminos~iurenstoffwechsel an der isolert perfundierten Leber. Verh. dr. Ges. inn. Med. 69, 426-431.
MAINTENANCE
OF PLASMA
PROTEINS AND
AMINO
ACIDS BY EEL LIVER
175
GOLDSTEIN L., FORSTER R. P. & FAN~LLI G. M. (1964) Gill blood flow and ammonia excretion in the marine teleost, Myoxoeephalus seorpius. Comp. Biochem. Physiol. 12, 489-499. KEYS A. B. (1933) T h e mechanism of adaptation to varying salinity in the common eel, and the general problem of osmotic regulation in fishes. Proc. R. Soc. B 112, 184-199. McBEAN R. L., NEPPEL M. J. & GOLDSTEIN L. (1966) Glutamate dehydrogenase and ammonia production in the eel (Anguilla rostrata). Comp. Biochem. Physiol. 18, 909-920. McC~'qcE R. A. (1944) T h e chemistry of growth and the food value of the common eel (AnguiUa anguiUa L.). Biochem. a%38, 474-480. MADDEN S. C. & WHIPPLE G. (1940) Plasma proteins; their source, production and utilisation. Physiol. Rev. 20, 194-217. MADDOCK S. & SWDBEEO A. (1938) T h e effect of total removal of the liver of the monkey. Am. ft. Physiol. 121, 203-208. MANN F. C. (1927) T h e effects of complete and of partial removal of the liver. Medicine, Baltimore 6, 419-511. MILLER L. L. (1962) T h e role of the liver and the non-hepatic tissues in the regulation of free amino acid levels in the blood. In Amino Acid Pools. p. 708. Elsevier, Amsterdam. MILLER L. L. & BALE W. F. (1954) Synthesis of all plasma protein fractions except gamma globulins by the liver. J . exp. Med. 99, 125-132. MILLER L. L., BL~" C. G., WATSON M. L. & BALE W. F. (1951) T h e dominant role of the liver in plasma protein synthesis. J . exp. Med. 94, 431-453. PEQUIN L. (1962) Les teneurs en azote ammoniacal du sang chez la carpe (Cyprinus carpio). C. r. Acad. Sci., Paris 255, 1795-1797. PEQUIN L. & SERFATY A. (1963) L'excrdtion ammoniacal chez un tdldostdan dulcicole, Cyprinus carpio L. Comp. Biochem. Physiol, 10, 315-324. PEQUIN L. & SERFATYA. (1966) Acide glutamique et excrdtion azotde chez la carpe commune (Cyprinus carpio L.). Comp. Biochern. Physiol. 18, 141-149. PETERS T . JR. & ANFINSEN C. B. (1950) Production of radioactive serum albumin by liver slices. J . biol. Chem. 186, 805-813. 1L~COSEN D. & JOHNSTONE D. B. (1961) Estimation of protein in cellular material. Nature, Loud. 191, 492-493. ROSEN H. (1957) A modified ninhydrin colorimetric analysis for amino acids. Archs Biochem. Biophys. 67, 10-15. VAN SLYKE D. D. & M~/~R G. M. (1912) T h e fate of protein digestion products in the body. .7. biol. Chem. 16, 213-229. SMITH H. W. (1929) T h e excretion of ammonia and urea by the gills of fish. J. biol. Chem. 81, 727-742. SVEDBERG A., MADDOCK S. & DRURY D, D. (1938) T h e effect of total removal of the liver in the rabbit. Am. J. Physiol. 121, 209-214. TARVER H. & REINHARDT W. O. (1947) Methionine labelled with radioactive sulfur as an indicator of protein formation in the hepatectomised dog. J. biol. Chem. 167, 395-400. WOOD J. D. (1950) Nitrogen excretion in some marine teleosts. Can. jY. Biochem. Physiol. 36, 1237-1242.
T H E ROLE OF T H E LIVER IN T H E M A I N T E N A N C E OF PLASMA PROTEINS AND A M I N O ACIDS IN T H E EEL, A N G U I L L A A N G U I L L A L., W I T H REFERENCE TO A M I N O ACID D E A M I N A T I O N ALWYN J E A N E T T E KENYON Department of Zoology, The University, Sheffield 10, England
(Received 27 ~anuary 1967) A b s t r a c t - - 1 . The effect of hepatectomy on plasma proteins and amino acids
and ammonia excretion in the freshwater eel (Anguilla anguilla L.) has been investigated. 2. Removal of the liver was followed by a 25% fall in the concentration of plasma proteins and a 65% rise in the concentration of plasma amino acids, measured 10 days after operation. 3. Eel liver does not appear to be essential in amino acid deamination, as ammonia excretion continues unchanged in hepatectomized eels. However, excess exogenous amino acids cannot be deaminated in the absence of the liver. 4. The function of the eel liver is discussed and compared with that of the mammal. INTRODUCTION
IN A REVIEWon mammalian plasma proteins, Madden & Whipple (1940) suggested that the liver was the site of plasma protein synthesis. Later, Tarver & Reinhardt (1947), comparing the incorporation of asS-labelled methionine into the tissues of intact and hepatectomized dogs, showed that the liver produces most plasma proteins. Miller et al. (1951) and Miller & Bale (1954), working with perfused liver, and Peters & Anfinsen (1950), using liver slices, have confirmed this in the rat. Removal of the liver brings about a sharp rise in the concentration of all plasma amino acids and the cessation of urea production in dogs (Bollman et al., 1926), rabbits (Svedberg et al., 1938) and monkeys (Maddock & Svedberg, 1938). As there is no appreciable rise in ammonia in the blood or urine of these animals, it appears that deamination ceases in the absence of the liver. Smith (1929) showed that the freshwater goldfish (Carassius auratus) and carp (Cyprinus carpio) excrete nitrogen mainly in the form of ammonia. Marine teleosts also excrete 80 per cent of the total waste nitrogen as ammonia (Wood, 1950). Goldstein et al. (1964), McBean et al. (1966) and Pequin & Serfaty (1963, 1966) consider that the liver is the site of ammonia formation in the teleosts Myoxocephalus scorpius, Anguilla rostrata and Cyprinus carpio respectively. The purpose of the experiments reported here was to investigate the role of the liver of the freshwater 169
170
ALWYN JEANETTE KENYON
eel (Anguilla anguilla L.) in the maintenance of plasma proteins and amino acids. From observations on the effect of hepatectomy on ammonia excretion it was hoped to obtain some information as to the possible site of amino acid deamination in the eel. MATERIALS AND METHODS Freshwater yellow or silver eels, weighing 150-250 g, were obtained from Billingsgate market. They were kept, without food, in tanks of aerated Sheffield tap water at 16°C during the course of the experiments.
Hepatectomy Eels were anaesthetized in MS 222 (Sandoz Ltd., approximately 1 g/l). Twenty minutes was normally sufficient time to ensure that the animal remained anaesthetized during the operation. A ventral incision was made, extending from just behind the heart to about 1 cm behind the posterior end of the liver. The mesentery attaching the liver to the dorsal body wall was slit and ligatures were tied loosely round the hepatic portal vein and the hepatic vein, where they entered the liver. As much blood as possible was squeezed out of the liver before securely tying off the blood vessels. The liver and gall bladder were cut away, making sure that no tissue remained attached to the cut ends of the main blood vessels. The body wall was stitched together, with the edges of the wound closely apposed to prevent any leakage of water into the body. Sham-operated eels were treated in the same way except that the liver and gall bladder were left intact in the animal.
Preparation of plasma Ten days after operation, eels were anaesthetized in MS 222 and the skin on the ventral surface in the region of the heart was removed. The posterior part of the tongue muscle was cut away to expose the heart and a slit made in the pericardium. Blood was withdrawn from the conus arteriosus into a heparinized syringe, centrifuged immediately and the plasma retained.
Plasma proteins Plasma was diluted 1 : 2 0 with water and the total protein concentration estimated by the biuret method (Racusen & Johnstone, 1961).
Ninhydrin-positive material in the plasma One-tenth millilitre of 50% trichloroacetic acid (TCA) was added to 1.0 ml fresh plasma which was then centrifuged. Total ninhydrin-positive material in the supernatant was estimated by the method of Rosen (1957). TCA was removed from pooled samples from sixteen control eels and eighteen hepatectomized eels. The samples were then chromatographed on an automatic amino acid analyser (Evans Electroselenium Ltd.) as follows. (1) Acidic and neutral amino acids; 1-0-ml samples of eel plasma were run on a 150-cm column at 30-50°C at a pH of 3-25-4.25.
MAINTENANCE OF PLASMA PROTEINS AND AMINO ACIDS BY EEL LIVER
171
(2) Basic amino acids; 1.0-ml samples of eel plasma were run on a 15-cm column at 50°C at a pH of 5.28.
Ammonia excretion Sham-operated and hepatectomized eels were kept in 400 ml of Sheffield tap water overnight (16 hr). Ammonia was estimated on 1.0-ml samples of water using either Nessler's reagent or the method of Chaney & Marbach (1962).
RESULTS
Effect of hepatectomy on plasma proteins and amino acids Ten days after operation the concentration of total plasma proteins in ten hepatectomized eels was significantly lower, 53.3 + 6.33 mg/ml, than that of the twelve sham-operated controls, 72.75 + 1.69 mg/ml (Table 1). The concentration of plasma amino acids (total ninhydrin-positive material) had risen by 65 per cent from 9.96 + 1-09/~moles/ml in four control eels, to 16.50 + 3.71/zmoles/ml in four hepatectomized eels (Table 1). Analysis of individual amino acids showed that the concentration of all of them had increased after hepatectomy. There was a very marked rise in the amount of serine, tyrosine and histidine in the plasma (Table 2). Summation of the quantities of individual amino acids measured with the analyser gave figures for total amino acids which were 80 per cent lower in control animals and 60 per cent lower in hepatectomized animals than the values obtained from measuring total ninhydrin-positive material. TABLE 1--EFFECT OF HEPATECTOMY ON PLASMA PROTEINS AND AMINO ACIDS IN THE FRESHWATER YELLOW EEL Plasma proteins (mg/ml)
Sham-operated Hepatectomized * P = 0"01.
72-75 + 1"68 53"3 + 6'33*
Plasma amino acids (/zmoles/ml)
9"96 + 1-09 16"50 + 3"71t
t P = 0"01.
Proteins were estimated by the biuret method as described in the text. Total ninhydrinpositive material was estimated on protein-free samples of eel plasma by the method of Rosen (1957).
Effect of hepatectomy on total ammonia excretion The daily ammonia excretion was measured for up to 7 days after operation. There was no significant difference between ammonia excretion of sham-operated and hepatectomized eels. Intraperitoneal injection of L-c~-alanine [200 mg (2"0 ml) -1 (150 g)-l] into sham-operated eels produced a 43 per cent rise in the rate of ammonia excretion (Table 3). The rise was apparent 2 hr after injection
172
ALWYN JEANETTE KENYON TABLE 2--EFFECT OF HEPATECTOMY ON PLASMA AMINO ACIDS IN THE YELLOW EEL
C o n c e n t r a t i o n of a m i n o acid (mM) C Taurine Lysine Valine Isoleucine Leucine Alanine Phenylalanine Glycine G l u t a m i c acid Threonine Serine Arginine Tyrosine Histidine Methionine A s p a r t i c acid Total
H
0"290 0-281 0"162 0" 155 0-143 0-130 0" 100 0"094 0"080" 0'056 0'053 0"044 0"026 0"024* 0'024 0-017
0"962 0"644 0"298" 0-190" 0"233 * 0"689 * 0"352" 0-423 * 0-285 * 0"232" 0'585 * 0" 173 0"265" 0"262 * 0"128" 0"073 *
1"679
5"794
H/C 3-32 2"29 1"84 1-23 1 "63 5" 30 3"52 4-50 3"56 4"14 11-04
3" 93 10-19 10.92 5-33 4'29
F i g u r e s are m e a n s of t h r e e or two (*) e s t i m a t i o n s . Pooled samples f r o m sixteen c o n t r o l (C) a n d e i g h t e e n h e p a t e c t o m i z e d eels (H) were c h r o m a t o g r a p h e d o n an a u t o m a t i c a m i n o acid analyser ( E v a n s E l e c t r o s e l e n i u m Ltd.). Acidic a n d n e u t r a l a m i n o acids were s e p a r a t e d o n a 1 5 0 - c m c o l u m n at 3 0 - 5 0 ° C a n d p H 3-25-4-25. Basic a m i n o acids were s e p a r a t e d o n a 1 5 - c m c o l u m n at 50°C a n d p H 5"28.
TABLE 3--EFFECT OF HEPATECTOMY ON AMMONIA EXCRETION IN THE
YELLOW EEL Ammonia excretion * ( m g . h r -1 kg - t b o d y wt.)
Sham-operated Hepatectomized
C
A
2"12 _+0-21 1-78 +_0'10
3"73 _+0"22 1.76 _+0"26
* M e a n s + S.E. for n i n e a n i m a l s . A n i m a l s were i n j e c t e d w i t h e i t h e r 0 - 9 % NaC1 (C) or L - a - a l a n i n e in 0"9% N a C I [200 m g (2"0 ml) -1 (150 g ) - l ] (A). Eels were k e p t in jars in 400 m l aerated Sheffield tap water. S a m p l e s of w a t e r were t a k e n for a m m o n i a e s t i m a t i o n u s i n g N e s s l e r ' s r e a g e n t or b y t h e m e t h o d of C h a n e y & M a r b a c h (1962).
MAINTENANCE OF PLASMA PROTEINS AND AMINO ACIDS BY I ~ L LIVER
173
and continued at an almost constant rate for 25 hr (Fig. 1). It was calculated that 24 hr after injection, ammonia equivalent to 16 per cent of the exogenous alanine had been excreted. There was no change in ammonia excreted by hepatectomized eels injected with the same dose of L-a-alanine (Fig. 2). IO0--
g 75 |
~
°~°
} o
0
5I
I I I0 1. 5 Time, hr
I 20
I :>5
FIG. 1, Effect of an intraperitoneal injection of L-o~-alanine on ammonia excretion in the sham-operated freshwater eel. © ~ 0 , control injection of 0"9% NaCI. • • L-c~-alanine (200 mg/150 g body wt). I00
.~
--
75
3
-~ .o o
5
I0 Time,
15
20
25
hr
FIG. 2. Effect of an intraperltoneal injection of L-o~-alanine on ammonia excretion in the hepatectomized freshwater eel. © ©, control injection of 0.9% NaCI. • • , L-~-alanine (200 mg/150 g body wt).
174
ALWYN JEANETTE KENYON
DISCUSSION There appears to be a basic similarity in the part played by the liver of teleosts and mammals in the formation of plasma proteins. Studies on hepatectomized dogs, reviewed by Madden & Whipple (1940), showed that in the absence of the liver, plasma protein levels fall. Essentially the same effect was found after removal of the liver of the eel. The eels used in the present work were not fed and gradually lost weight. Teleosts excrete the majority of their total waste nitrogen in the form of ammonia (Smith, 1929; Wood, 1950) which in eels is equivalent to a loss in body weight of approximately 0.27 g (100 g)-i day-1. A starved eel loses approximately 0.35 per cent of its body weight per day (Keys, 1933) and, as about 15 per cent of the body is protein (McCance, 1944), the ammonia excretion observed in the present work can be accounted for by catabolism of the tissues. In mammals, the sharp rise in the concentration of amino acids in plasma and tissues after hepatectomy indicates that protein catabolism has ceased at the level of deamination (Mann, 1927). However, removal of the liver in eels did not alter the rate of ammonia excretion. Even up to 7 days after operation, the rate of ammonia excretion was not significantly different from that of sham-operated control animals. The experiments of van Slyke & Meyer (1912) and Gerok et al. (1963) suggest that mammalian liver regulates plasma amino acid levels. An increase in plasma amino acids is counteracted by deamination in the liver, while a decrease causes the liver to release amino acids into the blood. There is evidence (Miller, 1962) that extrahepatic tissues of the rat are capable of oxidizing a large group of "nonessential" amino acids. This may also apply to fish, for Pequin (1962) reported high concentrations of ammonia in blood coming from the kidney of the carp (Cyprinus carpio). It therefore seems possible that, in the non-feeding hepatectomized eel, the extrahepatic tissues could maintain the normal rate of deamination at a higher steady-state concentration of plasma amino acids. Amino acids, greatly in excess of the normal net rate of release from tissues, that are being utilized to provide energy (e.g. alanine injection in the present work) would normally be dealt with by the liver.
Acknowledgements--The author wishes to thank Professor I. Chester Jones, in whose department this work was carried out, for the provision of many facilities, Dr. D. Bellamy for his supervision and continual encouragement and Dr. W. Ferdinand of the Department of Biochemistry, the University of Sheffield, for carrying out the amino acid analyses. This work was carried out during the tenure of an S.R.C. Research Studentship. REFERENCES BOLLMANJ. L., MANNF. C. & MAGATHT. B. (1926) Studies on the physiology of the liver-15. Effect of total removal of the liver on deaminisation. Am. J. Physiol. 78, 258-269. CHANEYA. L. & MARBACHE. P. (1962) Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130-132. GrROK W., MITZ~T H. J. & SCHIMASSEKH. (1963) Untersuchungen zum Aminos~iurenstoffwechsel an der isolert perfundierten Leber. Verh. dr. Ges. inn. Med. 69, 426-431.
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