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The origin of serum amylase electrophoretic studies of isoamylases of the serum, liver and other tissues of adult and infant rats
441
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26629
T H E O R I G I N OF SERUM AMYLASE. E L E C T R O P H O R E T I C S T U D I E S OF ISOAMYLASES OF T H E SERUM, L I V E R AND O T H E R TISSUES OF A D U L T AND I N F A N T RATS
KERIE
H A M M E R T O N A~XDM I C H A E L M E S S E R
Department of Biochemistry, The University of Sydney, N.S.W. 2oo6 (Australia) (Received M a r c h 5th, 1971)
SUMMARY
To obtain information on the tissue origin of serum amylase, partially purified preparations of rat liver and serum amylase and extracts of various tissues were subjected to cellulose acetate electrophoresis followed b y detection of the amylolytic activity on starch-agar plates. A method is described for the 6oo-fold purification of detergent-solubilised liver amylase. The pancreas showed three bands of amylase activity migrating towards the cathode, while the parotid gland revealed five to six anodic bands. In adult rats, the serum and urine showed isoamylase patterns which were the same as those of the liver, and similar to those of the parotid. Isoamylases of the pancreatic type appeared in the serum and urine only after ligation of the common bile duct. In newborn rats, the serum and liver contained isoamylases of the pancreatic as well as parotid type but the pancreatic type disappeared from both serum and liver b y the third day after birth. There was a Ioo-fold increase in the levels of parotid gland amylase after weaning, which was not reflected in the levels of serum amylase. These results support the view that the liver is the major source of serum amylase in the normal rat.
INTRODUCTION
Although it has been known for over a century that human serum contains an amylolytic enzyme which is excreted in tile urine, the tissue origin of this enzyme remains in doubt. In 1941 , SOMOGYI1 concluded that under normal physiological conditions neither of the two chief amylase synthesising organs, namely the pancreas and the salivary glands, could be implicated. This subject was again investigated and reviewed in 1959 b y J A N O W l T Z AND D R E I L I N G ~, who postulated that the total amylolytic activity of the blood is the sum of the activities of several a-amylases of diverse origin, including the pancreas, salivary glands, liver and possibly also the fallopian tubes, striated muscle and adipose tissue. More recently, McGEACHIN et al.~, 4 and ARNOLD AND RUTTER5 made the imBiochim. Biophys. Acta, 244 (1971) 441-451
442
K. HAMMERTON, M. MESSER
portant observation that a-amylase is synthesised by the liver of the rat, and that the isolated liver secretes amylase into the perfusion medium. Therefore these authors suggested that in the normal rat the liver is a source of serum amylase, but were unable definitely to conclude that the liver is the sole or major source or to exclude either the pancreas or the salivary glands as possible contributary sources. Recent studies demonstrating the existence of electrophoretic variants of amylase that are tissue specific suggested a promising approach to elucidating the origin of serum amylase. In the mammalian species that have been investigated, including man6, 7, mouse 8 and rabbit" the amylases of the pancreas and salivary glands could be separated from each other by agar, acrylamide or cellulose acetate electrophoresis. Human serum and urine have been shown to contain two electrophoretic fractions corresponding to the salivary and pancreatic types, respectively 7. In view of these studies the question whether liver amylase has unique electrophoretic properties is relevant to the problem of the origin of serum amylase. This question has remained unresolved 1°- 12. In our work the electrophoretic isoamylase patterns exhibited by serum, urine, the liver, parotid gland, pancreas and small intestine have been studied and compared in both adult and infant rats. METHODS
Amylase assay Amylase activity was determined by the method of BERNFELD et al. 13 using the stable starch substrate solution recently described by STRUMEYER14. For the determination of liver amylase, the assay mixture contained o . i % Triton X-Ioo detergent as activator 15. One unit of amylase activity is defined as i.o/,mole of maltose equivalent liberated from starch per minute at 20 ° . Specific amylase activity was determined using the method of LOWRY et al. le for tile estimation of protein, bovine serum albumin being used as the standard. It has been observed by THEODORAND BIRNBAUM17 that human serum and hog pancreatic amylase activities increase on dilution. A similar "dilution effect" was observed in the present work with rat amylases. Electrophoretic separation of amylase isoenzymes This was done on cellulose acetate with the Beckman Microzone Model i o i electrophoresis apparatus. The buffer solution used was 0.02 M sodium potassium phosphate, pH 7.5, containing 0.02% NaC1. To obtain best results it was found important to de-aerate the cellulose acetate strip by placing it on the buffer solution under a low vacuum for IO rain, after which it was blotted to remove excess solution and placed in the electrophoresis apparatus. A total of 0.75/*1 of each sample was placed on the strip with the 0.25 #1 applicator. Electrophoresis was done at room temperature using a constant voltage of 200 V for 9° rain. The amylolytic bands were detected by placing the cellulose acetate strip on a starch-agar plate 18which was then maintained at 37 ° for 30-60 rain. After removal of the strip the plate was developed by immersion in 70% ethanol containing 5% acetic acid followed by a solution of I2 in o.I M KI. The electrophoretic patterns were photographed over a black velvet cloth using high contrast film. Slides to be photographed were not stained with I2. Biochim. Biophys. Acta, 244 (1971) 441-451
ORIGIN OF SERUM AMYLASE
443
As noted by POORT AND VAN VENROOY TM,precipitation of the starch by ethanolacetic acid begins at the surface of the starch-agar film and continues downwards with increasing fixation time. A short fixation time therefore reveals minor bands which disappear on prolonged fixation; on the other hand, longer fixation permits a more exact localisation of the major bands. In some of the photographs presented in this paper, the fixation time was such as to show up the major bands to maximum advantage; we have therefore supplemented these photographs with diagrams illustrating the minor bands. The amylolytic bands obtained on electrophoresis of solubilised amylase from a crude liver homogenate, or of untreated serum, were found to be distorted or displaced by other proteins present in the tissue preparation. For this reason rat liver and serum amylases had to be partially purified before electrophoresis (see below).
Partial purification of rat liver amylase Male Wistar rats were anaesthetised with ether and thoroughly bled from the jugular vein in order to reduce the blood content of the livers 1~. During removal of the livers extreme care had to be taken to prevent contamination with pancreatic tissue. The organs were homogenised in 3 vol. (v/w) of 0.25 M sucrose containing I mM CaCI~, and the homogenate centrifuged at 105000 × g for 60 rain. Of the total liver amylase, between 70 and 90% was found to be localised in the particulate fraction following ultracentrifugation. The remaining amylase in the supernatant fraction was discarded on the assumption that this fraction would contain all the amylase derived from blood contained in the liver, since it could be calculated from the experimentally determined mean level of amylase in blood (2.6 units/ml) and from an assumed value of 9 ml of blood per IOO g of liver from a well-bled rat 19, that of the total liver amylase activity, 7% would be due to serum amylase. In three separate trials the liver of an anaesthetised rat was perfused with 0.25 M sucrose solution via a catheter inserted into the inferior vena cava, in order to remove all the blood contained in the vascular system. The perfusate was collected from the portal vein and perfusion was continued under hydrostatic pressure until the liver had attained a bloodless appearance (500 ml). It was found that the supernatant fraction of homogenates of perfused livers contained only between IO and 40% of the amylase activity of the supernatant fraction of unperfused livers, while the particulate fraction contained between 50 and 80% of that of unperfused livers. Therefore perfusion resulted in a considerable loss of tile liver amylase, and for this reason the livers used for the purification were not perfnsed. The particulate fraction was resuspended in 6 vol. (w/v, with respect to the weight of fresh tissue) of 20 mM Tris-maleate, pH 7.0, containing 20 mM CaCI~, 20 mM NaC1 and either o.I% Triton X-Ioo or 0.4% digitonin, and allowed to stand at 5 ° for 20 h. The preparation was then incubated at 37 ° for 30 rain. This treatment, which solubilised at least 80% of the particulate amylase, is a modification of the solubilisation procedure of MORDOH et al. ~5. It was found that the degree of solubilisation depended on the protein concentration of the suspended particulate fraction; for maximum solubilisation this had to be less than 20 mg/ml. The solubilised enzyme was dialysed for 12 h against 50 mM Tris-maleate, pH 7.0, containing 20 mM CaC12 and 20 mM NaC1; any sediment which formed was removed. 2 vol. of ethanol (previously chilled to - - 2 0 °) were then added slowly under Biochim. Biophys. Acta, 244 (1971) 441 451
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K. HAMMERTON, M. MESSER
stirring to t h e ice-cold solution. The m i x t u r e was allowed to s t a n d for 3o min in an ice b a t h a n d t h e n centrifuged at IOOOO × g for 2o min. The s e d i m e n t was s u s p e n d e d in 8o ml of i o mM T r i s - m a l e a t e , p H 7.o, c o n t a i n i n g IO mM CaC12 a n d IO mM NaC1, a n d allowed to s t a n d at 5 ° for 12 h. The suspension was centrifuged at IOOOO ~< g for 3o min a n d the s u p e r n a t a n t solution d i a l y s e d a g a i n s t 2 mM T r i s - m a l e a t e , p H 7.o, c o n t a i n i n g 2 mM CaC12 a n d 2 mM NaC1. The d i a l y s e d solution was c o n c e n t r a t e d to a p p r o x i m a t e l y o n e - q u a r t e r its volume b y p e r v a p o r a t i o n from the dialysis b a g in front of a fan. The e n z y m e solution was t h e n s u b j e c t e d to gel filtration on a column of Sephadex G - I o o (5o em × 2.5 cm) ~°. The elution was done w i t h o . 9 % NaC1 at a flow r a t e of lO-2O ml/h. The p r o t e i n c o n t e n t of the fractions (4.o ml) was m e a s u r e d b y a b s o r b a n c e at 28o nm. Over 9 o % of t h e t o t a l p r o t e i n was e l u t e d in the first IOO ml following the v o i d v o l u m e ; the a m y l a s e a c t i v i t y a p p e a r e d as a b r o a d a s y m m e t r i c a l p e a k in the second IOO ml. The fractions c o n t a i n i n g a m y l a s e a c t i v i t y were pooled, d i a l y s e d against I mM T r i s - m a l e a t e , p H 7.o, c o n t a i n i n g I mM CaC12 a n d I mM NaC1, a n d c o n c e n t r a t e d to a p p r o x i m a t e l y o n e - t e n t h t h e i r p o o l e d v o l u m e b y p e r v a p o r a t i o n . The purified e n z y m e solution was s t a b l e when s t o r e d for u p to t h r e e weeks at 5 ° . TABLE 1 PARTIAL PURIFICATION
OF RAT LIVER AMYLASE
Volume (ml) Crude homogenate 750 105000 xg pellet 15°0 AfterTritonX-ioosoluhilisation 141o After ethanol fractionation 18 After Sephadex G-ioo filtration 27
Total protein (rag)
Total amylase activity (units)
Specific amylase activity (units/ mg protein)
Purification factor
49200 365 °0 11 200 2050 74
625
O.Ol3 o.o13 0.040 0.23 8.3
I i 3 18 640
475 447 47 ° 613
Table I s u m m a r i s e s a t y p i c a l purification of a m y l a s e from 30 r a t livers, in which a 64o-fold purification was achieved. The t a b l e indicates an a p p a r e n t increase in t o t a l a m y l a s e a c t i v i t y after S e p h a d e x G - I o o filtration. A possible e x p l a n a t i o n for this a n o m a l o u s result is the " d i l u t i o n effect" p r e v i o u s l y n o t e d (see METHODS), as a result of which strict c o m p a r i s o n s b e t w e e n a m y l a s e activities of different p r e p a r a t i o n s are difficult if n o t impossible. A l t e r n a t i v e l y , the result could be due to the r e m o v a l of an a m y l a s e i n h i b i t o r d u r i n g gel filtration. Partial purification of serum amylase R a t b l o o d o b t a i n e d b y cardiac p u n c t u r e was allowed to clot a n d t h e n centrifuged. The s e r u m was t r e a t e d with e t h a n o l a n d p a r t i a l l y purified on S e p h a d e x G - I o o as described for tile liver. A 4oo-fold purification of sertun a m y l a s e was o b t a i n e d . Urine Urine collected from r a t s m a i n t a i n e d in m e t a b o l i s m cages was d i a l y s e d a g a i n s t 2 mM T r i s - m a l e a t e , p H 7.0, c o n t a i n i n g 2 mM CaC12 a n d 2 mM NaC1 a n d t h e n conc e n t r a t e d Io-fold b y p e r v a p o r a t i o n . U n d i a l y s e d urine was s u b j e c t to interference b y salts during electrophoresis, a n d could n o t be a s s a y e d for a m y l a s e b y the Bernfeld Biochim. Biophys. Acta, 244 (1971) 441-451
445
ORIGIN OF SERUM AMYLASE
method due to very high blanks. The dialysed, concentrated urine required no further treatment prior to electrophoresis.
Parotid gland and pancreas Over 9o% of the amylase activity of these organs was solubilised by homogenisation in 0.2 M Tris-maleate, p H 8.0, containing I mM CaC12 and 0.02% Triton X - I o o 21. Because of the high amylase activity of these tissues no further treatment was necessary before electrophoresis. Small intestine Small intestine was washed out with ice-cold saline and the mucosa removed with a glass rod. The weighed mueosa was homogenised in 3 vol. of 0.25 M sucrose solution containing I mM CaC12, and the homogenate centrifuged at 105000 x g for 60 rain. The supernatant fraction was concentrated by ethanol precipitation and by pervaporation as described above. The particulate fraction was treated with Triton X - I o o to solubilise the amylase and subjected to the purification procedure described for liver amylase. RESULTS
Amylase activities of tissues of adult and infant rats Our results for the assay of amylase activities of crude homogenates of the pancreas, salivary glands, small intestinal mucosa and liver, and of the serum of adult and infant rats are presented in Table I I and Fig. i (see also Table I). In infant rats TABLE
]I
AMYLASE OF INFANT
ACTIVITIES AND
Mean values; number
Tissue
OF THE
ADULT
PANCREAS,
SALIVARY
GLANDS
AND
MUCOSA OF THE
of r a t s in p a r e n t h e s e s .
Amylase activity (units/g of fresh tissue) infant rats Days after birth." io 2o 3o
Salivary glands Parotid Submaxillary Major sublingual Pancreas Small intestine
SMALL INTESTINE
RATS
3.,5 (5) --1 0 3 0 0 (5) 38 (2)
27 (5)
Adult rats
2 8 0 0 (5) --
61oo (io) 1. 7 ( i o ) 2. 3 (IO)
--
--
7600
65 (2)
--
97
--
(io) (2)
the amylase activity of the pancreas was higher than that found in adult rats. By contrast, the parotid gland contained very low levels of amylase up to the 2oth day after birth, but these rapidly increased during the weaning period from the 2oth to 3oth days (Table II). The submaxillary and sublingual glands of adult rats contained very low levels of amylase as compared with the parotid gland. There was a three-fold increase in both serum and liver amylase activities during the first 24 h after birth (Fig. i). This was followed by a decrease in activity in both tissues during the next few days. After the i o t h day, the levels of amylase activity in both liver and serum gradually increased to adult levels. Biochim. Biophys. Acta, 2 4 4 (1971) 4 4 1 - 4 5 1
446
K. HAMMERTON, M. MESSER
E 2
m
,}
~4
tw iI
> t
CL ol
>~ > 0
< 50~'ado~t Age (days) Fig. i. Changes in the levels of r a t liver and s e r u m amylase activities during p o s t n a t a l developmeut. E a c h point is the m e a n value obtained using four litters; livers and sera, respectively, of individuals of each litter were pooled. The bars indicate the range of values obtained for each age group. ~?, liver; Q, serum.
Amylase isoenzymes of adult rat tissues A comparison of the patterns of amylase isoenzymes obtained after cellulose acetate electrophoresis of extracts of the pancreas, parotid gland, partially purified liver, and of partially purified serum of adult rats is presented in Fig. 2. The pancreas exhibited three bands of activity located on the cathodic side of the origin. The presence of two major and one minor amylase isoenzyme in rat pancreas has been previously observed by POORT AND VAN VENROOY 18, who used agar gel electrophoresis. The parotid gland showed 5-6 amylase bands migrating toward the anode. Fresh parotid gland extract showed one major and four minor bands, whereas extracts which had been stored at 4 ° for several days exhibited the two major and four minor bands illustrated in Fig. 2. Five isoamylases have been observed by WOLF AND TAYLOR~2 after acrylamide gel electrophoresis of rat parotid saliva. The partially purified liver preparation exhibited one major band corresponding to the least anodic major band of the parotid, plus three minor bands which also corresponded to those of the parotid. The liver pattern differed from that of the parotid in that it did not show any bands corresponding to the two least anodic parotid isoenzymes. No amylase of the pancreatic type could be detected. (The possibility was considered that the absence of pancreatic type amylase from the liver might be due to selective inactivation of this type during the solubilisation or purification procedure. However when pancreatic extract was added to the particulate liver fraction and the mixture carried through the purification procedure, the pancreatic type could be easily detected). Electrophoretic patterns for liver amylase identical with those in Fig. 2 were obtained when the amylase had been solubilised with either Triton X-Ioo or digitonin and through all stages of the purification procedure. Partially purified serum amylase preparations gave electrophoretic patterns which were similar to those of the liver, except that they contained an extra minor anodic isoenzyme. This isoenzyme, however, appeared only after the final step in the purification procedure, i.e. after gel filtration. Biochim. Biophys. Acta, 244 (1971) 44t-451
ORIGIN OF SERUM AMYLASE
[
HII
,o,[ HHIIHH I
447
H IHI
I
I' I
Fig. 2. (a) Electrophoretic separation of isoamylases of rat tissues. I, pancreas ; 2, parotid gland ; 3, liver; 4, serum. (b) Diagrammatic representation of the isoamylase patterns of (a) observed during fixation of the starch agar plates (see METHODS). Urinary and liver isoamylases were electrophoretically identical, both exhibiting the four bands of the parotid type shown in Fig. 2. Thus, rat urine did not contain a n y amylase of the pancreatic type, nor did it contain the sixth most anodic b a n d which appeared in serum after gel filtration. In the mucosa of the small intestine, 60% of the amylase activity was found in the supernatant fraction following ultracentrifugation (mean value for 2 rats). This fraction contained mainly pancreatic t y p e amylase, but also showed some of the parotid type. The solubilised particulate fraction consisted almost wholly of the pancreatic type, a trace only of the parotid type being observed.
Effect of ligature of the common bile duct on serum and urinary isoamylases Since no amylase of the pancreatic type was found in normal rat serum or urine, it was of interest to discover whether pancreatic amylase would appear under conditions resembling h u m a n pancreatitis. To this end a ligature was applied to the Biochim. Biophys. Acta, 244 (1971) 441-451
448
K. HAMMERTON, M. MESSER
common bile duct using the "procedure B" of HERRIOTT AND PALMER~a*. Samples of urine were collected over a period of 12 h after which the animals were killed. Their sera were then collected and examined electrophoretically without prior purification. No changes were observed in urinary amylase I h after application of the ligature (Fig. 3); after 4 h, however, amylase of the pancreatic type appeared in the urine, which increased progressively at 8 and 12 h. No amylase of the pancreatic type was seen in the urine of animals on which sham operations had been performed. After 12 h the serum contained a significant amount of amylase of the pancreatic type, in addition to the parotid type seen in normal serum. These results show that pancreatic amylase can enter the circulation under abnormal conditions (cf. MIHOL6I~ AND VE£1EREK24).
Fig. 3- E l e c t r o p h o r e t i c s e p a r a t i o n of i s o a m y l a s e s of s e r u m a n d uri ne a t different t i m e s a f t e r l i g a t u r e of t h e c o m m o n bile duct. A, B, C, D: S e r u m o b t a i n e d i, 4, 8 a n d I2 h, r e s p e c t i v e l y , a f t e r l i g a t u r e . E : S e r u m o b t a i n e d I2 h a f t e r l i g a t u r e . F, G: Urine a n d serum, r e s p e c t i v e l y , o b t a i n e d i2 h a f t e r s h a m o p e r a t i o n .
Amylase isoenzymes of infant rat tissues The electrophoretic isoarnylase patterns of the pancreas and parotid glands of pre-weaning rats were found to be same as those of adult rats. However, electrophoresis of urine and partially purified liver and serum of newborn and 1-day-old rats showed that these tissues contained both the pancreatic and parotid type amylases (Fig. 4). The pancreatic type had almost disappeared in 2-day-old rats and was not present in the liver, serum and urine of 3-day-old rats. Thus, the electrophoretic patterns of the liver, serum and urine remained unchanged from the third day after birth. The small intestinal mucosa of Io-day -old rats differed from that of adult rats in that the supernatant and particulate fractions contained only amylase of the pancreatic type, the parotid type being absent. Since the parotid glands of io-day-old rats contained very little amylase as compared with those of adult rats (Table II), this difference suggests that the presence of parotid type amylase in the small intestine of adult rats m a y be the result of adsorption of salivary amylase derived from the parotid gland 25. * L i g a t u r e of t h e c o m m o n bile d u c t p r o d u c e s s i m u l t a n e o u s p a n c r e a t i c a n d b i l i a r y o b s t r u c t i o n since in t h e r a t t h e p a n c r e a t i c d u c t s e n t e r t h e c o m m o n bile d u c t a l ong its course.
Biochim. Biophys. Acta, 244 (1971) 441 4,51
ORIGIN OF SERUM AMYLASE
449
Fig. 4- Changes in the isoamylase p a t t e r n s of rat liver and s e r u m during the first 3 days after birth, i, 3: Livers from 1-day-old and 3-day-old rats, respectively. 2, 4: Serum from 1-day-old a n d 3-day-old rats, respectively. DISCUSSION
In this work we have compared the electrophoretic isoamylase patterns of various tissues of the rat to obtain information on the origin of serum amylase. Similar comparisons have been made previously 7, but these did not include the liver, presumably because studies of liver amylase presented special difficulties. Liver amylase is localised mainly in the microsomal fraction 26 and therefore needs to be solubilised. The recent finding of ~{ORDOH et al. 15 that rat liver amylase can be solubilised with detergent made it possible to overcome this difficulty. Furthermore the liver enzyme is present in low concentrations as compared with pancreatic, parotid and small intestinal amylases, and it must therefore be partially purified before electrophoresis to prevent interference with isoamylase patterns by non-amylase proteins. (This problem is also encountered with serum). In our studies the pancreatic and parotid amylases of the rat could be readily differentiated since they moved in opposite directions on the cellulose acetate strip. By contrast, various workers*,7, 27-29 have found that the isoamylases of human saliva and pancreas differ only slightly in mobility. Nevertheless human serum and urinary amylases could be separated into two fractions, one of slower cathodic mobility being of the salivary type, while the other was of the pancreatic type. Serum amylase activity of the pancreatic type was found to be increased in acute pancreatitis, while amylase activity of the salivary type was increased in cases of mumps29, ~. These observations supported the view that normal human serum amylase is of both pancreatic and salivary origin. Our results definitely exclude the pancreas as a source of serum amylase in the normal rat since no amylase of the pancreatic type was found in rat serum or urine, Biochim. Biophys..4cta, 244 (1971) 441-451
45 °
K. HAMMERTON, M. MESSER
except after ligature of the common bile duct. Similarly, the small intestine is unlikely to be a source of serum amylase al since the intestinal mucosa contained predominantly (adult rats) or exclusively (infant rats) amylase of the pancreatic type. Since rat serum and urine contained amylase of the parotid type only, it is possible that the parotid gland is a source of serum amylase. Although the isoamylase patterns of the serum and parotid differed insofar as the former lacked the two least anodic parotid bands, this difference was not sufficient to permit a clear differentiation between them. However, our studies on infant rats tend to exclude the salivary glands as a source of serum amylase. The parotid glands of preweanling rats contained very little amylase as compared with those of adult rats*. Despite the hundred-fold increase in the amylase content of the parotid gland which occurred between the 2oth and 3oth days after birth (Table II), the level of serum amylase remained almost constant over this period. On the other hand, several observations implicate the liver as an important source of serum amylase in the rat. The electrophoretic isoamylase patterns of the serum and urine were identical with those of the liver in rats of all ages. In newborn and 1-day-old rats, the liver contained both the parotid and the pancreatic types of amylase isoenzymes. The latter disappeared by the third day, after which the liver isoenzyme pattern was the same as that found in adult rats. This striking change in the liver isoenzyme patterns during the immediate postnatal period was mirrored in the serum and urine which, like the liver, contained the pancreatic type on the first but not the third day after birth, or thereafter. Furthermore, there was an increase in the levels of both serum and liver amylase levels during the first 24 h after birth, followed by a marked decrease on the third day. Thereafter the levels gradually increased to adult levels. The present results, together with previous studies showing that the perfused liver synthesises and secretes amylase into the perfusate 3-5 support the view that the liver is the sole or major source of serum amylase in the rat. The data of other workers on the amylases of human tissues ~8-3° make it hazardous to generalise from the rat to other animal species. Preliminary studies with tissues from the mouse, guinea pig, rabbit, cat and dog suggest, however, that liver and serum isoamylases are electrophoretically identical in these animals a3. The possibility remains that skeletal muscle, adipose tissue, the kidneys or other organs contribute to normal levels of serum amylase. In the absence of evidence that such organs are active in synthesisingamylase the liver remains the most likely source. ACKNOWLEDGEMENTS
This work was supported by a grant from the Australian Research Grants Committee. K.H. is a recipient of a Commonwealth Postgraduate Research Studentship.
* The o b s e r v a t i o n t h a t the parotid gland oi infant r a t s contained very little amylase does not a p p e a r to h a v e been r e p o r t e d previously, b u t is consistent with the work of REDMAN AND SCREEBX¥ 32 who, using morphological criteria, observed a rapid m a t u r a t i o n of the parotid gland cells between 18 and 2 5 d a y s after birth, i.e. during the w e a n i n g period.
Biochim. Biophys. Mcta, 24-4 (I97 l) 441-451
ORIGIN OF SERUM AMYLASE
45I
REFERENCES i M. SOMOGYI Arch. Intern. Med., 67 (1941) 665. 2 H . D . JANOWITZ AND D . A . DREILING, A m . . ] . ~/Ied., 27 (1959) 924.
3 4 5 6 7 8 9 io Ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33
R. L. McGEACHIN, B. A. POTTER AND A. DESPOPOULOS, Arch. Biochem. Biophys., 90 (196o) 319. R. L. McGEACHIN, B. A. POTTER AND A. DESPOPOULOS, Arch. Biochem. Biophys., 98 (1962) 89. M. ARNOLD AND W. J. RUTTER, J. Biol. Chem., 238 (1963) 2760. J. IfAMAR~T AND R. LAXOV~-, Plumangenetik, I (1965) 579. S. E. Aw, J. R. HOBBS AND I. D. P. WOOTTON, Gut, 8 (1967) 4o2. H. SICK AND J. T. NIELSEN, Hereditas, 51 (1964) 291. G. M. MALAClNSKI AND W. J. RUTTER, Biochemistry, 8 (1969) 4392. R. R. JOSEPH, E. OLIVERO AND •. RESSLER, Gastroenterology, 51 (1966) 377. J. O. O. HOEKE, C. YOORRIPS AND J. DE WAEL, Clin. Chim. Acta, 16 (1967) 171. N. RESSLER, Clin. Chim. Acta, 17 (1967) 307. P. BERNFELD in S. P. COLOWICK AND N. O. I{APLAN, Methods in En~ymology, Vol. I, Academic Press, New York, 1955, p. 149D. H. STRUMEYER Anal. Biochem., 19 (1967) 61. J. MORDOH, C. R. HRISMAN, A. J. PARODI AND L. F. LELOIR, Arch. Biochem. Biophys., 127 (1968) 193. O. ]7I. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. E. THEODOR AND D. BIRNBAUM, ,J. Lab. Clin. Med., 63 (1964) 879. C. POORT AND ~V. J. W. VAN VENROOY, Nature, 204 (1964) 684. N. CASTAGNA,Nature, 205 (1965) 905. P. WILDING, Clin. Chim. Acta, 8 (1963) 918. J. L. VAN LENCKER AND R. L. HOLTZER, J. Biol. Chem., 234 (1959) 2359. R. O. WOLF AND L. L. TAYLOR, Nature, 213 (1967) 1128. B. A. HERRIOTT AND A. A. PALMER, Australian J. Exp. Biol., 42 (1964) 27. M. MIHOL~I~ AND B. VE(~EREK, Clin. Chim. Acta, 16 (1964) 27. A. M. UGOLEV, Physiol. Rev., 45 (1965) 555. R. W. BROSEMER AND W. J. RUTTER, J. Biol. Chem., 236 (1961) 1253. S. IqORBY, Exptl. Cell Research, 36 (1964) 663. J. E. ]DERK, S. ]-IAYASHI, R. L. SEARCY AND N. C. I-IIGHTOWER, Am. J. Dig. Dis., I I (1966) 695. F. A. DE LA LANDE AND B. BOETTCHER, Enzymologia, 37 (1969) 335. S. E. Aw, Nature, 2c9 (1966) 298. lXT.HIATT, Ann. Surg., 154 (1961) 864. R. S. REDMAN AND L. N. SCREEBNY, J. Cell Biol., 46 (197 o) 81. H. HAMMERTON AND M. MESSER, Proc. Australian Biochem. Soc., 3 (197 o) 15.
Biochim. Biophys. Acta, 244 (1971) 441-451
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26629
T H E O R I G I N OF SERUM AMYLASE. E L E C T R O P H O R E T I C S T U D I E S OF ISOAMYLASES OF T H E SERUM, L I V E R AND O T H E R TISSUES OF A D U L T AND I N F A N T RATS
KERIE
H A M M E R T O N A~XDM I C H A E L M E S S E R
Department of Biochemistry, The University of Sydney, N.S.W. 2oo6 (Australia) (Received M a r c h 5th, 1971)
SUMMARY
To obtain information on the tissue origin of serum amylase, partially purified preparations of rat liver and serum amylase and extracts of various tissues were subjected to cellulose acetate electrophoresis followed b y detection of the amylolytic activity on starch-agar plates. A method is described for the 6oo-fold purification of detergent-solubilised liver amylase. The pancreas showed three bands of amylase activity migrating towards the cathode, while the parotid gland revealed five to six anodic bands. In adult rats, the serum and urine showed isoamylase patterns which were the same as those of the liver, and similar to those of the parotid. Isoamylases of the pancreatic type appeared in the serum and urine only after ligation of the common bile duct. In newborn rats, the serum and liver contained isoamylases of the pancreatic as well as parotid type but the pancreatic type disappeared from both serum and liver b y the third day after birth. There was a Ioo-fold increase in the levels of parotid gland amylase after weaning, which was not reflected in the levels of serum amylase. These results support the view that the liver is the major source of serum amylase in the normal rat.
INTRODUCTION
Although it has been known for over a century that human serum contains an amylolytic enzyme which is excreted in tile urine, the tissue origin of this enzyme remains in doubt. In 1941 , SOMOGYI1 concluded that under normal physiological conditions neither of the two chief amylase synthesising organs, namely the pancreas and the salivary glands, could be implicated. This subject was again investigated and reviewed in 1959 b y J A N O W l T Z AND D R E I L I N G ~, who postulated that the total amylolytic activity of the blood is the sum of the activities of several a-amylases of diverse origin, including the pancreas, salivary glands, liver and possibly also the fallopian tubes, striated muscle and adipose tissue. More recently, McGEACHIN et al.~, 4 and ARNOLD AND RUTTER5 made the imBiochim. Biophys. Acta, 244 (1971) 441-451
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K. HAMMERTON, M. MESSER
portant observation that a-amylase is synthesised by the liver of the rat, and that the isolated liver secretes amylase into the perfusion medium. Therefore these authors suggested that in the normal rat the liver is a source of serum amylase, but were unable definitely to conclude that the liver is the sole or major source or to exclude either the pancreas or the salivary glands as possible contributary sources. Recent studies demonstrating the existence of electrophoretic variants of amylase that are tissue specific suggested a promising approach to elucidating the origin of serum amylase. In the mammalian species that have been investigated, including man6, 7, mouse 8 and rabbit" the amylases of the pancreas and salivary glands could be separated from each other by agar, acrylamide or cellulose acetate electrophoresis. Human serum and urine have been shown to contain two electrophoretic fractions corresponding to the salivary and pancreatic types, respectively 7. In view of these studies the question whether liver amylase has unique electrophoretic properties is relevant to the problem of the origin of serum amylase. This question has remained unresolved 1°- 12. In our work the electrophoretic isoamylase patterns exhibited by serum, urine, the liver, parotid gland, pancreas and small intestine have been studied and compared in both adult and infant rats. METHODS
Amylase assay Amylase activity was determined by the method of BERNFELD et al. 13 using the stable starch substrate solution recently described by STRUMEYER14. For the determination of liver amylase, the assay mixture contained o . i % Triton X-Ioo detergent as activator 15. One unit of amylase activity is defined as i.o/,mole of maltose equivalent liberated from starch per minute at 20 ° . Specific amylase activity was determined using the method of LOWRY et al. le for tile estimation of protein, bovine serum albumin being used as the standard. It has been observed by THEODORAND BIRNBAUM17 that human serum and hog pancreatic amylase activities increase on dilution. A similar "dilution effect" was observed in the present work with rat amylases. Electrophoretic separation of amylase isoenzymes This was done on cellulose acetate with the Beckman Microzone Model i o i electrophoresis apparatus. The buffer solution used was 0.02 M sodium potassium phosphate, pH 7.5, containing 0.02% NaC1. To obtain best results it was found important to de-aerate the cellulose acetate strip by placing it on the buffer solution under a low vacuum for IO rain, after which it was blotted to remove excess solution and placed in the electrophoresis apparatus. A total of 0.75/*1 of each sample was placed on the strip with the 0.25 #1 applicator. Electrophoresis was done at room temperature using a constant voltage of 200 V for 9° rain. The amylolytic bands were detected by placing the cellulose acetate strip on a starch-agar plate 18which was then maintained at 37 ° for 30-60 rain. After removal of the strip the plate was developed by immersion in 70% ethanol containing 5% acetic acid followed by a solution of I2 in o.I M KI. The electrophoretic patterns were photographed over a black velvet cloth using high contrast film. Slides to be photographed were not stained with I2. Biochim. Biophys. Acta, 244 (1971) 441-451
ORIGIN OF SERUM AMYLASE
443
As noted by POORT AND VAN VENROOY TM,precipitation of the starch by ethanolacetic acid begins at the surface of the starch-agar film and continues downwards with increasing fixation time. A short fixation time therefore reveals minor bands which disappear on prolonged fixation; on the other hand, longer fixation permits a more exact localisation of the major bands. In some of the photographs presented in this paper, the fixation time was such as to show up the major bands to maximum advantage; we have therefore supplemented these photographs with diagrams illustrating the minor bands. The amylolytic bands obtained on electrophoresis of solubilised amylase from a crude liver homogenate, or of untreated serum, were found to be distorted or displaced by other proteins present in the tissue preparation. For this reason rat liver and serum amylases had to be partially purified before electrophoresis (see below).
Partial purification of rat liver amylase Male Wistar rats were anaesthetised with ether and thoroughly bled from the jugular vein in order to reduce the blood content of the livers 1~. During removal of the livers extreme care had to be taken to prevent contamination with pancreatic tissue. The organs were homogenised in 3 vol. (v/w) of 0.25 M sucrose containing I mM CaCI~, and the homogenate centrifuged at 105000 × g for 60 rain. Of the total liver amylase, between 70 and 90% was found to be localised in the particulate fraction following ultracentrifugation. The remaining amylase in the supernatant fraction was discarded on the assumption that this fraction would contain all the amylase derived from blood contained in the liver, since it could be calculated from the experimentally determined mean level of amylase in blood (2.6 units/ml) and from an assumed value of 9 ml of blood per IOO g of liver from a well-bled rat 19, that of the total liver amylase activity, 7% would be due to serum amylase. In three separate trials the liver of an anaesthetised rat was perfused with 0.25 M sucrose solution via a catheter inserted into the inferior vena cava, in order to remove all the blood contained in the vascular system. The perfusate was collected from the portal vein and perfusion was continued under hydrostatic pressure until the liver had attained a bloodless appearance (500 ml). It was found that the supernatant fraction of homogenates of perfused livers contained only between IO and 40% of the amylase activity of the supernatant fraction of unperfused livers, while the particulate fraction contained between 50 and 80% of that of unperfused livers. Therefore perfusion resulted in a considerable loss of tile liver amylase, and for this reason the livers used for the purification were not perfnsed. The particulate fraction was resuspended in 6 vol. (w/v, with respect to the weight of fresh tissue) of 20 mM Tris-maleate, pH 7.0, containing 20 mM CaCI~, 20 mM NaC1 and either o.I% Triton X-Ioo or 0.4% digitonin, and allowed to stand at 5 ° for 20 h. The preparation was then incubated at 37 ° for 30 rain. This treatment, which solubilised at least 80% of the particulate amylase, is a modification of the solubilisation procedure of MORDOH et al. ~5. It was found that the degree of solubilisation depended on the protein concentration of the suspended particulate fraction; for maximum solubilisation this had to be less than 20 mg/ml. The solubilised enzyme was dialysed for 12 h against 50 mM Tris-maleate, pH 7.0, containing 20 mM CaC12 and 20 mM NaC1; any sediment which formed was removed. 2 vol. of ethanol (previously chilled to - - 2 0 °) were then added slowly under Biochim. Biophys. Acta, 244 (1971) 441 451
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K. HAMMERTON, M. MESSER
stirring to t h e ice-cold solution. The m i x t u r e was allowed to s t a n d for 3o min in an ice b a t h a n d t h e n centrifuged at IOOOO × g for 2o min. The s e d i m e n t was s u s p e n d e d in 8o ml of i o mM T r i s - m a l e a t e , p H 7.o, c o n t a i n i n g IO mM CaC12 a n d IO mM NaC1, a n d allowed to s t a n d at 5 ° for 12 h. The suspension was centrifuged at IOOOO ~< g for 3o min a n d the s u p e r n a t a n t solution d i a l y s e d a g a i n s t 2 mM T r i s - m a l e a t e , p H 7.o, c o n t a i n i n g 2 mM CaC12 a n d 2 mM NaC1. The d i a l y s e d solution was c o n c e n t r a t e d to a p p r o x i m a t e l y o n e - q u a r t e r its volume b y p e r v a p o r a t i o n from the dialysis b a g in front of a fan. The e n z y m e solution was t h e n s u b j e c t e d to gel filtration on a column of Sephadex G - I o o (5o em × 2.5 cm) ~°. The elution was done w i t h o . 9 % NaC1 at a flow r a t e of lO-2O ml/h. The p r o t e i n c o n t e n t of the fractions (4.o ml) was m e a s u r e d b y a b s o r b a n c e at 28o nm. Over 9 o % of t h e t o t a l p r o t e i n was e l u t e d in the first IOO ml following the v o i d v o l u m e ; the a m y l a s e a c t i v i t y a p p e a r e d as a b r o a d a s y m m e t r i c a l p e a k in the second IOO ml. The fractions c o n t a i n i n g a m y l a s e a c t i v i t y were pooled, d i a l y s e d against I mM T r i s - m a l e a t e , p H 7.o, c o n t a i n i n g I mM CaC12 a n d I mM NaC1, a n d c o n c e n t r a t e d to a p p r o x i m a t e l y o n e - t e n t h t h e i r p o o l e d v o l u m e b y p e r v a p o r a t i o n . The purified e n z y m e solution was s t a b l e when s t o r e d for u p to t h r e e weeks at 5 ° . TABLE 1 PARTIAL PURIFICATION
OF RAT LIVER AMYLASE
Volume (ml) Crude homogenate 750 105000 xg pellet 15°0 AfterTritonX-ioosoluhilisation 141o After ethanol fractionation 18 After Sephadex G-ioo filtration 27
Total protein (rag)
Total amylase activity (units)
Specific amylase activity (units/ mg protein)
Purification factor
49200 365 °0 11 200 2050 74
625
O.Ol3 o.o13 0.040 0.23 8.3
I i 3 18 640
475 447 47 ° 613
Table I s u m m a r i s e s a t y p i c a l purification of a m y l a s e from 30 r a t livers, in which a 64o-fold purification was achieved. The t a b l e indicates an a p p a r e n t increase in t o t a l a m y l a s e a c t i v i t y after S e p h a d e x G - I o o filtration. A possible e x p l a n a t i o n for this a n o m a l o u s result is the " d i l u t i o n effect" p r e v i o u s l y n o t e d (see METHODS), as a result of which strict c o m p a r i s o n s b e t w e e n a m y l a s e activities of different p r e p a r a t i o n s are difficult if n o t impossible. A l t e r n a t i v e l y , the result could be due to the r e m o v a l of an a m y l a s e i n h i b i t o r d u r i n g gel filtration. Partial purification of serum amylase R a t b l o o d o b t a i n e d b y cardiac p u n c t u r e was allowed to clot a n d t h e n centrifuged. The s e r u m was t r e a t e d with e t h a n o l a n d p a r t i a l l y purified on S e p h a d e x G - I o o as described for tile liver. A 4oo-fold purification of sertun a m y l a s e was o b t a i n e d . Urine Urine collected from r a t s m a i n t a i n e d in m e t a b o l i s m cages was d i a l y s e d a g a i n s t 2 mM T r i s - m a l e a t e , p H 7.0, c o n t a i n i n g 2 mM CaC12 a n d 2 mM NaC1 a n d t h e n conc e n t r a t e d Io-fold b y p e r v a p o r a t i o n . U n d i a l y s e d urine was s u b j e c t to interference b y salts during electrophoresis, a n d could n o t be a s s a y e d for a m y l a s e b y the Bernfeld Biochim. Biophys. Acta, 244 (1971) 441-451
445
ORIGIN OF SERUM AMYLASE
method due to very high blanks. The dialysed, concentrated urine required no further treatment prior to electrophoresis.
Parotid gland and pancreas Over 9o% of the amylase activity of these organs was solubilised by homogenisation in 0.2 M Tris-maleate, p H 8.0, containing I mM CaC12 and 0.02% Triton X - I o o 21. Because of the high amylase activity of these tissues no further treatment was necessary before electrophoresis. Small intestine Small intestine was washed out with ice-cold saline and the mucosa removed with a glass rod. The weighed mueosa was homogenised in 3 vol. of 0.25 M sucrose solution containing I mM CaC12, and the homogenate centrifuged at 105000 x g for 60 rain. The supernatant fraction was concentrated by ethanol precipitation and by pervaporation as described above. The particulate fraction was treated with Triton X - I o o to solubilise the amylase and subjected to the purification procedure described for liver amylase. RESULTS
Amylase activities of tissues of adult and infant rats Our results for the assay of amylase activities of crude homogenates of the pancreas, salivary glands, small intestinal mucosa and liver, and of the serum of adult and infant rats are presented in Table I I and Fig. i (see also Table I). In infant rats TABLE
]I
AMYLASE OF INFANT
ACTIVITIES AND
Mean values; number
Tissue
OF THE
ADULT
PANCREAS,
SALIVARY
GLANDS
AND
MUCOSA OF THE
of r a t s in p a r e n t h e s e s .
Amylase activity (units/g of fresh tissue) infant rats Days after birth." io 2o 3o
Salivary glands Parotid Submaxillary Major sublingual Pancreas Small intestine
SMALL INTESTINE
RATS
3.,5 (5) --1 0 3 0 0 (5) 38 (2)
27 (5)
Adult rats
2 8 0 0 (5) --
61oo (io) 1. 7 ( i o ) 2. 3 (IO)
--
--
7600
65 (2)
--
97
--
(io) (2)
the amylase activity of the pancreas was higher than that found in adult rats. By contrast, the parotid gland contained very low levels of amylase up to the 2oth day after birth, but these rapidly increased during the weaning period from the 2oth to 3oth days (Table II). The submaxillary and sublingual glands of adult rats contained very low levels of amylase as compared with the parotid gland. There was a three-fold increase in both serum and liver amylase activities during the first 24 h after birth (Fig. i). This was followed by a decrease in activity in both tissues during the next few days. After the i o t h day, the levels of amylase activity in both liver and serum gradually increased to adult levels. Biochim. Biophys. Acta, 2 4 4 (1971) 4 4 1 - 4 5 1
446
K. HAMMERTON, M. MESSER
E 2
m
,}
~4
tw iI
> t
CL ol
>~ > 0
< 50~'ado~t Age (days) Fig. i. Changes in the levels of r a t liver and s e r u m amylase activities during p o s t n a t a l developmeut. E a c h point is the m e a n value obtained using four litters; livers and sera, respectively, of individuals of each litter were pooled. The bars indicate the range of values obtained for each age group. ~?, liver; Q, serum.
Amylase isoenzymes of adult rat tissues A comparison of the patterns of amylase isoenzymes obtained after cellulose acetate electrophoresis of extracts of the pancreas, parotid gland, partially purified liver, and of partially purified serum of adult rats is presented in Fig. 2. The pancreas exhibited three bands of activity located on the cathodic side of the origin. The presence of two major and one minor amylase isoenzyme in rat pancreas has been previously observed by POORT AND VAN VENROOY 18, who used agar gel electrophoresis. The parotid gland showed 5-6 amylase bands migrating toward the anode. Fresh parotid gland extract showed one major and four minor bands, whereas extracts which had been stored at 4 ° for several days exhibited the two major and four minor bands illustrated in Fig. 2. Five isoamylases have been observed by WOLF AND TAYLOR~2 after acrylamide gel electrophoresis of rat parotid saliva. The partially purified liver preparation exhibited one major band corresponding to the least anodic major band of the parotid, plus three minor bands which also corresponded to those of the parotid. The liver pattern differed from that of the parotid in that it did not show any bands corresponding to the two least anodic parotid isoenzymes. No amylase of the pancreatic type could be detected. (The possibility was considered that the absence of pancreatic type amylase from the liver might be due to selective inactivation of this type during the solubilisation or purification procedure. However when pancreatic extract was added to the particulate liver fraction and the mixture carried through the purification procedure, the pancreatic type could be easily detected). Electrophoretic patterns for liver amylase identical with those in Fig. 2 were obtained when the amylase had been solubilised with either Triton X-Ioo or digitonin and through all stages of the purification procedure. Partially purified serum amylase preparations gave electrophoretic patterns which were similar to those of the liver, except that they contained an extra minor anodic isoenzyme. This isoenzyme, however, appeared only after the final step in the purification procedure, i.e. after gel filtration. Biochim. Biophys. Acta, 244 (1971) 44t-451
ORIGIN OF SERUM AMYLASE
[
HII
,o,[ HHIIHH I
447
H IHI
I
I' I
Fig. 2. (a) Electrophoretic separation of isoamylases of rat tissues. I, pancreas ; 2, parotid gland ; 3, liver; 4, serum. (b) Diagrammatic representation of the isoamylase patterns of (a) observed during fixation of the starch agar plates (see METHODS). Urinary and liver isoamylases were electrophoretically identical, both exhibiting the four bands of the parotid type shown in Fig. 2. Thus, rat urine did not contain a n y amylase of the pancreatic type, nor did it contain the sixth most anodic b a n d which appeared in serum after gel filtration. In the mucosa of the small intestine, 60% of the amylase activity was found in the supernatant fraction following ultracentrifugation (mean value for 2 rats). This fraction contained mainly pancreatic t y p e amylase, but also showed some of the parotid type. The solubilised particulate fraction consisted almost wholly of the pancreatic type, a trace only of the parotid type being observed.
Effect of ligature of the common bile duct on serum and urinary isoamylases Since no amylase of the pancreatic type was found in normal rat serum or urine, it was of interest to discover whether pancreatic amylase would appear under conditions resembling h u m a n pancreatitis. To this end a ligature was applied to the Biochim. Biophys. Acta, 244 (1971) 441-451
448
K. HAMMERTON, M. MESSER
common bile duct using the "procedure B" of HERRIOTT AND PALMER~a*. Samples of urine were collected over a period of 12 h after which the animals were killed. Their sera were then collected and examined electrophoretically without prior purification. No changes were observed in urinary amylase I h after application of the ligature (Fig. 3); after 4 h, however, amylase of the pancreatic type appeared in the urine, which increased progressively at 8 and 12 h. No amylase of the pancreatic type was seen in the urine of animals on which sham operations had been performed. After 12 h the serum contained a significant amount of amylase of the pancreatic type, in addition to the parotid type seen in normal serum. These results show that pancreatic amylase can enter the circulation under abnormal conditions (cf. MIHOL6I~ AND VE£1EREK24).
Fig. 3- E l e c t r o p h o r e t i c s e p a r a t i o n of i s o a m y l a s e s of s e r u m a n d uri ne a t different t i m e s a f t e r l i g a t u r e of t h e c o m m o n bile duct. A, B, C, D: S e r u m o b t a i n e d i, 4, 8 a n d I2 h, r e s p e c t i v e l y , a f t e r l i g a t u r e . E : S e r u m o b t a i n e d I2 h a f t e r l i g a t u r e . F, G: Urine a n d serum, r e s p e c t i v e l y , o b t a i n e d i2 h a f t e r s h a m o p e r a t i o n .
Amylase isoenzymes of infant rat tissues The electrophoretic isoarnylase patterns of the pancreas and parotid glands of pre-weaning rats were found to be same as those of adult rats. However, electrophoresis of urine and partially purified liver and serum of newborn and 1-day-old rats showed that these tissues contained both the pancreatic and parotid type amylases (Fig. 4). The pancreatic type had almost disappeared in 2-day-old rats and was not present in the liver, serum and urine of 3-day-old rats. Thus, the electrophoretic patterns of the liver, serum and urine remained unchanged from the third day after birth. The small intestinal mucosa of Io-day -old rats differed from that of adult rats in that the supernatant and particulate fractions contained only amylase of the pancreatic type, the parotid type being absent. Since the parotid glands of io-day-old rats contained very little amylase as compared with those of adult rats (Table II), this difference suggests that the presence of parotid type amylase in the small intestine of adult rats m a y be the result of adsorption of salivary amylase derived from the parotid gland 25. * L i g a t u r e of t h e c o m m o n bile d u c t p r o d u c e s s i m u l t a n e o u s p a n c r e a t i c a n d b i l i a r y o b s t r u c t i o n since in t h e r a t t h e p a n c r e a t i c d u c t s e n t e r t h e c o m m o n bile d u c t a l ong its course.
Biochim. Biophys. Acta, 244 (1971) 441 4,51
ORIGIN OF SERUM AMYLASE
449
Fig. 4- Changes in the isoamylase p a t t e r n s of rat liver and s e r u m during the first 3 days after birth, i, 3: Livers from 1-day-old and 3-day-old rats, respectively. 2, 4: Serum from 1-day-old a n d 3-day-old rats, respectively. DISCUSSION
In this work we have compared the electrophoretic isoamylase patterns of various tissues of the rat to obtain information on the origin of serum amylase. Similar comparisons have been made previously 7, but these did not include the liver, presumably because studies of liver amylase presented special difficulties. Liver amylase is localised mainly in the microsomal fraction 26 and therefore needs to be solubilised. The recent finding of ~{ORDOH et al. 15 that rat liver amylase can be solubilised with detergent made it possible to overcome this difficulty. Furthermore the liver enzyme is present in low concentrations as compared with pancreatic, parotid and small intestinal amylases, and it must therefore be partially purified before electrophoresis to prevent interference with isoamylase patterns by non-amylase proteins. (This problem is also encountered with serum). In our studies the pancreatic and parotid amylases of the rat could be readily differentiated since they moved in opposite directions on the cellulose acetate strip. By contrast, various workers*,7, 27-29 have found that the isoamylases of human saliva and pancreas differ only slightly in mobility. Nevertheless human serum and urinary amylases could be separated into two fractions, one of slower cathodic mobility being of the salivary type, while the other was of the pancreatic type. Serum amylase activity of the pancreatic type was found to be increased in acute pancreatitis, while amylase activity of the salivary type was increased in cases of mumps29, ~. These observations supported the view that normal human serum amylase is of both pancreatic and salivary origin. Our results definitely exclude the pancreas as a source of serum amylase in the normal rat since no amylase of the pancreatic type was found in rat serum or urine, Biochim. Biophys..4cta, 244 (1971) 441-451
45 °
K. HAMMERTON, M. MESSER
except after ligature of the common bile duct. Similarly, the small intestine is unlikely to be a source of serum amylase al since the intestinal mucosa contained predominantly (adult rats) or exclusively (infant rats) amylase of the pancreatic type. Since rat serum and urine contained amylase of the parotid type only, it is possible that the parotid gland is a source of serum amylase. Although the isoamylase patterns of the serum and parotid differed insofar as the former lacked the two least anodic parotid bands, this difference was not sufficient to permit a clear differentiation between them. However, our studies on infant rats tend to exclude the salivary glands as a source of serum amylase. The parotid glands of preweanling rats contained very little amylase as compared with those of adult rats*. Despite the hundred-fold increase in the amylase content of the parotid gland which occurred between the 2oth and 3oth days after birth (Table II), the level of serum amylase remained almost constant over this period. On the other hand, several observations implicate the liver as an important source of serum amylase in the rat. The electrophoretic isoamylase patterns of the serum and urine were identical with those of the liver in rats of all ages. In newborn and 1-day-old rats, the liver contained both the parotid and the pancreatic types of amylase isoenzymes. The latter disappeared by the third day, after which the liver isoenzyme pattern was the same as that found in adult rats. This striking change in the liver isoenzyme patterns during the immediate postnatal period was mirrored in the serum and urine which, like the liver, contained the pancreatic type on the first but not the third day after birth, or thereafter. Furthermore, there was an increase in the levels of both serum and liver amylase levels during the first 24 h after birth, followed by a marked decrease on the third day. Thereafter the levels gradually increased to adult levels. The present results, together with previous studies showing that the perfused liver synthesises and secretes amylase into the perfusate 3-5 support the view that the liver is the sole or major source of serum amylase in the rat. The data of other workers on the amylases of human tissues ~8-3° make it hazardous to generalise from the rat to other animal species. Preliminary studies with tissues from the mouse, guinea pig, rabbit, cat and dog suggest, however, that liver and serum isoamylases are electrophoretically identical in these animals a3. The possibility remains that skeletal muscle, adipose tissue, the kidneys or other organs contribute to normal levels of serum amylase. In the absence of evidence that such organs are active in synthesisingamylase the liver remains the most likely source. ACKNOWLEDGEMENTS
This work was supported by a grant from the Australian Research Grants Committee. K.H. is a recipient of a Commonwealth Postgraduate Research Studentship.
* The o b s e r v a t i o n t h a t the parotid gland oi infant r a t s contained very little amylase does not a p p e a r to h a v e been r e p o r t e d previously, b u t is consistent with the work of REDMAN AND SCREEBX¥ 32 who, using morphological criteria, observed a rapid m a t u r a t i o n of the parotid gland cells between 18 and 2 5 d a y s after birth, i.e. during the w e a n i n g period.
Biochim. Biophys. Mcta, 24-4 (I97 l) 441-451
ORIGIN OF SERUM AMYLASE
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