Distribution of α-Amylase Activity in Selected Broiler Tissues1

Distribution of a-Amylase Activity in Selected Broiler Tissues1 D. P. RODEHEAVER and R. D. WYATT2 Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication April 22, 1985)

1986 Poultry Science 65:325-329 INTRODUCTION Because a-amylases of different origins respond differently t o e x p e r i m e n t a l conditions (Meites and Rogols, 1 9 6 8 ) , it has been necessary t o first delineate t h e biochemical req u i r e m e n t s and characteristics of t h e e n z y m e u n d e r study. Most a-amylases of animal origin have been found t o share t h e same biochemical properties as h u m a n and p o r c i n e a-amylases ( T h o m a et al, 1 9 7 1 ) . Laws and Moore ( 1 9 6 3 ) established t h e chlorine r e q u i r e m e n t and specific p H o p t i m u m of chick pancreatic a-amylase, and a r e c e n t s t u d y investigated o t h e r c o n d i t i o n s of assay for p o u l t r y a-amylase (Rodeheaver and Wyatt, 1 9 8 4 ) . T h e variation in animal a-amylases arises primarily from t h e site and rate of e n z y m e synthesis, and change in e n z y m e levels w i t h age (Meites and Rogols, 1 9 7 1 ) . A few studies have r e p o r t e d changes in pancreatic a-amylase levels of t h e developing chick e m b r y o (Marchaim a n d Kulka, 1 9 6 7 ; Heller a n d Kulka, 1 9 6 8 ) . A n o t h e r s t u d y dealt with variation in activity levels of t h e chick pancreas from 1 t o 30 days of age (Laws and Moore, 1 9 6 3 ) . O t h e r investigations

1 Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.

have examined t h e presence of a-amylase in various tissues of some avian species (McGeachin et al., 1 9 5 8 ; J e r r e t t and G o o d g e , 1 9 7 3 ; Sturkie, 1 9 7 6 ) a n d t h e pancreatic a-amylase isoenzymes (Lehrner a n d Malacinski, 1 9 7 5 ) . T h e objectives of this s t u d y w e r e t o d e t e r m i n e t h e relative level of a-amylase activity in various tissues of broilers at selected ages, t o d e t e r m i n e t h e effect of certain tissue p r e p a r a t i o n techniques o n a p p a r e n t a-amylase activity, and to investigate t h e electrophoretic p a t t e r n s of a-amylase activity of t h e serum, liver, and pancreas. MATERIALS AND METHODS Male H u b b a r d broilers were m a i n t a i n e d u n d e r o p t i m a l environmental c o n d i t i o n s and fed t h e standard, n o n m e d i c a t e d University of Georgia broiler starter diet ad libitum in all e x p e r i m e n t s . All serum samples were o b t a i n e d b y intravenous e x t r a c t i o n from t h e wing vein. Bile samples were collected directly from t h e gall bladder w i t h a needle a n d syringe. Whole liver a n d pancreas were carefully excised, rinsed twice in separate volumes of Earle's Salt Solut i o n (Earle, 1 9 4 3 ) t o remove excess b l o o d , b l o t t e d , and weighed. Liver and pancreas samples were homogenized individually for 45 sec w i t h a P o l y t r o n homogenizer ( B r i n k m a n n I n s t r u m e n t s , Westbury, N Y ) in a p p r o p r i a t e v o l u m e s of Earle's Salt S o l u t i o n (Laws and M o o r e , 1 9 6 3 ) , t h e n sonicated for 4 5 sec (Sonic

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ABSTRACT In an examination of broiler a-amylase, significant variation in the serum enzyme activity level was noted, adult levels were lower than those of young chicks. Analysis of a-amylase activity in various body fluids and tissues of 11-day and 7-week-old broilers indicated that the liver cannot be considered a source of a-amylase, although there was activity in both liver tissue and bile of 10 units/g wet weight and 35 units/100 ml, respectively. Fluid from the oral cavity had low levels of a-amylase activity, <100 units/100 ml, which decreased with age, indicating that the salivary glands may synthesize some a-amylase but are not a primary source. Sonication of the pancreatic homogenates was found to significantly increase the apparent activity of a-amylase 35-fold over unsonicated homogenates. The pancreas was the major source of a-amylase with activities ranging from 89 X 10 2 to 445 X 10 2 units/g wet weight. The level of activity increased with age of the bird. The electrophoretic zymograms of serum, liver, and pancreatic homogenates indicate a similar pancreatic origin for the a-amylase found in each tissue or fluid. (Key words.- broiler, tissue a-amylase, pancreatic a-amylase, electrophoresis)

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RODEHEAVER AND WYATT of the clear bands were measured immediately after separation. Statistical Analysis. All data were subjected to an analysis of variance and the means partitioned using a t-test for differences among several means (Bruning and Kintz, 1968). RESULTS Hepatic Intracellular and Extracellular aAmylase. A very low hepatic a-amylase activity of <100 units/100 ml of diluted homogenate or <10 units/g wet tissue (Fig. 1) was observed. Additionally, liver a-amylase activities were not significantly changed by sonication of the homogenate. The insoluble and soluble liver homogenate fractions showed no significant difference in the a-amylase activity associated with each and demonstrated very low activity levels. In contrast to liver homogenates, the pancreas had very high levels of a-amylase activity, and sonication of the homogenates produced a significant increase in the observed activity (Fig. 1). Homogenization of pancreatic samples yielded a mean a-amylase activity of 750 units/g wet tissue, while sonicated homogenates had a mean activity of 26,335 units/g wet tissue. Variation in Serum a-Amylase with Age. Male broiler chicks exhibited a significant decrease in serum a-amylase activity for the first 2 weeks of age (269 units/100 ml) compared to the activity of 321 units/100 ml at one day of age (Table 1). At the 3rd week, there

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FIG. 1. Effect of sonication on measured aamylase activity in pancreatic and liver homogenates of broiler chicks; H = homogenated, H/S = homogenated sonicated.

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Dismembrator Model 300, Fisher Scientific). All samples were maintained at 4 C and aamylase activity determined by the Amylochrome method (Roche Diagnostics, NJ). Hepatic Intracellular and Extracellular aAmylase. Livers of 13-day-old broilers were prepared as described, except aliquots of individual liver homogenates were assayed for a-amylase activity before and after sonication. Both soluble and insoluble fractions of the sonicated liver homogenates were also assayed for enzyme activity. Whole pancreas samples from 30 20-day-old broilers were prepared in an identical manner and assayed before and after sonication. Variation in Serum a-Amylase with Age. Thirty-two day-old male Hubbard broilers were raised t o 7 weeks of age. Serum was obtained at 1, 2, 3, 5, and 7 weeks of age. Serum samples were obtained via cardiac puncture from 23 day-old male broiler hatchmates for the 0-week a-amylase activity determination. a-Amylase Activity in Body Fluids and Tissues. Serum, mouth fluid, bile, liver, and pancreas samples were collected from 10 11-day-old broilers. One hr after the birds had been removed from feed, mouth fluid samples were obtained by thorough swabbing of the oral cavity with preweighed nonabsorbant cotton swabs. Each swab was reweighed to determine the quantity of mouth fluid removed. The mouth fluid was immediately assayed for a-amylase activity by immersion of the sample end of the swab into the preincubated substrate solution. Activity was calculated for standard .1-ml sample size by correction for weight of the sample. Similar fluid and tissue samples were obtained from 32 7-week-old male broiler hatchmates and assayed for a-amylase activity. Electrophoretic Separation of a-Amylase. Serum, liver, and pancreatic samples from 12 3-week-old broilers were prepared as described above. The liver and pancreatic homogenates were centrifuged at 10,000 X g for 30 min and the supernatant saved as the sample fraction. The samples were separated on a 7% polyacrylamide disc gel in a .02 M Tris-.19 M glycine buffer with a 2 mA/gel current for 15 min, followed by 3 mA/gel for 45 min with the anode as the origin. After electropheresis, the gels were incubated in a 1% starch solution and immersed in a KI-I stain (Boettcher and de la Lande, 1969). The gels were maintained at 4 C following incubation. The width and distance

POULTRY a-AMYLASE

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TABLE 1. Serum ot-amylase activity of male broilers at different ages Age.wk 0

1

a-Amylase activity, 321.3 t 9.6 b 269.3 ± 6.9 a units/100 ml n 23 32

2

3

265.2 + 7 . i a 32

7

5

373.8 + 25.9 a 318.8 ± 15.5 b 30

245.4 ± 15.5 a 32

30

a,bValues (mean + SEM) with different superscripts are significantly different (P<.05).

Electrophoresis. Electrophoresis of pancreas homogenates and serum resulted in detection of four bands of a-amylase activity arbitrarily designated as 1, 2, 3, and 4 from the anode (Fig. 2). The bands present in the pancreatic homogenates exhibited mean migration distances of 20.1, 22.0, 24.6, and 26.5 mm, respectively. The a-amylase bands of activity in the serum showed mean migration distances statistically the same as those in the pancreas. The liver exhibited only two bands of activity which corresponded to Bands 2 and 3 of the pancreas, with mean migration distances not significantly different from either the pancreas or the serum. The width of the bands was a reflection of degree of a-amylase activity, the pancreas having the widest bands.

DISCUSSION The body fluids and tissues of the male broilers examined had measurable levels of a-amylase activity although all levels were low compared to the activity in the pancreas. Bile

TABLE 2. a-Amylase activity in body fluids and tissues of 11-day and 7-week-old male broilers Age Source

11 Days

7 Weeks

Serum, units/100 ml Mouth fluid, units/100 ml Bile, units/100 ml Liver Units/g wet wt Units/g dry wt Pancreas Units X 102 /g wet wt Units X 10 2 /gdrywt

270.8 ± 7.0a 81.7 ± 15.1 a 32.1 ± 8.3 a

245.4 ± 15.5a 36.3 ± 9.9b 38.7 ± 7.1 a

1.3 ±

.la

89.2 ± 3.6b

1.4 ± 4.9 ±

.la .3

445.0 ± 29.2a 1424.5 ± 92.5

Values (mean ± SEM) with different superscripts within rows are significantly different (P<.05).

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was a significant rise in serum a-amylase above earlier levels to 374 units/100 ml, followed by a consistant decline until the mean a-amylase activity at 7 weeks of age was statistically the same as that during the first 2 weeks posthatch. a-Amylase Activity in Body Fluids and Tissues. Measurable levels of a-amylase activity were found in all body fluids and tissues assayed (Table 2). Both bile and liver samples had very low a-amylase activities, with no significant change in activity between 11 days and 7 weeks of age. a-Amylase activity in the mouth fluid of 11-day-old birds was approximately 30% that of the serum, but at 7 weeks of age, this activity level had decreased significantly to less than 15% of the corresponding serum a-amylase activity. The serum activities were not significantly different between the two sample times. The pancreas exhibited the highest degree of tissue a-amylase activity at both ages. A significant increase at 7 weeks, of nearly five times the activity present at 11 days of age, was found. At 20 days of age, an intermediate level was noted.

RODEHEAVER AND WYATT

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FIG. 2. Electrophoretic bands of a-amylase activity present in the serum, liver, and pancreas of broiler chicks.

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exhibited the lowest mean activity value, with no difference between samples from 11-day-old and 7-week-old chicks. The biliary amylase activity was somewhat lower than those reported by Farner (1943) and, in comparison to pancreatic activity, did not represent a significant reservoir of a-amylase for starch digestion. This reflects a possible filtration and sequestering of blood a-amylase by the liver into the bile. Mouth fluid samples had low, but measurable, a-amylase activity with levels at 7 weeks of age being about half those of 11day-old chicks. Jerrett and Goodge (1973) had not found amylase activity in broiler salivary glands, yet, because there was no sangual contamination in the samples from this present study, it was felt that the activity observed here resulted from limited synthesis and secretion of a-amylase into the oral cavity by the salivary glands. Serum a-amylase activities varied significantly with age of the broilers. The highest level was observed at 3 weeks of age and lower levels at 7 weeks. The differing serum a-amylase levels at various ages of the chick may be influenced by nonparallel changes in pancreatic production and secretion. The pancreas was chosen for evaluation because it is the primary source of a-amylase in poultry (Moran, 1982). The liver was also evaluated due to conflicting data regarding the presence or absence of a-amylase in the liver of many animals (McGeachin et al, 1958; Messer and Dean, 1975; Takeuchi et al, 1975). In the present study, we were concerned with the presence and level of a-amylase activity in the liver and pancreas and the extracellular or intracellular nature of the enzyme. The liver was found to contain a low level of a-amylase activity, which was distributed uniformly between the various fractions of liver tissue. No

difference was found between the hepatic enzyme activities at 11 days and 7 weeks of age. Sonication had no effect on whole homogenate activity, indicating either that homogenization alone adequately disrupted cellular membranes thereby liberating the enzyme, or that the a-amylase was extracellular. Based on the low levels of hepatic a-amylase compared to serum a-amylase levels, and the failure of sonication to liberate additional enzyme activity, residual hepatic blood may have been the source of a-amylase activity. The pancreas of broilers exhibited the highest a-amylase activity of all the tissues examined. Sonication increased the measured activity more than 35-fold over that of the unsonicated homogenates, confirming the intracellular nature of the pancreatic a-amylase (Bird, 1971). It, therefore, appears that the enzyme is synthesized and stored intracellularly in the pancreas, and subsequently secreted into the duodenum for digestion. Normal broiler pancreatic a-amylase activity showed a progressive rise in activity from 11 to 49 days of age. This was opposite to the findings of Laws and Moore (1963), although Marchaim and Kulka (1967) and Heller and Kulka (1968) found increasing levels of pancreatic a-amylase and other enzymes in the chick embryo. The increasing levels of activity observed here may be a normal response to increasing metabolic needs of the bird. Through electrophoresis, it is possible to compare the a-amylases of different origins, each of which will exhibit a distinct zymogram. Using this technique, human serum has been shown to be of both salivary and pancreatic origin (Takeuchi et al, 1974), while the rat liver amylase appears to be a complex of the serum a-amylase and glycogen (Takeuchi et al, 1975). Recognizing that the pancreas is a major site of a-amylase synthesis, the serum and liver aamylases were compared to the pancreas to determine the zymogram pattern. While the liver did not show Bands 1 and 4, it is felt that this was due to the extremely low level of a-amylase activity present in the liver. The two bands that were exhibited in the liver, as well as the four bands present in the serum, had mean migration distances no different than the corresponding pancreatic enzyme bands. This supports the conclusion that the serum aamylase is of pancreatic origin and that aamylase activity appears in the liver as a result of filtration from the blood. It is possible

POULTRY a-AMYLASE t h a t t h e a-amylase, b y s o m e m e c h a n i s m , diffuses directly from t h e pancreas into t h e b l o o d and t h u s does n o t show t h e altered electrophoretic z y m o g r a m s of t h e intestinal a-amylase observed b y Lehrner and Malacinski (1976).

REFERENCES

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Bird, F. H., 1971. Distribution of trypsin and amylase activities in the duodenum of the domestic fowl. Br. Poult. Sci. 12:373-378. Boettcher, B., and F. A. de la Lande, 1969. Electrophoresis of human salivary amylase in gel slabs. Anal. Biochem. 28:510-514. Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott, Foresman, and Co., Glenview, IL. Earle, W. R., 1943. Propagation of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in living cells. J. Natl. Cancer Inst. 4:165-212. Farner, D. S., 1943. Biliary amylase in the domestic fowl. Biol. Bull. 84:240-243. Heller, H., and R. G. Kulka, 1968. Amylase isoenzymes in the developing chick pancreas. Biochim. Biophys. Acta. 165:393-397. Jerrett, S. A., and W. R. Goodge, 1973. Evidence for amylase in avian salivary glands. J. Morphol. 139:27-46. Laws, B. M., and J. H. Moore, 1963. Some observations on the pancreatic amylase and intestinal maltase of the chick. Can. J. Biochem. Physiol. 41:2107-2121. Lehrner, L. M., and G. M. Malacinski, 1975. Biochemical genetics of a-amylase isozymes of the chicken pancreas. Biochem. Genet. 13:145—173. Lehrner, L. M., and G. M. Malacinski, 1976. Analysis

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