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Amylase distribution in extrapancreatic, extrasalivary tissues
ARCHIVES
OF
BIOCHEMISTRY
AND
76, 403-411
BIOPHYSICS
Amylase Distribution Extrasalivary
(1958)
in Extrapancreatic, Tissue& 2
Robert L. McGeachin, John R. Gleason3 and Morgan R. Adams3’ 4 From the Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky Received
August
30, 1957
INTRODUCTION
Formation of amylase by the pancreas in all animals and the salivary glands in some is well recognized. It is found in the plasma and extracellular fluid of all animals although no specific function of amylase in such fluids has been discovered. While earlier workers assumed that the sources of serum and extracellular amylase are the pancreas and salivary glands, evidence is accumulating that there is an extrapancreatic, extrasalivary source (or sources) of amylase which serves to maintain serum nmylase levels. Among others, Markowitz et aE. (1) and Reid et al. (2) showed that the dog, which produces no salivary amylase, maintains a normal serum amylase concentration if treated with insulin following complete pancreatectomy. Roe, Smith, and Treadwell (3) reported that depancreatized rats maintained normal serum amylase levels. It has also been shown that removal of the salivary glands in the rat does not markedly affect serum amylase (4). At various times the liver (3), the lungs (5), and the intestinal tract (6) have all been suggested as possible additional sites of amylase production. We shall present additional data on the amylase contents of various 1 This work was partially supported by research grants (C-2601 and C-2601C) from the National Cancer Instit’ute, National Institutes of Health, U. S. Public Health Service. 2 A preliminary report on part of this work was presented at the 1957 meeting of the American Society of Biological Chemists, R. L. McGeachin and J. R. Glesson, Federation Proc. 16, 219 (1957). 3 Student Research Scholars, 1955-56. 4 With the technical assistance of Katherine B. Daniel. 403
404
MCGEACHIN, GLEASON AND ADAMS
tissues and organs, including those of the salivarectomized-pancreatectomized rat, and some calculations indicating that, the liver and muscle in several species contain intracellular amylase. METHODS AND MATERIALS Theanimalsused in this study, rats, mice, guinea pigs, dogs, chickens, hamsters, and cats, were killed by being bled to death under ether anesthesia. With the exception of the serum, the tissues and organs were immediately frozen at -18°C. and kept frozen in covered containers until just before using. The duodenum and colon in each case were washed free of food and feces prior to freezing. The serum amylase determination for each animal was carried out first in order to have a guide for the proper dilution of the tissue extracts. Amylase extracts were prepared by homogenizing in a Waring blendor in a 2’ cold room the thawed, minced tissues with appropriate measured quantities (usually making a 1:50 or 1: 100 dilution) of 0.9% sodium chloride. The homogenates were then centrifuged, and the supernatant fluids were used in the amylase determinations. Amylase analyses were carried out using Van Loon et al.% (7) amyloclastic method with a phosphate buffer unless otherwise noted. For comparative purposes, a modification of the method using 0.02 M pH 7.6 Verona1 buffer in place of the 0.02 M pH 7.0 phosphate buffer was used in some analyses. In this comparison (Table II) of phosphate and Verona1 buffers for tissues of the rat and mouse, 1:lOO dilutions of the tissue extracts were used except for the brain, 1:lO; duodenum, l:lOOO-1:2000; the pancreas, l:lOO,OOO; and the muscle of the rat, 1:lO. Except for the muscle, these extracts showed no evidence of phosphorylase activity in either the pH 7.0 phosphate buffer-starch mixture or the pH 7.6 Verona1 buffer-starch. The tissue extracts tested showed only a slight loss in amylase activity when dialyzed at 2°C. for 30 hr. in Visking cellulose casings against 0.9% sodium chloride. This small loss of activity can be accounted for quantitatively by the slight increase in volume of the extracts during dialysis. Analysis of the dialyzed extracts showed that they contain less than 5 pg. P/ml. Treatment of the original extracts with corn starch (8) removed all amylase activity as tested by Van Loon’s method. The procedure for pancreatectomy of rats under Nembutal (30 mg./kg.) anesthesia used was similar to that of Treadwell and Roe (9) employing small cotton pledgets wrapped tightly on the end of toothpicks to remove the pancreas by rubbing the pancreatic tissue away from the mesentery and neighboring organs. Two weeks later the 9 rats surviving out of the original 20 were subjected to salivarectomy. This was accomplished in the following manner: A longitudinal midline incision was made on the anterior aspect of the neck extending from the inferior border of the mandible to the suprasternal notch. By alternating blunt and sharp dissection, the salivary glands were freed from the underlying tissue and removed. Three weeks later blood was drawn from the five rats surviving both operations for Nelson glucose determinations. Their mouths were rinsed with 0.9% sodium chloride, and the rinsings were immediately reaspirated into the same micropipet used for the addition. These rinsings were tested qualitatively for amylase activity. The rats were then sacrificed, and blood and various tissues,
AMYLASE
405
DISTRIBUTION
including mesentery from the area where the pancreas had been, were removed for analysis as mentioned before. In obtaining blood immediately before and after it passed through the lungs, catheters were placed in the right auricles and the arches of the aorta of six anesthetized, heparinized dogs, and blood samples were simultaneously drawn in each pair of catheters. RESULTS
It is apparent from Table I that amylase is widely distributed throughout the tissues and organs of the animals used. While the results on
rats are in general agreement with those of others (3, B), several points of disagreement are noted. Wiberg and Tuba stated that the spleen, heart, and brain of rats are completely devoid of demonstrable amylase action. We have found measurable amylase levels in these organs. The blood vessels of the brain apparently restrict entrance of amylase, and since the amylase content. of brain is much lower than the other organs, and since Wiberg and Tuba’s amylase unit equals 50 Van Loon units, it is possible that such low values were not readily distinguishable by their method. Such an explanation is not satisfactory for the spleen and the heart, however. For liver amylase in rats, our results were intermediate between those of Roe, Smith, and Treadwell (3), 3310 units/ 100 g., and of Wiberg and Tuba (6), 665 units/100 g. (both results translated into Van Loon units). Acting on a suggestion from Dr. B. W. Smith of George Washington University, we found that it was necessary to allow the livers to “age” by standing overnight before amylase analysis. The activities of five rat livers were an average of 27 % greater after standing overnight at -18°C. than when determined on the fresh livers immediately following removal from the animals. TABLE Amylase Average
I
Content of Tissues
amylase
units/100
and Organs
ml. or 100 g. tissue
Animal _______.-
Rat (14) Mouse (6) Guinea pig (4) Dog (3) Chicken (2) -
2730 760 36401790 2210 910 490 250 830 320
____-
570 830 320 108 131
_.-
500 800 310 121 198 271
1,750,ooo ?, 110,000 280,000 496,000 1,420,OOO __-
406
MCGEACHIN,
GLEASON
TABLE Comparative
AND
ADAMS
II
Amylase Activities of Tissues and Organs in pH 7.0 Phosphate Bu$er and pH Y.6 Verona1 Buffer
-
Rats (4)
Tissue or organ
Mice (5)
I
Amylase activities: units/100 ml. serum or 100g. tissue
-
7
pH 7.0 phosphate Serum
Lungs Spleen Heart Kidney Brain Duodenum Perirenal fat Colon Muscle Liver Pancreas
2010 663 543 464 652 52 66,400 644 3960 202 781 2,810,OOO
.-
-
pH 7.6 veranal
Ratio
pH 7.0 phosphate
1980 641 562 439 638 56 65,700 628 3880 134 737 2,474,OOO
.99 .97 1.03 .94 .98 1.08 .99 .98 .98 .66 .94 .88
3750 1690 1240 735 890 95 12,240
3360 1590 1210 710 840 91 12,500
.90 .94 .98 .97 .94 .96 1.02
1340 1040 1660 2,099,ooo
1310 900 1620 1,514,OOo
.98 .87 .98 .72
-
pH 7.6 VWXKil
-
Ratio
Since it is of prime importance in using an amyloclastic method for amylase determination to distinguish between phosphorolytic and hydrolytic starch degradation by the tissue extracts, the comparative analyses of amylase activities of tissues from the rat and mouse shown in Table II were carried out. Except for the muscle and the pancreas, these tissue amylase values are essentially the same for the two buffers. The determinations on serum, which contains no phosphorylase, at pH’s 7.0 and 7.6 establish the fact that amylase is equally active in both buffers. This has also been established for human serum. Under the conditions of Van Loon’s original method for amylase which employs a phosphate buffer, phosphorylase could conceivably be contributing to the amylase activities as measured. Also, even though no phosphate is present in the test system when Verona1 buffer is used except that very small amount endogenous to the diluted tissue extracts, the question has been raised as to whether phosphorylase might still be active here. In the 1: 100 dilutions of tissue extracts (except for muscle) the amount of inorganic phosphate was only about 0.002 mg. P/ml; not nearly enough to provide the necessary amount for phosphorolytic cleavage of starch in the Verona1 buffer system even if phosphorylase
AMYLASE
407
DISTRIBUTION
were active. Even if the concentration were ten times this amount, this would be less than a tenth the amount necessary to degrade half the starch in the usual test system. Dialysis of tissue extracts to remove phosphate did not diminish the amylase activities of these extracts as would have been the case if phosphorylase were contributing to their amylase activities. In summary, phosphorylase is not contributing to amylase activity when Verona1 buffer is used since (a) the amount of endogenous phosphate is too low to allow for phosphorylase activity; and (b) removal of phosphate by dialysis does not diminish the observed amylase activity. Thus, since phosphorylase is not contributing to amylase activity when Verona1 buffer is used and the activities in Verona1 and phosphate buffers are equal, phosphorylase is not active in these extracts even in the presence of phosphate. This could perhaps be due to the presence of phosphorylase-inactivating enzymes that exist (10) in most tissues. The lower amylase activity of the pancreatic extracts at pH 7.6 in both the rat and mouse may be caused by partial proteolytic destruction of nmylase at the higher pH or may indicate that, pancreatic amylase is different from the amylase in the serum and other tissues. Preliminary findings on the paper electrophoretic behavior of serum and pancreatic nmylases indicate that the latter may indeed be true. If one compares the tissue amylase to serum amylase ratios with the extracellular fluid percentages in the rat, mouse, guinea pig, and dog, it is seen (Table III) that in all these animals some of the liver amylase is intracellular. If the amylase in the organ were limited t,o the extracellular fluid and were present only because of diffusion from the blood, TABLE
Ratio of Tissue Amylase by Extracellular
III
to Serum Amylase Fluid Fraction
The value in parentheses are extracellular organs concerned. Organ
Liver Kidney Lung Heart Muscle Spleen Brain
Rat
2.67 0.71 0.51 0.73 0.46 0.51 0.11
(0.25) (0.45) (0.55) (0.25) (0.15) (0.41) (0.27)
Animal Mouse
2.10 0.55 0.89 0.88 2.09 0.56 0.10
Divided
fluid fractions for t,he t,issues and
Guinea
Pig
2.43 0.88 0.75 0.56 2.93 0.35 0 14
mg
1.50 0.93 0.94 1.00 1.30 0.52 0.21
(0.31) (0.45) (0.54) (0.24) (0.16) (0.42) (0.32)
408
MCQEACHIN,
GLEASON
AND ADAMS
then the ratio of tissue amylase to serum amylase divided by the extracellular fluid fraction for that organ could only approach unity as a maximum value. However, if any of the organ or tissue amylase is intracellular, this value may be more than one. Manery’s (11) data for extracellular fluid contents of the rat and dog were used, while values for the mouse and guinea pig (no data available) were assumed to be identical with those of the rat. Admittedly, there is the possibility that some of these data on extracellular fluid may be in error, but they seem to be the best estimates available. In Table III it is apparent that there is not only intracellular amylase in the livers of all four of these animals but also amylase in the muscle cells of the mouse, guinea pig, and dog. Saline rinsings of the mouths of the salivarectomized rats were tested qualitatively for amylase action. Whereas these rinsings showed no evidence of action on 0.5% starch after 5 hr., such rinsings from the mouths of normal rats were powerfully amylolytic, completely digesting 5 ml. of 0.5 % starch in 5-10 min. The blood glucose levels of the salivarectomized-pancreatectomized rats (Table I) indicate that they were diabetic, and analyses of tissues in the area from which the pancreas had been removed indicate approximately 98 % removal of the pancreas, e.g., (2,750,OOO- 54,000)/2,750,000 = 0.98. In the five rats that survived both salivarectomy and pancreatectomy, it is evident (Table IV) that removal of both pancreas and salivary glands does not remove the source(s) of serum and tissue amylase. In these rats, not only is amylase still present in the serum and tissues, but also the levels are normal except in the gastrointestinal tract. In seven TABLE
IV
Amylase Content of Tissues and Organs of Pancreatectomized-Salivarectomized Rats
Rat
1 2 3 4 5 Av. for 5 Av. for normals
“;gdI case
Serum -~-__
271 376 337 444 357 100
-
Amylase activity:
units/l00
ml. or 100 g.
-
PUKI~S
g&
35,500 1520 2580 54,600 5090 3130 71,800 2030 2970 22,600 2600 61,00011,800 2450 49,ooo 5110 2750 2730 2,750,00063,600
lxSdney
Colon _-
920 420 1600 1560 1200 1140 870
1140 2630 840 1160 1170 1390 3500 -
Liver
Spleen
Lung
_---
--
2160 2350 2240 2700 2700 2430 1820
-
1110 400 570 590 890 710 570
980 960 1150 950 1020 1010 760
AMYLASE
409
DISTRIBUTION
TABLE V Amylase in Blood before and after Passing the Lungs of Dogs Serum amylase of blood from: Right auricle Arch of aorta Difference Dog 1
2 3 4 5 6 Av.
704 644 652 526 632 1007
712 649 654 527 624 1006
+8 +5
+2 +1 -8 -1 +1
other salivarectomized-pancreatectomized rats obtained at a later date, the serum amylase levels were only slightly subnormal (ZOOO2500) 3 weeks following the operations. However, these rats had still not completely recovered from the surgery and were not eating quite normally. It was postulated that if the lungs are a source of amylase production as has been suggested (5), amylase might be secreted into the blood during its passage through the lung. In Table V, the serum amylase contents of blood from the right auricles and the arches of the aorta indicate no significant detectable addition of amylase to pulmonary blood. It is, of course, possible that very small amounts per unit time might be secreted, and, with the large blood flow through the lungs, a considerable quantity of amylase could be added to the blood without the A-V difference being detectable. Our observations do not agree with Takano’s report (12) that in rabbits left cardiac blood has a stronger amylolytic power than right cardiac blood. Five human lung samples obtained from autopsy specimens all had amylase levels (250-950) well above the normal range for human serum, but the delays between the deaths of the patients, the autopsies, and obtaining the specimens make the validity of such values doubtful. Unfortunately, no blood samples were available for these patients. Analyses of the lungs and sera from the cat and hamster showed serum amylase to lung amylase ratios similar to the other animals listed. DISCUSSION
It is significant that when both known sources of amylase production, the pancreas and the salivary glands, are removed from the rat, amylase does not disappear from the serum and tissues. When the animals have recovered from the trauma of surgery and are eating normally, amylase
410
MCGEACHIN,
GLEASON
AND
ADAMS
levels are found to be within the normal range. In those animals who recover only partially from the surgery or who are not eating normally, serum amylase levels may be somewhat subnormal but are still appreciable. Roe, Smith, and Treadwell (3) showed that amylase excretion continued unchanged in their pancreatectomized rats. Calculations indicate that if all sources of serum, tissue, and urinary amylase were removed, such amylase should all be cleared from the rat within 3 days. When serum amylase remains at normal or slightly subnormal levels 3 weeks after removal of both pancreas and salivary glands, this is conclusive evidence that there must be some extrapancreatic, extrasalivary source of amylase formation in the rat, confirming previous suggestions (3, 6, 13) that such a source must exist. The data on the tissue amylase to serum amylase ratios compared to the extracellular fluid fractions for various tissues and organs indicate that in the rat, mouse, guinea pig, and dog, there is intracellular amylase in the liver. For the mouse, and guinea pig (and perhaps the dog), there is also intracellular muscle amylase. The presence of an intracellular concentration of an enzyme of course does not prove that the enzyme is synthesized in that cell but does indicate this possibility. While Somogyi (13) and Cori, Cori, and Schmidt (14) indicated that liver has no intracellular amylase, Roe, Smith, and Treadwell state (3) that, under certain conditions, the liver may contain 5-10 times as much amylase as can be accounted for by the blood in the organ. Preliminary results in this laboratory on isolated, washed, whole liver cells indicate that these cells do contain amylase. When these facts are considered together with the data indicating that low serum amylase levels are associated with liver damage (15), the liver as a site of amylase formation seemsa likely possibility. This is presently the subject of further investigation. Wiberg and Tuba (6) suggested the intestinal mucosa as a possible site of amylase formation. Our data confirm this possibility since there is amylase still to be found in the gastrointestinal tract following pancreatectomy and salivarectomy. Takano’s (5) suggestion that amylase is formed in the lungs seemsof doubtful validity. SUMMARY
1. The distribution of amylase in the serum and tissues of pancreatectomized-salivarectomized rats is essentially the same as in the normal except for the gastrointestinal tract. 2. Calculations from tissue amylase to serum amylase ratios and
AMYLASE DISTRIBUTION
111
extracellular fluid contents indicate that the liver in the rat, mouse, guinea pig, and dog, and muscle in the mouse and guinea pig have intracellular amylase. 3. Tissue and serum amylase activities are essentially the same in pH 7.0 phosphate and pH 7.6 Verona1 buffers, except for muscle and pancreas. 4. No detectable amylase is added to the blood passing through the ungs of dogs. REFERENCES 1. MARKOWITZ, J., AND HOUGH, H. B., AWL. J. Physiol. 76, 571 (1926). 2. REID, E., &UIGLEY, J. P., AND MYERS, V. C., J. Riol. Chern. 99, 615 (1933). 3. ROE, J. H., JR., SMITH, B. W., AND TREADWELL, C. I~., I’roc. Sot. h’zptl. Riol. Med. 87, 79 (1954). 4. MCGEACHIN, R. L., AND GLEASON, J. R., Science 123, 841 (1956). 5. TAKANO, T., J. Oriental Afed. 28, 1231 (1938). 6. WIBERG, G. S., AND TUBA, J., Can. J. Biochem. Physiol. 33, 817 (1955). 7. VAN LOON, E. J., LIEENS, M. R., AND SEGER, A. J., Am. J. Clin. Puthol. 22, 1134 (1952). 8. CORI, G. T., AND LARNER, J., J. Biol. Chem. 186, 17 (1951). 9. TREADWELL, C. R., AND ROE, J. H., Proc. Sot. Exptl. Biol. Med. 86,878 (1954). 10. SUTHERLAND, E. W., in “A Symposium on the Clinical and Biological Aspects of Carbohydrate Utilization in Health and Disease” (Najjar, V. A,, ed.), p. 25, The Johns Hopkins Press, Baltimore, 1952. 11. MANERY, J. F., Physiol. Revs. 34, 334 (1954). 12. TAKANO, T., J. Oriental Med. 28, 1263 (1938). 13. SOMOGYI, M., A.M.A. Arch. Znternal Med. 67, 665 (1941). 14. CORI, C. F., CORI, G. T., AND SCHMIDT, G., J. Biol. Chem. 129, 631 (1939). 15. GRAY, 8. H., PROBSTEIN, J. G., AND HEIFETZ, C. J., A.M.A. Arch. Internal Med. 67, 805 (1941).
OF
BIOCHEMISTRY
AND
76, 403-411
BIOPHYSICS
Amylase Distribution Extrasalivary
(1958)
in Extrapancreatic, Tissue& 2
Robert L. McGeachin, John R. Gleason3 and Morgan R. Adams3’ 4 From the Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky Received
August
30, 1957
INTRODUCTION
Formation of amylase by the pancreas in all animals and the salivary glands in some is well recognized. It is found in the plasma and extracellular fluid of all animals although no specific function of amylase in such fluids has been discovered. While earlier workers assumed that the sources of serum and extracellular amylase are the pancreas and salivary glands, evidence is accumulating that there is an extrapancreatic, extrasalivary source (or sources) of amylase which serves to maintain serum nmylase levels. Among others, Markowitz et aE. (1) and Reid et al. (2) showed that the dog, which produces no salivary amylase, maintains a normal serum amylase concentration if treated with insulin following complete pancreatectomy. Roe, Smith, and Treadwell (3) reported that depancreatized rats maintained normal serum amylase levels. It has also been shown that removal of the salivary glands in the rat does not markedly affect serum amylase (4). At various times the liver (3), the lungs (5), and the intestinal tract (6) have all been suggested as possible additional sites of amylase production. We shall present additional data on the amylase contents of various 1 This work was partially supported by research grants (C-2601 and C-2601C) from the National Cancer Instit’ute, National Institutes of Health, U. S. Public Health Service. 2 A preliminary report on part of this work was presented at the 1957 meeting of the American Society of Biological Chemists, R. L. McGeachin and J. R. Glesson, Federation Proc. 16, 219 (1957). 3 Student Research Scholars, 1955-56. 4 With the technical assistance of Katherine B. Daniel. 403
404
MCGEACHIN, GLEASON AND ADAMS
tissues and organs, including those of the salivarectomized-pancreatectomized rat, and some calculations indicating that, the liver and muscle in several species contain intracellular amylase. METHODS AND MATERIALS Theanimalsused in this study, rats, mice, guinea pigs, dogs, chickens, hamsters, and cats, were killed by being bled to death under ether anesthesia. With the exception of the serum, the tissues and organs were immediately frozen at -18°C. and kept frozen in covered containers until just before using. The duodenum and colon in each case were washed free of food and feces prior to freezing. The serum amylase determination for each animal was carried out first in order to have a guide for the proper dilution of the tissue extracts. Amylase extracts were prepared by homogenizing in a Waring blendor in a 2’ cold room the thawed, minced tissues with appropriate measured quantities (usually making a 1:50 or 1: 100 dilution) of 0.9% sodium chloride. The homogenates were then centrifuged, and the supernatant fluids were used in the amylase determinations. Amylase analyses were carried out using Van Loon et al.% (7) amyloclastic method with a phosphate buffer unless otherwise noted. For comparative purposes, a modification of the method using 0.02 M pH 7.6 Verona1 buffer in place of the 0.02 M pH 7.0 phosphate buffer was used in some analyses. In this comparison (Table II) of phosphate and Verona1 buffers for tissues of the rat and mouse, 1:lOO dilutions of the tissue extracts were used except for the brain, 1:lO; duodenum, l:lOOO-1:2000; the pancreas, l:lOO,OOO; and the muscle of the rat, 1:lO. Except for the muscle, these extracts showed no evidence of phosphorylase activity in either the pH 7.0 phosphate buffer-starch mixture or the pH 7.6 Verona1 buffer-starch. The tissue extracts tested showed only a slight loss in amylase activity when dialyzed at 2°C. for 30 hr. in Visking cellulose casings against 0.9% sodium chloride. This small loss of activity can be accounted for quantitatively by the slight increase in volume of the extracts during dialysis. Analysis of the dialyzed extracts showed that they contain less than 5 pg. P/ml. Treatment of the original extracts with corn starch (8) removed all amylase activity as tested by Van Loon’s method. The procedure for pancreatectomy of rats under Nembutal (30 mg./kg.) anesthesia used was similar to that of Treadwell and Roe (9) employing small cotton pledgets wrapped tightly on the end of toothpicks to remove the pancreas by rubbing the pancreatic tissue away from the mesentery and neighboring organs. Two weeks later the 9 rats surviving out of the original 20 were subjected to salivarectomy. This was accomplished in the following manner: A longitudinal midline incision was made on the anterior aspect of the neck extending from the inferior border of the mandible to the suprasternal notch. By alternating blunt and sharp dissection, the salivary glands were freed from the underlying tissue and removed. Three weeks later blood was drawn from the five rats surviving both operations for Nelson glucose determinations. Their mouths were rinsed with 0.9% sodium chloride, and the rinsings were immediately reaspirated into the same micropipet used for the addition. These rinsings were tested qualitatively for amylase activity. The rats were then sacrificed, and blood and various tissues,
AMYLASE
405
DISTRIBUTION
including mesentery from the area where the pancreas had been, were removed for analysis as mentioned before. In obtaining blood immediately before and after it passed through the lungs, catheters were placed in the right auricles and the arches of the aorta of six anesthetized, heparinized dogs, and blood samples were simultaneously drawn in each pair of catheters. RESULTS
It is apparent from Table I that amylase is widely distributed throughout the tissues and organs of the animals used. While the results on
rats are in general agreement with those of others (3, B), several points of disagreement are noted. Wiberg and Tuba stated that the spleen, heart, and brain of rats are completely devoid of demonstrable amylase action. We have found measurable amylase levels in these organs. The blood vessels of the brain apparently restrict entrance of amylase, and since the amylase content. of brain is much lower than the other organs, and since Wiberg and Tuba’s amylase unit equals 50 Van Loon units, it is possible that such low values were not readily distinguishable by their method. Such an explanation is not satisfactory for the spleen and the heart, however. For liver amylase in rats, our results were intermediate between those of Roe, Smith, and Treadwell (3), 3310 units/ 100 g., and of Wiberg and Tuba (6), 665 units/100 g. (both results translated into Van Loon units). Acting on a suggestion from Dr. B. W. Smith of George Washington University, we found that it was necessary to allow the livers to “age” by standing overnight before amylase analysis. The activities of five rat livers were an average of 27 % greater after standing overnight at -18°C. than when determined on the fresh livers immediately following removal from the animals. TABLE Amylase Average
I
Content of Tissues
amylase
units/100
and Organs
ml. or 100 g. tissue
Animal _______.-
Rat (14) Mouse (6) Guinea pig (4) Dog (3) Chicken (2) -
2730 760 36401790 2210 910 490 250 830 320
____-
570 830 320 108 131
_.-
500 800 310 121 198 271
1,750,ooo ?, 110,000 280,000 496,000 1,420,OOO __-
406
MCGEACHIN,
GLEASON
TABLE Comparative
AND
ADAMS
II
Amylase Activities of Tissues and Organs in pH 7.0 Phosphate Bu$er and pH Y.6 Verona1 Buffer
-
Rats (4)
Tissue or organ
Mice (5)
I
Amylase activities: units/100 ml. serum or 100g. tissue
-
7
pH 7.0 phosphate Serum
Lungs Spleen Heart Kidney Brain Duodenum Perirenal fat Colon Muscle Liver Pancreas
2010 663 543 464 652 52 66,400 644 3960 202 781 2,810,OOO
.-
-
pH 7.6 veranal
Ratio
pH 7.0 phosphate
1980 641 562 439 638 56 65,700 628 3880 134 737 2,474,OOO
.99 .97 1.03 .94 .98 1.08 .99 .98 .98 .66 .94 .88
3750 1690 1240 735 890 95 12,240
3360 1590 1210 710 840 91 12,500
.90 .94 .98 .97 .94 .96 1.02
1340 1040 1660 2,099,ooo
1310 900 1620 1,514,OOo
.98 .87 .98 .72
-
pH 7.6 VWXKil
-
Ratio
Since it is of prime importance in using an amyloclastic method for amylase determination to distinguish between phosphorolytic and hydrolytic starch degradation by the tissue extracts, the comparative analyses of amylase activities of tissues from the rat and mouse shown in Table II were carried out. Except for the muscle and the pancreas, these tissue amylase values are essentially the same for the two buffers. The determinations on serum, which contains no phosphorylase, at pH’s 7.0 and 7.6 establish the fact that amylase is equally active in both buffers. This has also been established for human serum. Under the conditions of Van Loon’s original method for amylase which employs a phosphate buffer, phosphorylase could conceivably be contributing to the amylase activities as measured. Also, even though no phosphate is present in the test system when Verona1 buffer is used except that very small amount endogenous to the diluted tissue extracts, the question has been raised as to whether phosphorylase might still be active here. In the 1: 100 dilutions of tissue extracts (except for muscle) the amount of inorganic phosphate was only about 0.002 mg. P/ml; not nearly enough to provide the necessary amount for phosphorolytic cleavage of starch in the Verona1 buffer system even if phosphorylase
AMYLASE
407
DISTRIBUTION
were active. Even if the concentration were ten times this amount, this would be less than a tenth the amount necessary to degrade half the starch in the usual test system. Dialysis of tissue extracts to remove phosphate did not diminish the amylase activities of these extracts as would have been the case if phosphorylase were contributing to their amylase activities. In summary, phosphorylase is not contributing to amylase activity when Verona1 buffer is used since (a) the amount of endogenous phosphate is too low to allow for phosphorylase activity; and (b) removal of phosphate by dialysis does not diminish the observed amylase activity. Thus, since phosphorylase is not contributing to amylase activity when Verona1 buffer is used and the activities in Verona1 and phosphate buffers are equal, phosphorylase is not active in these extracts even in the presence of phosphate. This could perhaps be due to the presence of phosphorylase-inactivating enzymes that exist (10) in most tissues. The lower amylase activity of the pancreatic extracts at pH 7.6 in both the rat and mouse may be caused by partial proteolytic destruction of nmylase at the higher pH or may indicate that, pancreatic amylase is different from the amylase in the serum and other tissues. Preliminary findings on the paper electrophoretic behavior of serum and pancreatic nmylases indicate that the latter may indeed be true. If one compares the tissue amylase to serum amylase ratios with the extracellular fluid percentages in the rat, mouse, guinea pig, and dog, it is seen (Table III) that in all these animals some of the liver amylase is intracellular. If the amylase in the organ were limited t,o the extracellular fluid and were present only because of diffusion from the blood, TABLE
Ratio of Tissue Amylase by Extracellular
III
to Serum Amylase Fluid Fraction
The value in parentheses are extracellular organs concerned. Organ
Liver Kidney Lung Heart Muscle Spleen Brain
Rat
2.67 0.71 0.51 0.73 0.46 0.51 0.11
(0.25) (0.45) (0.55) (0.25) (0.15) (0.41) (0.27)
Animal Mouse
2.10 0.55 0.89 0.88 2.09 0.56 0.10
Divided
fluid fractions for t,he t,issues and
Guinea
Pig
2.43 0.88 0.75 0.56 2.93 0.35 0 14
mg
1.50 0.93 0.94 1.00 1.30 0.52 0.21
(0.31) (0.45) (0.54) (0.24) (0.16) (0.42) (0.32)
408
MCQEACHIN,
GLEASON
AND ADAMS
then the ratio of tissue amylase to serum amylase divided by the extracellular fluid fraction for that organ could only approach unity as a maximum value. However, if any of the organ or tissue amylase is intracellular, this value may be more than one. Manery’s (11) data for extracellular fluid contents of the rat and dog were used, while values for the mouse and guinea pig (no data available) were assumed to be identical with those of the rat. Admittedly, there is the possibility that some of these data on extracellular fluid may be in error, but they seem to be the best estimates available. In Table III it is apparent that there is not only intracellular amylase in the livers of all four of these animals but also amylase in the muscle cells of the mouse, guinea pig, and dog. Saline rinsings of the mouths of the salivarectomized rats were tested qualitatively for amylase action. Whereas these rinsings showed no evidence of action on 0.5% starch after 5 hr., such rinsings from the mouths of normal rats were powerfully amylolytic, completely digesting 5 ml. of 0.5 % starch in 5-10 min. The blood glucose levels of the salivarectomized-pancreatectomized rats (Table I) indicate that they were diabetic, and analyses of tissues in the area from which the pancreas had been removed indicate approximately 98 % removal of the pancreas, e.g., (2,750,OOO- 54,000)/2,750,000 = 0.98. In the five rats that survived both salivarectomy and pancreatectomy, it is evident (Table IV) that removal of both pancreas and salivary glands does not remove the source(s) of serum and tissue amylase. In these rats, not only is amylase still present in the serum and tissues, but also the levels are normal except in the gastrointestinal tract. In seven TABLE
IV
Amylase Content of Tissues and Organs of Pancreatectomized-Salivarectomized Rats
Rat
1 2 3 4 5 Av. for 5 Av. for normals
“;gdI case
Serum -~-__
271 376 337 444 357 100
-
Amylase activity:
units/l00
ml. or 100 g.
-
PUKI~S
g&
35,500 1520 2580 54,600 5090 3130 71,800 2030 2970 22,600 2600 61,00011,800 2450 49,ooo 5110 2750 2730 2,750,00063,600
lxSdney
Colon _-
920 420 1600 1560 1200 1140 870
1140 2630 840 1160 1170 1390 3500 -
Liver
Spleen
Lung
_---
--
2160 2350 2240 2700 2700 2430 1820
-
1110 400 570 590 890 710 570
980 960 1150 950 1020 1010 760
AMYLASE
409
DISTRIBUTION
TABLE V Amylase in Blood before and after Passing the Lungs of Dogs Serum amylase of blood from: Right auricle Arch of aorta Difference Dog 1
2 3 4 5 6 Av.
704 644 652 526 632 1007
712 649 654 527 624 1006
+8 +5
+2 +1 -8 -1 +1
other salivarectomized-pancreatectomized rats obtained at a later date, the serum amylase levels were only slightly subnormal (ZOOO2500) 3 weeks following the operations. However, these rats had still not completely recovered from the surgery and were not eating quite normally. It was postulated that if the lungs are a source of amylase production as has been suggested (5), amylase might be secreted into the blood during its passage through the lung. In Table V, the serum amylase contents of blood from the right auricles and the arches of the aorta indicate no significant detectable addition of amylase to pulmonary blood. It is, of course, possible that very small amounts per unit time might be secreted, and, with the large blood flow through the lungs, a considerable quantity of amylase could be added to the blood without the A-V difference being detectable. Our observations do not agree with Takano’s report (12) that in rabbits left cardiac blood has a stronger amylolytic power than right cardiac blood. Five human lung samples obtained from autopsy specimens all had amylase levels (250-950) well above the normal range for human serum, but the delays between the deaths of the patients, the autopsies, and obtaining the specimens make the validity of such values doubtful. Unfortunately, no blood samples were available for these patients. Analyses of the lungs and sera from the cat and hamster showed serum amylase to lung amylase ratios similar to the other animals listed. DISCUSSION
It is significant that when both known sources of amylase production, the pancreas and the salivary glands, are removed from the rat, amylase does not disappear from the serum and tissues. When the animals have recovered from the trauma of surgery and are eating normally, amylase
410
MCGEACHIN,
GLEASON
AND
ADAMS
levels are found to be within the normal range. In those animals who recover only partially from the surgery or who are not eating normally, serum amylase levels may be somewhat subnormal but are still appreciable. Roe, Smith, and Treadwell (3) showed that amylase excretion continued unchanged in their pancreatectomized rats. Calculations indicate that if all sources of serum, tissue, and urinary amylase were removed, such amylase should all be cleared from the rat within 3 days. When serum amylase remains at normal or slightly subnormal levels 3 weeks after removal of both pancreas and salivary glands, this is conclusive evidence that there must be some extrapancreatic, extrasalivary source of amylase formation in the rat, confirming previous suggestions (3, 6, 13) that such a source must exist. The data on the tissue amylase to serum amylase ratios compared to the extracellular fluid fractions for various tissues and organs indicate that in the rat, mouse, guinea pig, and dog, there is intracellular amylase in the liver. For the mouse, and guinea pig (and perhaps the dog), there is also intracellular muscle amylase. The presence of an intracellular concentration of an enzyme of course does not prove that the enzyme is synthesized in that cell but does indicate this possibility. While Somogyi (13) and Cori, Cori, and Schmidt (14) indicated that liver has no intracellular amylase, Roe, Smith, and Treadwell state (3) that, under certain conditions, the liver may contain 5-10 times as much amylase as can be accounted for by the blood in the organ. Preliminary results in this laboratory on isolated, washed, whole liver cells indicate that these cells do contain amylase. When these facts are considered together with the data indicating that low serum amylase levels are associated with liver damage (15), the liver as a site of amylase formation seemsa likely possibility. This is presently the subject of further investigation. Wiberg and Tuba (6) suggested the intestinal mucosa as a possible site of amylase formation. Our data confirm this possibility since there is amylase still to be found in the gastrointestinal tract following pancreatectomy and salivarectomy. Takano’s (5) suggestion that amylase is formed in the lungs seemsof doubtful validity. SUMMARY
1. The distribution of amylase in the serum and tissues of pancreatectomized-salivarectomized rats is essentially the same as in the normal except for the gastrointestinal tract. 2. Calculations from tissue amylase to serum amylase ratios and
AMYLASE DISTRIBUTION
111
extracellular fluid contents indicate that the liver in the rat, mouse, guinea pig, and dog, and muscle in the mouse and guinea pig have intracellular amylase. 3. Tissue and serum amylase activities are essentially the same in pH 7.0 phosphate and pH 7.6 Verona1 buffers, except for muscle and pancreas. 4. No detectable amylase is added to the blood passing through the ungs of dogs. REFERENCES 1. MARKOWITZ, J., AND HOUGH, H. B., AWL. J. Physiol. 76, 571 (1926). 2. REID, E., &UIGLEY, J. P., AND MYERS, V. C., J. Riol. Chern. 99, 615 (1933). 3. ROE, J. H., JR., SMITH, B. W., AND TREADWELL, C. I~., I’roc. Sot. h’zptl. Riol. Med. 87, 79 (1954). 4. MCGEACHIN, R. L., AND GLEASON, J. R., Science 123, 841 (1956). 5. TAKANO, T., J. Oriental Afed. 28, 1231 (1938). 6. WIBERG, G. S., AND TUBA, J., Can. J. Biochem. Physiol. 33, 817 (1955). 7. VAN LOON, E. J., LIEENS, M. R., AND SEGER, A. J., Am. J. Clin. Puthol. 22, 1134 (1952). 8. CORI, G. T., AND LARNER, J., J. Biol. Chem. 186, 17 (1951). 9. TREADWELL, C. R., AND ROE, J. H., Proc. Sot. Exptl. Biol. Med. 86,878 (1954). 10. SUTHERLAND, E. W., in “A Symposium on the Clinical and Biological Aspects of Carbohydrate Utilization in Health and Disease” (Najjar, V. A,, ed.), p. 25, The Johns Hopkins Press, Baltimore, 1952. 11. MANERY, J. F., Physiol. Revs. 34, 334 (1954). 12. TAKANO, T., J. Oriental Med. 28, 1263 (1938). 13. SOMOGYI, M., A.M.A. Arch. Znternal Med. 67, 665 (1941). 14. CORI, C. F., CORI, G. T., AND SCHMIDT, G., J. Biol. Chem. 129, 631 (1939). 15. GRAY, 8. H., PROBSTEIN, J. G., AND HEIFETZ, C. J., A.M.A. Arch. Internal Med. 67, 805 (1941).