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Electrophoretic Study of Human Isoamylases
Vol. 51, No.3 Printed in U.S.A.
GASTROENTEROLOGY
Copyright
© 1966 by The Williams & Wilkins Co.
ELECTROPHORETIC STUDY OF HUMAN ISOAMYLASES A new saccharogenic staining method and p1·eliminary l'esults RAMON R. JosEPH, M.D., EDWIN OLIVERO, M.S., AND NEWTON RESSLER, PH.D.
Depa1·tments of Medicine and Pathology, Wayne County General Hospi tal, Eloise , and University of Michigan M edical School, Ann Arbor, Michigan
The concept of isozymes, or heterogeneous forms of enzymes, having the same substrate specificity and catalyzing the same reaction has been well established for a number of enzymes in clinical medicine. 1 Attempts to extend this concept to amylase have been handicapped by reports yielding conflicting data. 2 In early reports of heterogeneous amylases in human serum, 1vicGeachin and Lewis 3 reported that amylase found in normal human serum had an electrophoretic mobility similar to serum albumin, while sera from patients with pancreatitis had amylase activity corresponding in mobility to serum y-globulin. These findings were confirmed by Dreiling et al. 4 However, Wilding 5 and Ujihira et al. 6 reported amylase activity only in the y-globulin fraction in both normal sera and sera from patients with pancreatitis. Searcy et al.7 have demonstrated that the discrepancies reported may be related to whether an amyloclastic or saccharogenic method was used to estimate amylase activity and have suggested that the results reported by those investigators using an amyloclastic method were partly artifactual. All authors to date agree that in sera from patients with pancreatitis most of the amylase activity is in the y-globulin fraction . Berk and Searcy 2 have summarized the evidence, which indicates that at least part of the amylase activity in the yglobulin fraction originates from the pancreas, but were unable to distinguish this
pancreatic amylase from other amylases having similar electrophoretic mobility. In order to differentiate small differences in electrophoretic mobility more accurately, a method has been developed by which amylase activity can be visualized directly on the electrophoretic plate utilizing a modification of the coupled glucose oxidase-peroxidase-chromogen reaction. 8 Materials and Method Electrophoresis. Glass plates (25 X 15 X
Received April 2, 1966. Accepted April 22, 1966. Address requests for reprints to: Dr. Ramon R. Joseph, Gastroenterology Section, Wayne County General Hospital, Eloise, Michigan 48132. This study was supported in part by a grant from the United States Public Health Service.
0.5 em) containing a thin layer of 0.4% agar (Difco N able agar) were prepared, and agar electrophoresis was conducted as described by Ressler and Moy,• using 0.05 M phosphate buffer at pH 7.25. Approximately 1-gm sections, of human liver and pancreas, obtained from fresh autopsy specimens, were homogenized in 2 ml of 0.9% saline. Serum and saliva were obtained from laboratory personnel, and serum was obtained from 10 hospitalized patients with pancreatitis and from 9 patients without pancreatitis whose sera had high amylase values (3 patients with renal failure, 2 patients with hepatic cirrhosis, 1 patient with hepatitis, 1 patient with perforated duodenal ulcer, and 2 patients with unknown or uncertain diagnoses). Small filter paper strips (1 em X 0.1 em, Whatman no. 17) were saturated with tissue homogenate, serum, or saliva and carefully imbedded in the agar on the glass plates. The electrophoretic cell was covered with plastic wrap to prevent evaporation. Electrophoresis was conducted at 75 v overnight (10 to 14 hr) at room temperature. A sample of serum stained with bromphenol blue was included on each plate to serve as a marker to note the distance of migration. It was found advisable to include controls of liver and pancreatic tissue when analyzing unknown sera. Reagents. Glucose oxidase reagent: One vial of o-dianisidine (Glucostat reduced chromogen
377
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JOSEPH ET AL.
containing o-dianisidine and peroxidase, Worthington Biochemical Corporation, Freehold, N. J.) was dissolved in 30 ml of distilled water. One vial of purified, polysaccharidase-free glucose oxidase (Glucostat special glucose oxidase, Worthington) was added to the solution, and then diluted to 45 ml. This stock solution can be kept fro zen for 1 week and thawed just before using. Iodine reagent : A 0.1 N stock iodine reagent \\·as prepared by adding 1.7835 g of KIOa and 22.5 g of KI to 400 ml of water. To this was slowly added 4.5 ml of concentrated HCl (sp. gr . 1.18), and the final solution was diluted to 500 ml with distilled water. Staining. After the albumin had migrated a sufficient distance to allow separation of plasma components, the current was disconnected and the glass plate removed. It was then placed in a shallow container and covered with a thin film of Glucostat reagent solution prepared by t hawing 10 ml of Glucostat reagent, and adding 10 mg of maltase (standardized, 600 paranit rophenyl gluconide tmits, Nutritional Biochemical Company, Cleveland) and 10 ml of 2% soluble starch reagent (Nutritional Bio-
Vol. 51, No . 3
chemical). The plate was then incubated at 37 C. The reaction was stopped after 30 min by rinsing with 4 M HCl. A blue precipitate formed at the site of amylase activity. Confirmatory studies. To confirm that the precipitates corresponded to amylase activity, five agar plates were prepared and subjected to electrophoresis simultaneously in a single cell. The experiment was run in duplicate. Two plates contained samples of liver, pancreas, and saliva. One of these was stained for amylase activity with the method described above, t he other was stained for amylase activity by a modification of the method of Wilding5 in which the agar plate was incubated with 1% soluble starch solution for 30 min at 37 C and then covered with stock iodine reagent. Amylase appeared as clear bands in the agar against a dark blue background. The remaining t hree plates contained samples of liver, pancreas, saliva, and pancreatitis serum . One of these plates was stained for protein with Amido black, one was stained for amylase with the glucose oxidase method described above, and the third was sectioned at 3-mm intervals between t he anode and the
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FIG. 1. Electrophoresis of human isoamylases. Plate on left is stained for protein with Amido black; plate on right is stained for amylase with glucose oxidase method. The straight line in middle of picture is the origin, and the anode is at bottom of picture. Specimens are, from left to right: liver homogenate (L), saliva (Sa), normal serum (S), and pancreas homogenate (P) . Note that faint amylase band (arrow) in normal serum has same mobility as amylase band in liver.
cathode and each individual section was analyzed for amylase activity by the dinitrosalicylic acid method of Ujihira et al." Results
A blue precipitate formed at the site of amylase activity. Under the conditions employed, all tissues showed bands of activity which migrated toward the cathode. Liver amylase moved farthest from the origin, followed by pancreatic amylase. Salivary amylase remained closest to the origin. The amylase activity of liver, pancreas, and saliva appeared to have slightly different electrophoretic mobilities (fig. 1). Sera from normal controls had a band of amylase activity similar in mobility to the band noted in liver. Sera from the 10 patients \Yith pancreatitis had a band of amylase activity corresponding in mobility to pancreatic amylase. Sera from the patients with renal failure · and liver disease
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ELECTROPHORETIC STUDY OF ISOAMYLASES
September 1966
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had amylase mobility similar to liver. One patient with a serum amylase value of 3780 Somogyi units, and no pancreatitis at postmortem examination, had serum amylase activity similar to liver amylase (fig. 2). All human amylase fractions had mobility roughly corresponding to y-globulin. Eluates from sections corresponding to the serum proteins showed activity in the y-globulin fraction for all tissues. Eluates from sections cut at 3-mm intervals confirmed that there were definite small differences between the various tissues, which ·were easily appreciated on the stained plates but not apparent if sections were made corresponding to the serum proteins (fig. 3). Liver amylase, which gave a band in the y-globulin zone on the plate stained with the glucose oxidase method, and had amylase activity in the y-globulin zone when eluted and assayed by the dinitrosalicylic
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Fra. 2. Isoamylase electrophoresis. Specimens are from a 51-year-old female patient who was admitted to hospital with diabetic acidosis, abdominal pain, and vascular collapse and who died in renal failure. Serum amylase 1 day before death was 3780 Somogyi units. At autopsy, no pancreatitis or liver disease was found. Plate on left is stained for proteins, plate on right is stained for amylase with glucose oxidase m ethod. Samples are, from left to right: marker (M), patient's liver homogenate (L), patient's pancreas homogenate (P), patient's serum (S), and normal serum control (Sc). Note that patient's serum amylase and normal serum amylase (closed arrows) have mobility similar to liver amylase (open arrow), and different from pancreas amylase.
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Fra. 3. Comparison of serum proteins, amylase activity by glucose oxidase staining method, and amylase activity determined biochemically. After electrophoresis, agar plate was sectioned at 3-mm intervals, and amylase activity eluted in saline and determined by dinitrosalicylic acid method. Relative positions of serum proteins and bands obtained by glucose oxidase method are superimposed at top of figure. Note that all amylases have mobility similar to ')'-globulin, that bands obtained with glucose oxidase staining method correlate exactly with amylase activity in eluates from corresponding sections, and that pancreatitis serum has both liver and pancreatic components with most amylase activity similar to pancreas. Small peak at origin is artifact due to amylase activity remaining on filter paper wicks after electrophoresis.
acid method, showed no reaction when stained with the iodine-starch-amyloclastic method (fig. 4). Pancreas and saliva gave bands with the iodine-starch method which were identical in position to the bands seen with the glucose oxidase method. Discussion
Advantages of new method. Most of the discrepancies found in previous reports of isoamylases have been the result of limitations of method. The amyloclastic method used by early investigators yielded baffling results in which the sum of the isoamylases
found in normal serum exceeded the total value found in the whole serum and necessitated the postulation of an "inhibiting factor" which was said to be present in normal serum but not in serum from patients with pancreatitis. 3 • 4 It was later shown that proteins, 5 hemoglobin, 10 dilution,11 and possibly other serum factors affected the starch-iodine color which was the basis of the reaction, and that heat inactivation of amylase activity actually enhanced the values obtained. 12 Available saccharogenic methods were not easily adaptable in our hands to the small quan-
September 1966
ELECTROPHORETIC STUDY OF ISOAMYLASES
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381
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Fw. 4. Isoamylase electrophoresis. Plate on left is stained by amyloclastic method, plate on right is stained by glucose oxidase saccharogenic method. Samples on each plate are, from left to right: liver homogenate (L), saliva (Sa), and pancreas homogenate (P). Note that liver homogenate gives definite amylase band with saccharogenic method, but no band with amyloclastic method.
tities of eluate available after electro- This was accomplished enzymatically with phoretic separation, and it was not until maltase. Lack of background staining inUjihira and colleagues 6 described a modi- dicated no significant amylase activity in fication of the dinitrosalicylic acid reac- the maltase. The reaction in the method tion that a saccharogenic reaction suitable used consisted of liberation of maltose by for our purposes was available. This latter amylase activity on a starch substrate, method, while extremely sensitive, still conversion of maltose to glucose by malthad the disadvantage that it was not ase, oxidation of glucose to gluconic acid possible to visualize directly the site of and hydrogen peroxide by glucose oxidase, amylase activity on the paper strips used. and production of a visible precipitate at In order to localize amylase activity, it the site of reaction by oxidation of the was necessary to make arbitrary sections o-dianisidine by peroxide in the presence of of the paper, elute amylase activity from peroxidase. It was found that this entire the sections, and compare the amylase reaction sequence could be accomplished in activity from the sections to strips of a single step. It is essential to use especially paper which were subjected to electro- purified glucose oxidase, since most comphoresis simultaneously and stained for mercially available glucose oxidases contain traces of polysaccharidases,1 3 which protein. Glucose oxidase has been used for the we found reacted with the substrate and enzymatic determination of glucose. 8 When gave an unsatisfactory background stain to coupled with a reduced chromogen such the entire plate. Significance of results. The normal huas o-dianisidine, it has the advantage of producing a visible blue precipitate. The man sera tested were found to have a single reaction also takes place at room tempera- zone of amylase activity by the glucose ture, a decided advantage for direct visual- oxidase method. This zone appeared to ization of amylase over other methods of have the same mobility as the single zone measuring glucose which require boiling at found when homogenate of human liver some stage during the assay. Since glucose was analyzed for isoamylase activity, and oxidase is specific for glucose, it was neces- corresponded in mobility to serum y-globusary to convert maltose formed by hy- lin, confirming the results of other workers drolysis of starch by amylase to glucose. using a saccharogenic method. 6• 7 The sera
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JOSEPH ET AL.
of our patients with pancreatitis, however, had amylase activity with less mobility toward the cathode than was noted in liver, £tnd was identical to the mobility noted in homogenate of human pancreas. Human saliva had amylase activity showing a mobility different from liver or pancreas, and different from normal or pancreatitic serum. Norby,l 4 utilizing similar electrophoretic methods, and identifying amylase by the amyloclastic method on plates, also found different mobilities for salivary and pancreatic amylase. Further study is needed to determine the reason why liver amylase is clearly demonstrable by saccharogenic methods, but differs from salivary and pancreatic amylase in failing to prevent the iodinestarch color reaction. Our results indicate that the method described is simple enough for routine clinical use and that determination of isoamylase activity may yield useful clinical information since it may be possible to differentiate pancreatic from nonpancreatic elevat ion of serum amylase. This distinction may be crucial in distinguishing acute pancreatitis from other intraabdominal catastrophies associated with high amylase values, in deciding whether a high amylase value in a patient with renal insufficiency is due to failure of the kidney to excrete liver amylase or to pancreatitis, and in distinguishing mumps pancreatitis from mumps parotitis. Studies are currently in progress to confirm these potential usages in a large series of cases, and to determine the isoamylase composition of muscle, Fallopian tube, and urine in normal and disease states. Summary
A new saccharogenic method is described for the direct visualization of isoamylases after agar electrophoresis. At least three different human isoamylases exist, originating, respectively, from liver, pancreas, and salivary gland. Normal human serum analyzed to date has amylase activity with mobility similar to liver amylase. Sera from 10 patients with acute pancreatitis had amylase activity similar to pancreatic amylase. All human isoamyl-
Vol. 51, No.3
ases detected to date have electrophoretic mobility in agar similar to y-globulin. REFERENCES 1. Kaplan, N. 0. 1963. Symposium on multiple
forms of enzymes and control mechanisms. I. Multiple forms of enzymes. Bact. R ev. 27: 155-169.2. Berk, J. E., and R. L. Searcy. 1965. I soen-
zymes of serum amylase in man. Gastroenterology 48 : 651-653. 3. McGeachin, R. L., and J.P. Lewis. 1959. Electrophoretic bepavior of serum amylase . J. Bioi. Chern. 234 : 795-798. 4. Dreiling, D. A., H . D. Janowitz, and L. J . Josephberg. 1963. Serum iso-amylases; an electrophoretic study of the blood amylase and the patterns observed in pancreatic disease. Ann . Intern. Med. 58: 235-244. 5. Wilding, P. 1963. Use of gel filtration in the study of human amylase. Clin. Chim. Acta 8: 918-924.
6. Ujihira, I., R. L. Searcy, J. E. Berk, and S. Hayashi. 1965. A saccharogenic method of estimating electrophoretic and chromatographic distribution of human serum amy lase. Clin. Chern. 11: 97-112. 7. Searcy, R. L., I. Ujihira, S. Hayashi, and J. E. Berk. 1964. An intrinsic disparity between amyloclastic and saccharogenic estimations of human serum isoamylase activities. Clin. Chim. Acta 9 : 505-508. 8. Saifer, A., and S. Gerstenfel. 1958. The photometric microdetermination of blood glucose with glucose oxidase. J. Lab. Clin. Med. 51: 448-460. 9. Ressler, N., and T. Moy. 1959. Simplified fluid
film method of electrophoresis. Clin. Chim. Acta 4: 901-904. 10. Reif, A. E., and D. C. Nabseth. 1962. Serum amylase determination by Somogyi's amyloclastic method with use of a photometric end point. Clin. Chern . 8: 113-129. 11. Theodor, E., and D. Birnbaum. 1964. The effect of dilution on the activity of amylase and its relation to the effect of electrophoresis. J. Lab. Clin. Med. 63: 879-884. 12. Berk, J . E., R. L. Searcy, S. Hayashi, and I. Ujihira. 1965. Distribution of serum amylase in man and animals ; electrophoretic and chromatographic studies. J . A. M . A. 192: 389-393. 13. Adams, E. D., Jr., R. L. Mast, and A. H. Free. 1960. Specificity of glucose oxidase. Arch. Biochem. Biophys. 91: 230-234. 14. Norby, S. 1964. Electrophoretic non-identity
of human salivary and pancreatic amylases. Exp. Cell Res. 36: 663-702.
GASTROENTEROLOGY
Copyright
© 1966 by The Williams & Wilkins Co.
ELECTROPHORETIC STUDY OF HUMAN ISOAMYLASES A new saccharogenic staining method and p1·eliminary l'esults RAMON R. JosEPH, M.D., EDWIN OLIVERO, M.S., AND NEWTON RESSLER, PH.D.
Depa1·tments of Medicine and Pathology, Wayne County General Hospi tal, Eloise , and University of Michigan M edical School, Ann Arbor, Michigan
The concept of isozymes, or heterogeneous forms of enzymes, having the same substrate specificity and catalyzing the same reaction has been well established for a number of enzymes in clinical medicine. 1 Attempts to extend this concept to amylase have been handicapped by reports yielding conflicting data. 2 In early reports of heterogeneous amylases in human serum, 1vicGeachin and Lewis 3 reported that amylase found in normal human serum had an electrophoretic mobility similar to serum albumin, while sera from patients with pancreatitis had amylase activity corresponding in mobility to serum y-globulin. These findings were confirmed by Dreiling et al. 4 However, Wilding 5 and Ujihira et al. 6 reported amylase activity only in the y-globulin fraction in both normal sera and sera from patients with pancreatitis. Searcy et al.7 have demonstrated that the discrepancies reported may be related to whether an amyloclastic or saccharogenic method was used to estimate amylase activity and have suggested that the results reported by those investigators using an amyloclastic method were partly artifactual. All authors to date agree that in sera from patients with pancreatitis most of the amylase activity is in the y-globulin fraction . Berk and Searcy 2 have summarized the evidence, which indicates that at least part of the amylase activity in the yglobulin fraction originates from the pancreas, but were unable to distinguish this
pancreatic amylase from other amylases having similar electrophoretic mobility. In order to differentiate small differences in electrophoretic mobility more accurately, a method has been developed by which amylase activity can be visualized directly on the electrophoretic plate utilizing a modification of the coupled glucose oxidase-peroxidase-chromogen reaction. 8 Materials and Method Electrophoresis. Glass plates (25 X 15 X
Received April 2, 1966. Accepted April 22, 1966. Address requests for reprints to: Dr. Ramon R. Joseph, Gastroenterology Section, Wayne County General Hospital, Eloise, Michigan 48132. This study was supported in part by a grant from the United States Public Health Service.
0.5 em) containing a thin layer of 0.4% agar (Difco N able agar) were prepared, and agar electrophoresis was conducted as described by Ressler and Moy,• using 0.05 M phosphate buffer at pH 7.25. Approximately 1-gm sections, of human liver and pancreas, obtained from fresh autopsy specimens, were homogenized in 2 ml of 0.9% saline. Serum and saliva were obtained from laboratory personnel, and serum was obtained from 10 hospitalized patients with pancreatitis and from 9 patients without pancreatitis whose sera had high amylase values (3 patients with renal failure, 2 patients with hepatic cirrhosis, 1 patient with hepatitis, 1 patient with perforated duodenal ulcer, and 2 patients with unknown or uncertain diagnoses). Small filter paper strips (1 em X 0.1 em, Whatman no. 17) were saturated with tissue homogenate, serum, or saliva and carefully imbedded in the agar on the glass plates. The electrophoretic cell was covered with plastic wrap to prevent evaporation. Electrophoresis was conducted at 75 v overnight (10 to 14 hr) at room temperature. A sample of serum stained with bromphenol blue was included on each plate to serve as a marker to note the distance of migration. It was found advisable to include controls of liver and pancreatic tissue when analyzing unknown sera. Reagents. Glucose oxidase reagent: One vial of o-dianisidine (Glucostat reduced chromogen
377
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JOSEPH ET AL.
containing o-dianisidine and peroxidase, Worthington Biochemical Corporation, Freehold, N. J.) was dissolved in 30 ml of distilled water. One vial of purified, polysaccharidase-free glucose oxidase (Glucostat special glucose oxidase, Worthington) was added to the solution, and then diluted to 45 ml. This stock solution can be kept fro zen for 1 week and thawed just before using. Iodine reagent : A 0.1 N stock iodine reagent \\·as prepared by adding 1.7835 g of KIOa and 22.5 g of KI to 400 ml of water. To this was slowly added 4.5 ml of concentrated HCl (sp. gr . 1.18), and the final solution was diluted to 500 ml with distilled water. Staining. After the albumin had migrated a sufficient distance to allow separation of plasma components, the current was disconnected and the glass plate removed. It was then placed in a shallow container and covered with a thin film of Glucostat reagent solution prepared by t hawing 10 ml of Glucostat reagent, and adding 10 mg of maltase (standardized, 600 paranit rophenyl gluconide tmits, Nutritional Biochemical Company, Cleveland) and 10 ml of 2% soluble starch reagent (Nutritional Bio-
Vol. 51, No . 3
chemical). The plate was then incubated at 37 C. The reaction was stopped after 30 min by rinsing with 4 M HCl. A blue precipitate formed at the site of amylase activity. Confirmatory studies. To confirm that the precipitates corresponded to amylase activity, five agar plates were prepared and subjected to electrophoresis simultaneously in a single cell. The experiment was run in duplicate. Two plates contained samples of liver, pancreas, and saliva. One of these was stained for amylase activity with the method described above, t he other was stained for amylase activity by a modification of the method of Wilding5 in which the agar plate was incubated with 1% soluble starch solution for 30 min at 37 C and then covered with stock iodine reagent. Amylase appeared as clear bands in the agar against a dark blue background. The remaining t hree plates contained samples of liver, pancreas, saliva, and pancreatitis serum . One of these plates was stained for protein with Amido black, one was stained for amylase with the glucose oxidase method described above, and the third was sectioned at 3-mm intervals between t he anode and the
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FIG. 1. Electrophoresis of human isoamylases. Plate on left is stained for protein with Amido black; plate on right is stained for amylase with glucose oxidase method. The straight line in middle of picture is the origin, and the anode is at bottom of picture. Specimens are, from left to right: liver homogenate (L), saliva (Sa), normal serum (S), and pancreas homogenate (P) . Note that faint amylase band (arrow) in normal serum has same mobility as amylase band in liver.
cathode and each individual section was analyzed for amylase activity by the dinitrosalicylic acid method of Ujihira et al." Results
A blue precipitate formed at the site of amylase activity. Under the conditions employed, all tissues showed bands of activity which migrated toward the cathode. Liver amylase moved farthest from the origin, followed by pancreatic amylase. Salivary amylase remained closest to the origin. The amylase activity of liver, pancreas, and saliva appeared to have slightly different electrophoretic mobilities (fig. 1). Sera from normal controls had a band of amylase activity similar in mobility to the band noted in liver. Sera from the 10 patients \Yith pancreatitis had a band of amylase activity corresponding in mobility to pancreatic amylase. Sera from the patients with renal failure · and liver disease
M M
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ELECTROPHORETIC STUDY OF ISOAMYLASES
September 1966
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had amylase mobility similar to liver. One patient with a serum amylase value of 3780 Somogyi units, and no pancreatitis at postmortem examination, had serum amylase activity similar to liver amylase (fig. 2). All human amylase fractions had mobility roughly corresponding to y-globulin. Eluates from sections corresponding to the serum proteins showed activity in the y-globulin fraction for all tissues. Eluates from sections cut at 3-mm intervals confirmed that there were definite small differences between the various tissues, which ·were easily appreciated on the stained plates but not apparent if sections were made corresponding to the serum proteins (fig. 3). Liver amylase, which gave a band in the y-globulin zone on the plate stained with the glucose oxidase method, and had amylase activity in the y-globulin zone when eluted and assayed by the dinitrosalicylic
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Fra. 2. Isoamylase electrophoresis. Specimens are from a 51-year-old female patient who was admitted to hospital with diabetic acidosis, abdominal pain, and vascular collapse and who died in renal failure. Serum amylase 1 day before death was 3780 Somogyi units. At autopsy, no pancreatitis or liver disease was found. Plate on left is stained for proteins, plate on right is stained for amylase with glucose oxidase m ethod. Samples are, from left to right: marker (M), patient's liver homogenate (L), patient's pancreas homogenate (P), patient's serum (S), and normal serum control (Sc). Note that patient's serum amylase and normal serum amylase (closed arrows) have mobility similar to liver amylase (open arrow), and different from pancreas amylase.
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JOSEPH ET AL. ALBUMIN
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ORIGIN (DISTANCE IN MM l
Fra. 3. Comparison of serum proteins, amylase activity by glucose oxidase staining method, and amylase activity determined biochemically. After electrophoresis, agar plate was sectioned at 3-mm intervals, and amylase activity eluted in saline and determined by dinitrosalicylic acid method. Relative positions of serum proteins and bands obtained by glucose oxidase method are superimposed at top of figure. Note that all amylases have mobility similar to ')'-globulin, that bands obtained with glucose oxidase staining method correlate exactly with amylase activity in eluates from corresponding sections, and that pancreatitis serum has both liver and pancreatic components with most amylase activity similar to pancreas. Small peak at origin is artifact due to amylase activity remaining on filter paper wicks after electrophoresis.
acid method, showed no reaction when stained with the iodine-starch-amyloclastic method (fig. 4). Pancreas and saliva gave bands with the iodine-starch method which were identical in position to the bands seen with the glucose oxidase method. Discussion
Advantages of new method. Most of the discrepancies found in previous reports of isoamylases have been the result of limitations of method. The amyloclastic method used by early investigators yielded baffling results in which the sum of the isoamylases
found in normal serum exceeded the total value found in the whole serum and necessitated the postulation of an "inhibiting factor" which was said to be present in normal serum but not in serum from patients with pancreatitis. 3 • 4 It was later shown that proteins, 5 hemoglobin, 10 dilution,11 and possibly other serum factors affected the starch-iodine color which was the basis of the reaction, and that heat inactivation of amylase activity actually enhanced the values obtained. 12 Available saccharogenic methods were not easily adaptable in our hands to the small quan-
September 1966
ELECTROPHORETIC STUDY OF ISOAMYLASES
(
381
...
Sa
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Fw. 4. Isoamylase electrophoresis. Plate on left is stained by amyloclastic method, plate on right is stained by glucose oxidase saccharogenic method. Samples on each plate are, from left to right: liver homogenate (L), saliva (Sa), and pancreas homogenate (P). Note that liver homogenate gives definite amylase band with saccharogenic method, but no band with amyloclastic method.
tities of eluate available after electro- This was accomplished enzymatically with phoretic separation, and it was not until maltase. Lack of background staining inUjihira and colleagues 6 described a modi- dicated no significant amylase activity in fication of the dinitrosalicylic acid reac- the maltase. The reaction in the method tion that a saccharogenic reaction suitable used consisted of liberation of maltose by for our purposes was available. This latter amylase activity on a starch substrate, method, while extremely sensitive, still conversion of maltose to glucose by malthad the disadvantage that it was not ase, oxidation of glucose to gluconic acid possible to visualize directly the site of and hydrogen peroxide by glucose oxidase, amylase activity on the paper strips used. and production of a visible precipitate at In order to localize amylase activity, it the site of reaction by oxidation of the was necessary to make arbitrary sections o-dianisidine by peroxide in the presence of of the paper, elute amylase activity from peroxidase. It was found that this entire the sections, and compare the amylase reaction sequence could be accomplished in activity from the sections to strips of a single step. It is essential to use especially paper which were subjected to electro- purified glucose oxidase, since most comphoresis simultaneously and stained for mercially available glucose oxidases contain traces of polysaccharidases,1 3 which protein. Glucose oxidase has been used for the we found reacted with the substrate and enzymatic determination of glucose. 8 When gave an unsatisfactory background stain to coupled with a reduced chromogen such the entire plate. Significance of results. The normal huas o-dianisidine, it has the advantage of producing a visible blue precipitate. The man sera tested were found to have a single reaction also takes place at room tempera- zone of amylase activity by the glucose ture, a decided advantage for direct visual- oxidase method. This zone appeared to ization of amylase over other methods of have the same mobility as the single zone measuring glucose which require boiling at found when homogenate of human liver some stage during the assay. Since glucose was analyzed for isoamylase activity, and oxidase is specific for glucose, it was neces- corresponded in mobility to serum y-globusary to convert maltose formed by hy- lin, confirming the results of other workers drolysis of starch by amylase to glucose. using a saccharogenic method. 6• 7 The sera
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of our patients with pancreatitis, however, had amylase activity with less mobility toward the cathode than was noted in liver, £tnd was identical to the mobility noted in homogenate of human pancreas. Human saliva had amylase activity showing a mobility different from liver or pancreas, and different from normal or pancreatitic serum. Norby,l 4 utilizing similar electrophoretic methods, and identifying amylase by the amyloclastic method on plates, also found different mobilities for salivary and pancreatic amylase. Further study is needed to determine the reason why liver amylase is clearly demonstrable by saccharogenic methods, but differs from salivary and pancreatic amylase in failing to prevent the iodinestarch color reaction. Our results indicate that the method described is simple enough for routine clinical use and that determination of isoamylase activity may yield useful clinical information since it may be possible to differentiate pancreatic from nonpancreatic elevat ion of serum amylase. This distinction may be crucial in distinguishing acute pancreatitis from other intraabdominal catastrophies associated with high amylase values, in deciding whether a high amylase value in a patient with renal insufficiency is due to failure of the kidney to excrete liver amylase or to pancreatitis, and in distinguishing mumps pancreatitis from mumps parotitis. Studies are currently in progress to confirm these potential usages in a large series of cases, and to determine the isoamylase composition of muscle, Fallopian tube, and urine in normal and disease states. Summary
A new saccharogenic method is described for the direct visualization of isoamylases after agar electrophoresis. At least three different human isoamylases exist, originating, respectively, from liver, pancreas, and salivary gland. Normal human serum analyzed to date has amylase activity with mobility similar to liver amylase. Sera from 10 patients with acute pancreatitis had amylase activity similar to pancreatic amylase. All human isoamyl-
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ases detected to date have electrophoretic mobility in agar similar to y-globulin. REFERENCES 1. Kaplan, N. 0. 1963. Symposium on multiple
forms of enzymes and control mechanisms. I. Multiple forms of enzymes. Bact. R ev. 27: 155-169.2. Berk, J. E., and R. L. Searcy. 1965. I soen-
zymes of serum amylase in man. Gastroenterology 48 : 651-653. 3. McGeachin, R. L., and J.P. Lewis. 1959. Electrophoretic bepavior of serum amylase . J. Bioi. Chern. 234 : 795-798. 4. Dreiling, D. A., H . D. Janowitz, and L. J . Josephberg. 1963. Serum iso-amylases; an electrophoretic study of the blood amylase and the patterns observed in pancreatic disease. Ann . Intern. Med. 58: 235-244. 5. Wilding, P. 1963. Use of gel filtration in the study of human amylase. Clin. Chim. Acta 8: 918-924.
6. Ujihira, I., R. L. Searcy, J. E. Berk, and S. Hayashi. 1965. A saccharogenic method of estimating electrophoretic and chromatographic distribution of human serum amy lase. Clin. Chern. 11: 97-112. 7. Searcy, R. L., I. Ujihira, S. Hayashi, and J. E. Berk. 1964. An intrinsic disparity between amyloclastic and saccharogenic estimations of human serum isoamylase activities. Clin. Chim. Acta 9 : 505-508. 8. Saifer, A., and S. Gerstenfel. 1958. The photometric microdetermination of blood glucose with glucose oxidase. J. Lab. Clin. Med. 51: 448-460. 9. Ressler, N., and T. Moy. 1959. Simplified fluid
film method of electrophoresis. Clin. Chim. Acta 4: 901-904. 10. Reif, A. E., and D. C. Nabseth. 1962. Serum amylase determination by Somogyi's amyloclastic method with use of a photometric end point. Clin. Chern . 8: 113-129. 11. Theodor, E., and D. Birnbaum. 1964. The effect of dilution on the activity of amylase and its relation to the effect of electrophoresis. J. Lab. Clin. Med. 63: 879-884. 12. Berk, J . E., R. L. Searcy, S. Hayashi, and I. Ujihira. 1965. Distribution of serum amylase in man and animals ; electrophoretic and chromatographic studies. J . A. M . A. 192: 389-393. 13. Adams, E. D., Jr., R. L. Mast, and A. H. Free. 1960. Specificity of glucose oxidase. Arch. Biochem. Biophys. 91: 230-234. 14. Norby, S. 1964. Electrophoretic non-identity
of human salivary and pancreatic amylases. Exp. Cell Res. 36: 663-702.