159
Clinica Chimica Acta, 89 (1978) 159-164 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 9654B
PANCREATITIS-LIKE
ISOAMYLASE
PATTERN
IN NORMAL
PERSONS
MAKOTO OTSUKI, HOSAI YUU, MITSUO MAEDA, SUSUMU SAEKI, KUNIYASU OKANO, TOM10 YAMASAKI, TSUTOMU KANDA, CHOITSU SAKAMOTO and SHIGEAKI BABA Second Department Kobe, 650 (Japan) (Received
April 14th,
of Internal Medicine, Kobe University School of Medicine, Ikuta-ku, 1978)
summary On the assumption that a rise in the pancreatic type isoamylases may not necessarily indicate underlying pancreatitis, genetic studies of human serum and urinary amylase isoenzymes have been performed with the use of electrophoresis
.
Although the preponderant increase in the two principal pancreatic isoamylases Amylase-1 and 2 has been accepted to be a specific index of pancreatic involvement, 1.68% of normal persons had Amylase-2 with an elevated amylase activity (named “Dominant Amylase-2”) up to the same levels as the major isoenzymes. Results of pancreozymin-secretin test and other laboratory findings of these persons with Dominant Amylase-2 were all within normal ranges. Pedigree studies confirmed an autosomal dominant mode of inheritance for this variant. The importance of serial determination and pedigree investigations has been shown to distinguish normal persons having Dominant Amylase-2 from patients with pancreatitis without elevated amylase activity. The existence of an inherited trait of pancreatitis-like isoamylase pattern in healthy individuals must be borne in mind before coming to a conclusion when amylase isoenzymes are used for clinical medicine, though preponderance of the pancreatic type isoenzymes in serum and urine has been revealed to be a characteristic finding in pancreatitis. Knowledge of amylase genetic polymorphism provides a scientific basis for amylase isoenzyme interpretation.
Introduction Hyperamylasemia in the absence of acute pancreatitis is well documented, while no significant elevation of serum amylase can be detected in approx-
160
imately 5% of cases of proven acute pancreatitis [ 11. We have shown the relation of isoamylase pattern to the extent of pancreatic inflammation and revealed the possibility of clarifying the severity of pancreatic diseases by electrophoretic amylase fractionations [Z], though the discrepancy between the degree of the rise in serum amylase activity and the extent of the damage to the pancreas is well known. The diagnosis of acute pancreatitis could be confirmed by findings of an elevated amylase activity in Amylase-1 and 2, not only in the hyperamylasemic state but also for a few days after returning to normal levels [2,3]. We, however, further demonstrated the existence of an inherited trait of the pancreatic isoamylases in healthy individuals and called attention to the genetic polymorphism of amylase isoenzyme in clinical medicine [ 21. Contrary to our observations, Berk and Fridhandler are tenacious to their hypothesis that preponderant increase in the pancreatic isoamylase provides support for the suspicion of underlying pancreatitis despite the lack of elevated serum amylase and lipase levels [4], and had some doubts about the occurrence of an inherited trait of the pancreatic isoamylases in healthy individuals [5]. Thus we have screened individual serum and urine samples of 4041 normal persons using a polyacrylamide electrophoretic system for the detection of variants. Materials and methods Serum and urine were obtained from 4041 normal persons (all Japanese) 16 to 59 years old without known diseases. 40 additional samples from patients with acute and chronic pancreatitis were also collected. Amylase isoenzymes were demonstrated by thin-layer sheet polyacrylamide electrophoresis, using methods previously described [ 6,7]. When individuals with variant patterns were found, family members were studied to define the heritability. Total serum and urinary amylase activity was measured by the chromogenic method using blue-dyed starch polymer [ 81 and creatinine concentrations were determined by the method of Jaffd [9] within 24 h of specimen collection. The amylase clearance to creatinine clearance (C,,,/C,,) ratio was measured based on the method of Levitt et al. [lo] and expressed as a percentage: amylase X urine creatiC,ln,lC,, = (urine amylase X serum creatinine)/(serum nine) X 100. Amylase isoenzyme activity in serum and urine was calculated by multiplying the percentage of an isoenzyme in the sample by the total amylase activity. Amylase isoenzyme clearance was calculated as follows; amylase isoenzyme to creatinine clearance ratio = (urine amylase X % isoenzyme in urine X serum creatinine)/(serum amylase X % isoenzyme in serum X urine creatinine) X 100 [ll]. Results and discussion Although the serum and urinary amylase isoenzymes of 98.3% of normal persons consisted of two major isoenzymes (Amylase-1 and 3) and two or three minor ones (Amylase-2, 5 and/or 7), four different patterns of amylase isoenzymes were observed as previously reported [ 6,7]. Of normal persons, 1.68% had Amylase-2 with an elevated amylase activity up to the same levels as the
161
major isoenzymes. We named this isoenzyme “Dominant Amylase-2” for the present consideration. Marked elevations of amylase activity in the two principal pancreatic isoamylases Amylase-1 and 2 were characteristic findings in acute pancreatitis [2,3,12,13]. This particular isoamylase pattern afforded a means for confirming the diagnosis of acute pancreatitis and enabled the diagnosis to be made several days after the onset of illness, since the average half-life of the elevated Amylase-2 in pancreatitis has been shown to be about 10 days [ 31. It is most important in clinical medicine to differentiate Dominant Amylase-2 in normal persons from Amylase-2 in pancreatitis, because the isoamylase pattern in both situations is similar, as demonstrated in Fig. 1.
( I) Acute Pancreatitis
(II)
Normal
Persons
Fig. 1. Changes of electrophoretic pattern of serum amylase isoenzymes in acute pancreatitis (1) and various representative patterns of amylase isoenzymes in normal persons (II). Preponderance of the two principal pancreatic type isoenzyme Amylase-1 and 2 was still observed in W4.5. though serum amylase activity returned to normal levels. Serum and urinary amylase isoenzyme pattern of 98.3% of 4041 normal persons is (10-4. In 1.88% of normal pemons, amylase activity of Amy&2 was increased (H-1.2.3) up to the same levels as Amylase-1 or 3. These isoamylase patterns in normal persons resemble to those of acute pancreatitis. The rest of normal persons (0.2%) had Amylase-1s [ 71.
162
Results of pancreozymin-secretin tests and other laboratory findings in these persons with Dominant Amylase-2 were all within normal ranges, though the preponderant increase in the pancreatic type isoenzymes has been accepted to be a specific index of pancreatic involvement. Total serum amylase activity in no individuals with Dominant Amylase-2 exceeded 186 Somogyi units/100 ml, which represents the mean + 3 S.D. of healthy subjects in our laboratory [ 141. Polyacrylamide gel electrophoresis was performed on pancreatic juice from individuals with Dominant Amylase-2 and from normal persons immediately after collection in ice-cold test-tubes with a double lumen Dreiling tube. Pancreatic juice in normal persons consisted of only one major isoenzyme corresponding to Amylase-1 in serum and urine and a few minor ones corresponding to Amylase-2 and 4. On the other hand, two major bands with the same intensity were identified in pancreatic juice in normal persons with Dominant Amylase-2; one corresponds to Amylase-1 and the other to Dominant Amylase-2 in serum and urine (Fig. 2). By simultaneous electrophoresis of all samples obtained during a pancreozymin-secretin test, no variations in mobility and intensity of these two isoenzymes were found. No isoenzymes corresponding to Dominant Amylase-2 were demonstrable in saliva in these subjects. These observations may indicate that Dominant Amylase-2 is of pancreatic origin and is not the created isoenzyme by posttranslation~ modification such as glycosidation and deamination of gene products [ 151, but is a genetically determined
Fig. 2. Amylaae isoenzymes in urine. serum and pancreatic juice obtained during pancreozyminsecretin test from individuals with Dominant Amylase-2. (A) Control urine and (B) specimens from a case with Dominant Amylase-2. Two major bands with the same intensity were identified in all samples obtained during pancraozymin+ecretin test: one corresponds to Amylase-1 and the other to Dominant Amylase-2 in serum and urine.
163
one. In support of this assumption are the results of family studies (Fig. 3). Individuals within each family were either normal or exhibited the Dominant Amylase-2 of the proband. In two families (Fig. 3B,C), X-linkage was excluded. In family B, II-8 transmitted the Dominant Amylase-2 to III-8 and III-lo. In family C, II-7 failed to transmit the trait to his daughter. Dominant Amylase-2 was noted in every generation in these families. No significant sex difference was apparent among normal and variant individuals. Since the population of individuals tested was homogeneous with regards to age, health and race, no attempt was made to distinguish differences which might be related to these factors. Within each family, the trait seems to be inherited as an autosomal codominant; evidence against X-linkage was observed. These findings strongly suggest the importance of serial determination and pedigree investigations of amylase isoenzymes to distinguish normal persons having Dominant Amylase-2 along with major isoenzyme Amylase-1 from pancreatitis with normal amylase activity . High resolution is the most important requirement for a clinically useful method for identification of the amylase isoenzyme, providing the ability to identify individual isoamylases of the pancreatic and the salivary type. On thin-layer sheet polyacrylamide electrophoresis not only the pancreatic and the salivary type isoenzymes but variant isoamylases will also routinely be detected. In contrast, failure of column chromatography to distinguish Amylase-2 from
(A)
(B)
Dominant
Anylase-2
I-= 0 not tested /P&arid Fig. 3. Pedigrees of families with Dominant Amylase-2. Dominant Amylase-2 was observed in the members shaded and noted in every generation.
164
the salivary type isoenzyme has been suggested [ 12,131, because of the electrophoretic and isoelectric similarity of both isoenzymes [13]. Column chromatography cannot provide simultaneous separation and direct comparability of samples and yields poor resolution of minor and variant isoamylases but provides only resolution of major pancreatic and salivary type isoenzymes. In support of poor resolution of column chromatography are the reports by Berk and his associates that both salivary and pancreatic isoamylases rise in acute pancreatitis [4,16,17] and that the human pancreas contains small amounts of the salivary type amylase [ 181. The mean amylase to creatinine clearance ratio for control and normal persons with Dominant Amylase-2 were 2.9 + 0.38 and 2.37 t 0.19, respectively. In patients with pancreatitis, it was 7.09 t 0.54. C,,,/C,, remained significantly elevated in patients with acute pancreatitis for longer than either serum or urine amylase values [ 191 as a particular isoamylase pattern [ 21. The findings of this study suggest that the amylase creatinine clearance ratio provides a dimension to differentiate Dominant Amylase-2 from the elevated Amylase-2 in pancreatitis. The ratio of urinary clearance of Dominant Amylase-2 (C._,,_z) to C,, was 2.3 + 0.24, which was less than Camy_r/Ccr (2.76 + 0.33), but consistently exceded that of Camy_s /C,, (1.69 t 0.23) of normal persons. Camy_l and Camy_s in acute pancreatitis were 6.98 + 0.71 and 5.0 ? 0.58 of C,,, respectively .
Although preponderance of the pancreatic type isoenzymes in serum and urine has been revealed to be a characteristic finding, the existence of an inherited trait of pancreatitis-like isoamylase pattern in healthy individuals must be borne in mind before coming to a conclusion when amylase isoenzymes are used in clinical medicine. Knowledge of the genetic polymorphism is of importance in clinical assessment of amylase isoenzymes in patients having an elevated Amylase-2 suggestive of pancreatitis. References 1 Kalliomaki. J.L. and Antila. L.E. (1971) J. Am. Geriatr. 19,517-525 2 Otsuki, M.. Yuu, H., Maeda. M., Yamasaki. T., Okano, J., Sakamoto, C. and Baba, S. (1977) Clin. Chim. Acta 79.1-6 3 Legaz. M.E. and Kenny, M.A. (1976) Clin. Chem. 22,57-62 4 Berk, J.E. and Fridhandler, L. (1975) Am. J. Gastroenterol. 63.457463 5 Berk. J.E. and Fridhandler, L. (1978) Clin. Chim. Acta 89,157 6 Otsuki, M., Saeki. S., Kondo, T.. Yuu, H. and Baba, S. (1974) Jap. J. Gastroenterol. 71, 1241-1248 7 Otsuki. M.. Saeki, S., Yuu, H., Maeda. M. and Baba. S. (1976) Clin. Chem. 22,439444 8 Ceska. M.. Birath, K. and Brown, B. (1969) Clin. Chim. Acta 26.437444 9 JaffB, M. (1886) Physiol. Chem. 10, 391-398 10 Levitt, M.D., Rapoport. M. and Cooperband. S.R. (1969) Ann. Intern. Med. 71.919-925 11 Long, E.B. and Grider, J.R. (1976) Gastroenterology 71, 589-593 12 Lehrner. L.M.. Ward, J.C., Kam. R.C., Ehrlich. C.E. and Merritt, A.D. (1976) Am. J. Clin. Pathol. 66, 576-587 13 Warshaw. A.L. and Lee, K.H. (1977) J. Surg. Res. 22.362-369 14 Otiuki. M.. Yuu, H., Maeda, M.. Saeki, S., Yamasaki, T. and Baba, S. (1977) Cancer 39,1656-1663 15 Kam. R.C.. Shulkin, J.C.. Merritt, A.,D. and Newell, R.C.(1973) Biochem. Genet. 10,341-350 16 Fridhandler. L.. Berk. J.E. and Ueda, M. (1972) Clin. Chem. 18.1493-1497 17 Berk, J.E. and Fridhandler. L. (1976) Mount Sinai J. Med. 43, 321337 18 Shimamura. J.. Fridhandler. L. and Berk. J.E. (1975) Gut 16.1006-1009 19 Murray, W.R. and MacKay. C. (1977) Br. J. Surg. 64.189-191
Clinica Chimica Acta, 89 (1978) 159-164 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 9654B
PANCREATITIS-LIKE
ISOAMYLASE
PATTERN
IN NORMAL
PERSONS
MAKOTO OTSUKI, HOSAI YUU, MITSUO MAEDA, SUSUMU SAEKI, KUNIYASU OKANO, TOM10 YAMASAKI, TSUTOMU KANDA, CHOITSU SAKAMOTO and SHIGEAKI BABA Second Department Kobe, 650 (Japan) (Received
April 14th,
of Internal Medicine, Kobe University School of Medicine, Ikuta-ku, 1978)
summary On the assumption that a rise in the pancreatic type isoamylases may not necessarily indicate underlying pancreatitis, genetic studies of human serum and urinary amylase isoenzymes have been performed with the use of electrophoresis
.
Although the preponderant increase in the two principal pancreatic isoamylases Amylase-1 and 2 has been accepted to be a specific index of pancreatic involvement, 1.68% of normal persons had Amylase-2 with an elevated amylase activity (named “Dominant Amylase-2”) up to the same levels as the major isoenzymes. Results of pancreozymin-secretin test and other laboratory findings of these persons with Dominant Amylase-2 were all within normal ranges. Pedigree studies confirmed an autosomal dominant mode of inheritance for this variant. The importance of serial determination and pedigree investigations has been shown to distinguish normal persons having Dominant Amylase-2 from patients with pancreatitis without elevated amylase activity. The existence of an inherited trait of pancreatitis-like isoamylase pattern in healthy individuals must be borne in mind before coming to a conclusion when amylase isoenzymes are used for clinical medicine, though preponderance of the pancreatic type isoenzymes in serum and urine has been revealed to be a characteristic finding in pancreatitis. Knowledge of amylase genetic polymorphism provides a scientific basis for amylase isoenzyme interpretation.
Introduction Hyperamylasemia in the absence of acute pancreatitis is well documented, while no significant elevation of serum amylase can be detected in approx-
160
imately 5% of cases of proven acute pancreatitis [ 11. We have shown the relation of isoamylase pattern to the extent of pancreatic inflammation and revealed the possibility of clarifying the severity of pancreatic diseases by electrophoretic amylase fractionations [Z], though the discrepancy between the degree of the rise in serum amylase activity and the extent of the damage to the pancreas is well known. The diagnosis of acute pancreatitis could be confirmed by findings of an elevated amylase activity in Amylase-1 and 2, not only in the hyperamylasemic state but also for a few days after returning to normal levels [2,3]. We, however, further demonstrated the existence of an inherited trait of the pancreatic isoamylases in healthy individuals and called attention to the genetic polymorphism of amylase isoenzyme in clinical medicine [ 21. Contrary to our observations, Berk and Fridhandler are tenacious to their hypothesis that preponderant increase in the pancreatic isoamylase provides support for the suspicion of underlying pancreatitis despite the lack of elevated serum amylase and lipase levels [4], and had some doubts about the occurrence of an inherited trait of the pancreatic isoamylases in healthy individuals [5]. Thus we have screened individual serum and urine samples of 4041 normal persons using a polyacrylamide electrophoretic system for the detection of variants. Materials and methods Serum and urine were obtained from 4041 normal persons (all Japanese) 16 to 59 years old without known diseases. 40 additional samples from patients with acute and chronic pancreatitis were also collected. Amylase isoenzymes were demonstrated by thin-layer sheet polyacrylamide electrophoresis, using methods previously described [ 6,7]. When individuals with variant patterns were found, family members were studied to define the heritability. Total serum and urinary amylase activity was measured by the chromogenic method using blue-dyed starch polymer [ 81 and creatinine concentrations were determined by the method of Jaffd [9] within 24 h of specimen collection. The amylase clearance to creatinine clearance (C,,,/C,,) ratio was measured based on the method of Levitt et al. [lo] and expressed as a percentage: amylase X urine creatiC,ln,lC,, = (urine amylase X serum creatinine)/(serum nine) X 100. Amylase isoenzyme activity in serum and urine was calculated by multiplying the percentage of an isoenzyme in the sample by the total amylase activity. Amylase isoenzyme clearance was calculated as follows; amylase isoenzyme to creatinine clearance ratio = (urine amylase X % isoenzyme in urine X serum creatinine)/(serum amylase X % isoenzyme in serum X urine creatinine) X 100 [ll]. Results and discussion Although the serum and urinary amylase isoenzymes of 98.3% of normal persons consisted of two major isoenzymes (Amylase-1 and 3) and two or three minor ones (Amylase-2, 5 and/or 7), four different patterns of amylase isoenzymes were observed as previously reported [ 6,7]. Of normal persons, 1.68% had Amylase-2 with an elevated amylase activity up to the same levels as the
161
major isoenzymes. We named this isoenzyme “Dominant Amylase-2” for the present consideration. Marked elevations of amylase activity in the two principal pancreatic isoamylases Amylase-1 and 2 were characteristic findings in acute pancreatitis [2,3,12,13]. This particular isoamylase pattern afforded a means for confirming the diagnosis of acute pancreatitis and enabled the diagnosis to be made several days after the onset of illness, since the average half-life of the elevated Amylase-2 in pancreatitis has been shown to be about 10 days [ 31. It is most important in clinical medicine to differentiate Dominant Amylase-2 in normal persons from Amylase-2 in pancreatitis, because the isoamylase pattern in both situations is similar, as demonstrated in Fig. 1.
( I) Acute Pancreatitis
(II)
Normal
Persons
Fig. 1. Changes of electrophoretic pattern of serum amylase isoenzymes in acute pancreatitis (1) and various representative patterns of amylase isoenzymes in normal persons (II). Preponderance of the two principal pancreatic type isoenzyme Amylase-1 and 2 was still observed in W4.5. though serum amylase activity returned to normal levels. Serum and urinary amylase isoenzyme pattern of 98.3% of 4041 normal persons is (10-4. In 1.88% of normal pemons, amylase activity of Amy&2 was increased (H-1.2.3) up to the same levels as Amylase-1 or 3. These isoamylase patterns in normal persons resemble to those of acute pancreatitis. The rest of normal persons (0.2%) had Amylase-1s [ 71.
162
Results of pancreozymin-secretin tests and other laboratory findings in these persons with Dominant Amylase-2 were all within normal ranges, though the preponderant increase in the pancreatic type isoenzymes has been accepted to be a specific index of pancreatic involvement. Total serum amylase activity in no individuals with Dominant Amylase-2 exceeded 186 Somogyi units/100 ml, which represents the mean + 3 S.D. of healthy subjects in our laboratory [ 141. Polyacrylamide gel electrophoresis was performed on pancreatic juice from individuals with Dominant Amylase-2 and from normal persons immediately after collection in ice-cold test-tubes with a double lumen Dreiling tube. Pancreatic juice in normal persons consisted of only one major isoenzyme corresponding to Amylase-1 in serum and urine and a few minor ones corresponding to Amylase-2 and 4. On the other hand, two major bands with the same intensity were identified in pancreatic juice in normal persons with Dominant Amylase-2; one corresponds to Amylase-1 and the other to Dominant Amylase-2 in serum and urine (Fig. 2). By simultaneous electrophoresis of all samples obtained during a pancreozymin-secretin test, no variations in mobility and intensity of these two isoenzymes were found. No isoenzymes corresponding to Dominant Amylase-2 were demonstrable in saliva in these subjects. These observations may indicate that Dominant Amylase-2 is of pancreatic origin and is not the created isoenzyme by posttranslation~ modification such as glycosidation and deamination of gene products [ 151, but is a genetically determined
Fig. 2. Amylaae isoenzymes in urine. serum and pancreatic juice obtained during pancreozyminsecretin test from individuals with Dominant Amylase-2. (A) Control urine and (B) specimens from a case with Dominant Amylase-2. Two major bands with the same intensity were identified in all samples obtained during pancraozymin+ecretin test: one corresponds to Amylase-1 and the other to Dominant Amylase-2 in serum and urine.
163
one. In support of this assumption are the results of family studies (Fig. 3). Individuals within each family were either normal or exhibited the Dominant Amylase-2 of the proband. In two families (Fig. 3B,C), X-linkage was excluded. In family B, II-8 transmitted the Dominant Amylase-2 to III-8 and III-lo. In family C, II-7 failed to transmit the trait to his daughter. Dominant Amylase-2 was noted in every generation in these families. No significant sex difference was apparent among normal and variant individuals. Since the population of individuals tested was homogeneous with regards to age, health and race, no attempt was made to distinguish differences which might be related to these factors. Within each family, the trait seems to be inherited as an autosomal codominant; evidence against X-linkage was observed. These findings strongly suggest the importance of serial determination and pedigree investigations of amylase isoenzymes to distinguish normal persons having Dominant Amylase-2 along with major isoenzyme Amylase-1 from pancreatitis with normal amylase activity . High resolution is the most important requirement for a clinically useful method for identification of the amylase isoenzyme, providing the ability to identify individual isoamylases of the pancreatic and the salivary type. On thin-layer sheet polyacrylamide electrophoresis not only the pancreatic and the salivary type isoenzymes but variant isoamylases will also routinely be detected. In contrast, failure of column chromatography to distinguish Amylase-2 from
(A)
(B)
Dominant
Anylase-2
I-= 0 not tested /P&arid Fig. 3. Pedigrees of families with Dominant Amylase-2. Dominant Amylase-2 was observed in the members shaded and noted in every generation.
164
the salivary type isoenzyme has been suggested [ 12,131, because of the electrophoretic and isoelectric similarity of both isoenzymes [13]. Column chromatography cannot provide simultaneous separation and direct comparability of samples and yields poor resolution of minor and variant isoamylases but provides only resolution of major pancreatic and salivary type isoenzymes. In support of poor resolution of column chromatography are the reports by Berk and his associates that both salivary and pancreatic isoamylases rise in acute pancreatitis [4,16,17] and that the human pancreas contains small amounts of the salivary type amylase [ 181. The mean amylase to creatinine clearance ratio for control and normal persons with Dominant Amylase-2 were 2.9 + 0.38 and 2.37 t 0.19, respectively. In patients with pancreatitis, it was 7.09 t 0.54. C,,,/C,, remained significantly elevated in patients with acute pancreatitis for longer than either serum or urine amylase values [ 191 as a particular isoamylase pattern [ 21. The findings of this study suggest that the amylase creatinine clearance ratio provides a dimension to differentiate Dominant Amylase-2 from the elevated Amylase-2 in pancreatitis. The ratio of urinary clearance of Dominant Amylase-2 (C._,,_z) to C,, was 2.3 + 0.24, which was less than Camy_r/Ccr (2.76 + 0.33), but consistently exceded that of Camy_s /C,, (1.69 t 0.23) of normal persons. Camy_l and Camy_s in acute pancreatitis were 6.98 + 0.71 and 5.0 ? 0.58 of C,,, respectively .
Although preponderance of the pancreatic type isoenzymes in serum and urine has been revealed to be a characteristic finding, the existence of an inherited trait of pancreatitis-like isoamylase pattern in healthy individuals must be borne in mind before coming to a conclusion when amylase isoenzymes are used in clinical medicine. Knowledge of the genetic polymorphism is of importance in clinical assessment of amylase isoenzymes in patients having an elevated Amylase-2 suggestive of pancreatitis. References 1 Kalliomaki. J.L. and Antila. L.E. (1971) J. Am. Geriatr. 19,517-525 2 Otsuki, M.. Yuu, H., Maeda. M., Yamasaki. T., Okano, J., Sakamoto, C. and Baba, S. (1977) Clin. Chim. Acta 79.1-6 3 Legaz. M.E. and Kenny, M.A. (1976) Clin. Chem. 22,57-62 4 Berk, J.E. and Fridhandler, L. (1975) Am. J. Gastroenterol. 63.457463 5 Berk. J.E. and Fridhandler, L. (1978) Clin. Chim. Acta 89,157 6 Otsuki, M., Saeki. S., Kondo, T.. Yuu, H. and Baba, S. (1974) Jap. J. Gastroenterol. 71, 1241-1248 7 Otsuki. M.. Saeki, S., Yuu, H., Maeda. M. and Baba. S. (1976) Clin. Chem. 22,439444 8 Ceska. M.. Birath, K. and Brown, B. (1969) Clin. Chim. Acta 26.437444 9 JaffB, M. (1886) Physiol. Chem. 10, 391-398 10 Levitt, M.D., Rapoport. M. and Cooperband. S.R. (1969) Ann. Intern. Med. 71.919-925 11 Long, E.B. and Grider, J.R. (1976) Gastroenterology 71, 589-593 12 Lehrner. L.M.. Ward, J.C., Kam. R.C., Ehrlich. C.E. and Merritt, A.D. (1976) Am. J. Clin. Pathol. 66, 576-587 13 Warshaw. A.L. and Lee, K.H. (1977) J. Surg. Res. 22.362-369 14 Otiuki. M.. Yuu, H., Maeda, M.. Saeki, S., Yamasaki, T. and Baba, S. (1977) Cancer 39,1656-1663 15 Kam. R.C.. Shulkin, J.C.. Merritt, A.,D. and Newell, R.C.(1973) Biochem. Genet. 10,341-350 16 Fridhandler. L.. Berk. J.E. and Ueda, M. (1972) Clin. Chem. 18.1493-1497 17 Berk, J.E. and Fridhandler. L. (1976) Mount Sinai J. Med. 43, 321337 18 Shimamura. J.. Fridhandler. L. and Berk. J.E. (1975) Gut 16.1006-1009 19 Murray, W.R. and MacKay. C. (1977) Br. J. Surg. 64.189-191
