Inhibitor Test for Amylase Isoenzymes

The percentage of pancreatic-type isoamylase activity and the ratio of pancreatic to salivary-type isoamylase activity were determined in five control and 35 hyperamylasemic serum samples by means of an established simplified chromatographic method and a recently described inhibitor test. There was good correlation in the results, except at very high and at very low levels of P-type isoamylase activity. At these extremes, the inhibitor method yields imprecise quantitative results. The inhibitor test findings may also prompt misleading conclusions when elevated serum amylase activity is due to undetected macroamylasemia.     

Where does serum amylase come from and where does it go?

The serum amylase concentration reflects the balance between the rates of amylase entry into and removal from the blood. Hyperamylasemia can result either from an increased rate of entry of amylase into the circulation and/or a decreased metabolic clearance of this enzyme. The pancreas and salivary glands have amylase concentrations that are several orders of magnitude greater than that of any other normal tissue, and these two organs probably account for almost all of the serum amylase activity in normal persons. A variety of techniques are now available to distinguish pancreatic from salivary-type isoamylase. Pancreatic hyperamylasemia results from an insult to the pancreas, ranging from trivial (cannulation of the pancreatic duct) to severe (pancreatitis). In addition, loss of bowel integrity (infarction or perforation) causes pancreatic hyperamylasemia due to absorption of amylase from the intestinal lumen. Hyperamylasemia due to salivary-type isoamylase is observed in conditions involving the salivary glands. In addition, this type of hyperamylasemia occurs in conditions in which there is no clinical evidence of salivary gland disease, such as chronic alcoholism, postoperative states (particularly postcoronary bypass), lactic acidosis, anorexia nervosa or bulimia, and malignant neoplasms that secrete amylase. Hyperamylasemia can also result from decreased metabolic clearance of amylase due to renal failure or macroamylasemia (a condition in which an abnormally high-molecular-weight amylase is present in the serum). Patients with abdominal pain and a markedly elevated serum amylase (more than three times the upper limit of normal) usually have acute pancreatitis, and additional serum enzyme testing is not helpful. Patients with smaller elevations of serum amylase often have conditions other than pancreatitis, and measurement of a serum enzyme more specific for the pancreas (pancreatitic isoamylase, lipase or trypsin) is frequently of diagnostic value in such patients.     

New dimensions in the laboratory diagnosis of pancreatic disease.

The laboratory diagnosis of pancreatic disease has been made more precise by certain modifications in older methods and by the introduction of a variety of new technical procedures. The principal human isoamylases may now be distinguished and their activities in serum and urine measured. A test has been devised which helps indicate the presence of acute pancreatitis by showing relatively increased excretion of amylase in the urine as compared with creatinine. The ratio of amylase to creatinine in the urine appears to be a good index of relative hyperamylasuria. A screening test for pancreatic-type hyperamylasuria has been formulated that allows increased urinary excretion of this isoamylase to be identified. These additions and developments have sharpened the interpretation of hyperamylasemia and hyperamylasuria and have added new dimensions to the laboratory diagnosis of pancreatic disease.     

Simulation of macroamylasaemia by salivary-type (`S type') hyperamylasaemia

Hyperamylasaemia with low or normal urinary amylase excretion in the face of normal renal function has been noted to be indirect evidence for the existence of macroamylasaemia. A more refined indicator is a low ratio of renal amylase clearance to creatinine clearance. This report describes observations made in three patients, each of whom displayed the indirect features suggestive of macroamylasaemia. In none of these patients, however, could a serum macroamylase be demonstrated by chromatography. Ultracentrifugation was also done in one of the patients and likewise failed to disclose a macroamylase in the serum. When the isozyme nature of the serum amylase in each of these patients was examined, there was found to be a marked rise in activity of the salivary-type isoamylase (;S-type amylase'). The pancreatic-type isoamylase (;P-type amylase') was normal in one patient and perhaps slightly above normal in the other two. These data indicate that (1) S-type hyperamylasaemia may exhibit the indirect criteria taken to indicate macroamylasaemia and thereby simulate the latter disorder; and (2) unequivocal identification of macroamylasaemia requires the direct demonstration of the presence in the serum of a macromolecular amylase complex.     

Amylase inhibitor from wheat: its action and clinical application

The electrophoretic and column chromatographic characteristics of an amylase inhibitor of wheat origin were investigated. Further, the clinical usefulness of this inhibitor for determining the ratio of pancreatic to salivary isoamylase activity in serum was evaluated. Amylase inhibitor inhibits the action of salivary alpha-amylase by making an amylase-inhibitor complex, which is easily separated into its individual component during electrophoresis with full recovery of amylase activity. Using the specific inhibitory effect of this inhibitor on salivary alpha-amylase activity, the ratio of pancreatic to salivary isoamylase activity (P/S) in serum was determined. There was a good correlation in P/S ratio in serum between the results obtained with the inhibitor method and those with electrophoretic method. The P/S ratio in sera from patients with acute pancreatitis was over 8.0, whereas that in sera from patients with salivary-type hyperamylasemia such as mumps, pulmonary diseases and following surgery was less than 0.1. However, hyperamylasemia due to macroamylase or renal failure could not be identified by the inhibitor method.     

Assay of Amylase and Isoamylase Activities in Serum and Urine

Certain modifications are described in assay methods previously reported from this laboratory for total amylase and isoamylase activities. These modifications provide more consistent results and achieve superior separation of pancreatic and salivary type (P- and S-type) isoamylases. The estimated range of values in the serum and urine of normal subjects using the modified assay procedures is presented for total amylase as well as for P- and S-type isoamylases.     

Sources of the serum isoamylases and their normal range of variation with age.

The isoamylases in various human tissue homogenates and body fluids were separated by agarose gel electrophoresis. Nothing suggested any significant production of amylase in the liver. Minute amounts of amylase belonging to the pancreatic group of isoamylases might be produced by the glands of the proximal duodenum. The specific group of isoamylases produced in the female genital tract could not be demonstrated in serum or urine. The activity of amylase in serum was derived from two groups of isoenzymes, one group originating from the salivary glands, the other from the pancreatic gland. The contribution of each of these two sources to the total serum amylase was determined from early foetal life to adult age. A very low activity of the salivary isoamylases was regularly found in serum from 14-week-old foetuses. The activity increased steadily with age and reached the normal adult level, about 80 U/l, at the age of 5 years. The pancreatic group of isoamylases in serum developed later; the majority of children below 3 months had no demonstrable pancreatic isoamylase activity. The activity rose slowly to reach adult level, about 80 U/l, at the age of 10 to 15 years. The activity did not vary with sex, and the diurnal variation of the isoamylase was negligible. In children with cystic fibrosis of the pancreas the activity of pancreatic isoamylases in serum was low.     

Isoamylases and their thermolability in serum and cyst fluid from patients with pancreatic pseudocysts

The isoamylase pattern in serum and the amylase thermolability have been suggested as screening tests for the development of pancreatic pseudocysts. To study whether serum reflects the contents of pseudocysts, we have investigated the isoamylases and their thermolability in cyst fluid and in serum from 13 patients with pancreatic pseudocysts. No significant correlation was found between the contents in serum and cyst fluid with regard to total amylase and isoamylase P2 and P3 or with regard to the thermolability of total amylase and isoamylase P2 and P3. Thus, serum does not reflect the cyst contents of isoamylases or their thermolability. Therefore these serum amylase determinations can hardly be expected to be useful in distinguishing patients developing pseudocysts among patients with pancreatitis.     

Differential Determination of Serum Isoamylase Using an Amylase Inhibitor and Its Clinical Application

Diagnostic significance of a simple and rapid screening procedure for determining the relative amounts of pancreatic and salivary isoamylase using an amylase inhibitor was evaluated in 242 subjects (controls 84, acute pancreatitis nine, chronic pancreatitis 28, pancreatic cancer 14, peptic ulcer 25, liver cirrhosis 15, cholelithiasis 24, irritable colon syndrome 13, diabetes mellitus 13, mumps seven, and chronic renal failure 10). Electrophoretically separated isoamylases of saliva and pure pancreatic juice were all inhibited at similar degrees to the corresponding unfractionated amylases. Total amylase and pancreatic isoamylase were elevated in all nine patients with acute pancreatitis. Pancreatic isoamylase was decreased in 12 of 28 patients (43%) with chronic pancreatitis and increased in nine of 14 patients (64%) with pancreatic cancer. The mean pancreatic isoamylase activity in the patients with acute pancreatitis was significantly higher (p less than 0.01), while that of chronic pancreatitis was significantly lower (p less than 0.05) when compared with controls. The inhibition method offers simple, rapid, and specific analysis of serum isoamylase for the differential diagnosis of hyperamylasemia in cases of emergency.     

Comparison of serum amylase pancreatic isoamylase and lipase in patients with hyperamylasemia

We compared results of measurements of total serum amylase, pancreatic isoamylase, and lipase measurements in patients with hyperamylasemia. Serial measurements of these three enzyme levels in patients recovering from acute pancreatitis indicated that pancreatic isoamylase and lipase were elevated above normal to a greater extent and remained elevated much longer than did the total amylase. This finding indicates an appreciable sensitivity advantage of the pancreatic isoamylase and lipase over total amylase measurement during the recovery phase of pancreatitis. Comparison of pancreatic isoamylase and lipase levels in selected sera indicated a good correlation (r=0.84) between these two measurements in patients who did not have macroamylasemia. Lipase was normal in sera with amylase elevations due solely to salivary isoamylase. Thus, in nonmacroamylsemic sera, pancreatic isoamylase and lipase appear to be roughly interchangeable markers of the level of pancreatic enzymes in the blood. An advantage of the lipase assay is that this enzyme is normal in hyperamylasemia caused by macroamylasemia, whereas the inhibitor assay indicates that the pancreatic isoamylase is elevated. Development of automated assays for either pancreatic isoamylase or lipase should lead to the routine use of one of these assays in place of the present reliance on total amylase measurements in the diagnosis of pancreatitis.Supported by Veterans Administration Research Funds and National Institutes of Health grant 13309-15.     

Acute alcohol intoxication: significance of the amylase level

To evaluate the effects of acute alcohol intoxication on serum amylase and isoamylase levels, 58 clinically intoxicated patients with blood alcohol levels greater than 100 mg/dL were studied. Comparisons were made to normal control and a sober chronic alcoholic group. Admitting serum isoamylase levels were determined by cellulose acetate membrane electrophoresis and serum amylase levels measured by the Amylochrome technique. The average blood alcohol level in the intoxicated group was 301 +/- 99 mg/dL. Thirty of the 58 patients had hyperamylasemia (greater than 207 IU). Twenty-five of these 30 patients had hyperamylasemia from nonpancreatic sources (increased salivary isoamylase). Two of the 30 patients had pancreatic hyperamylasemia and three patients had elevated levels of both isoamylases. Neither of the patients with pancreatic hyperamylasemia had clinical evidence of acute pancreatitis. Although nine of the 58 patients had abdominal pain and clinical symptoms suggestive of acute pancreatitis, none of these patients had elevated pancreatic isoamylase. The finding of hyperamylasemia in acutely intoxicated patients is common. This is most frequently due to a rise in the salivary (nonpancreatic) isoamylase. The reliability of the total serum amylase as an indication of pancreatic disease in the intoxicated patient is questioned.     

Occurrence and nature of hyperamylasemia in chronic alcoholics

The purpose of this work was: a) the prospective study of the prevalence of hyperamylasemia in 100 patients with chronic alcoholism; b) the determination of the serum isoamylase distribution in patients with hyperamylasemia by an inhibitor assay; c) the search of the origin of elevated serum isoamylase S. Moderate hyperamylasemia was found in 15 patients. The importance of alcohol abuse, the prevalences of cirrhosis and smokers were not statistically different from those observed in normoamylasemic patients. After one week of hospitalization, serum amylase was still elevated in 11 of 14 alcoholic patients. Hyperamylasemia was due to an increase in the isoamylase P in 5 cases, in the isoamylase S in 7 cases, and in both forms in 3 cases. Activities of serum lipase and isoamylase P were roughly parallel. Only two out of 8 patients with elevated isoamylase P had chronic pancreatitis. The salivary origin of elevated isoamylase S was suspected in only one out of 10 patients. This work shows that the origin of moderate hyperamylasemia, observed in alcoholic patients, is often extrapancreatic. It is suggested that the dosage of serum lipase simpler than that of isoamylases, may be routinely used in chronic alcoholic patients for diagnostic purposes.     

Hyperamylasaemia after duodenoscopy and retrograde cholangiopancreatography.

The salivary and pancreatic isoamylases of serum were determined separately in 234 cases of duodenoscopy and retrograde cholangiopancreatography. Successful pancreatic opacification was associated with pathologically high pancreatic serum amylase activities in 60% of the cases. Extensive opacification was associated with large increases of pancreatic serum isoamylases, the maximal rise recorded was 40 times the initial value. In spite of these striking chemical events only two patients developed clinical acute pancreatitis. There were some variations in pancreatic opacification and in the elevation of pancreatic serum amylase which seemed to depend upon the particular contrast material used. A rise of the salivary serum isoamylases caused pathologically high total serum amylase activities in 7% of the cases. High levels of pancreatic serum isoamylase activity before the time of examination did not result in any different pattern of hyperamylasaemia.     

Hyperamylasemia in patients with eating disorders

Hyperamylasemia, which has been reported in patients with the eating disorders anorexia nervosa and bulimia, generally has been thought to result from pancreatitis. To evaluate the mechanisms of hyperamylasemia, we measured amylase, lipase, and isoamylase activity in 17 consecutive patients admitted to the eating disorder unit. Six patients had elevated amylase activity, and 5 of these 6 had isolated increases in salivary isoamylase activity. Six other patients had normal serum total amylase activity but modest elevations in the salivary isoamylase fraction. No patient developed clinical evidence of pancreatitis during hospitalization. Thus, the hyperamylasemia in patients with anorexia and bulimia often is caused by increased salivary-type amylase activity. The appropriate diagnostic test for hyperamylasemia in patients with anorexia or bulimia is the simple measurement of serum lipase or pancreatic isoamylase activity. If these levels are found to be normal, further tests to exclude pancreatitis are unnecessary.     

Role of secondary hyperparathyroidism and liver function in hyperamylasemia in chronic renal failure

Elevated values of pancreatic-type amylase activity in serum were found in 59% of patients with liver cirrhosis not complicated with renal failure, in 67% of patients with chronic renal failure not complicated with hepatopathy and in 95% of patients with chronic renal failure complicated with hepatopathy. In all the three groups, a significant positive correlation was found between the pancreatic-type amylase and intestinal isoenzyme of serum alkaline phosphatase which is an asialoglycoprotein. However, in pancreatitis a prevalence of an increase in pancreatic-type amylase with respect to intestinal alkaline phosphatase was found. A multivariate analysis showed that in chronic renal failure not complicated with hepatopathy, and in chronic renal failure complicated with chronic liver disease, the changes in calcium homeostasis and also the liver disorder, respectively, contribute significantly to the above-normal values for pancreatic-type amylase.     

Mild pancreatic damage in acute viral hepatitis

Whether and to what extent the pancreas is involved in acute viral hepatitis is still unclear. In order to address this issue we evaluated serum and urinary amylase and isoamylase levels in 92 patients with acute viral hepatitis of different etiology and in 60 healthy volunteers. Furthermore, pancreatic structure and volume were evaluated by ultrasound scanning. Significant increase in serum and urinary pancreatic isoamylases was found in 12 and 35% of patients, respectively, in the early stage of the disease. Increase in serum pancreatic isoamylases was found only in patients suffering from B and non-A, non-B hepatitis. Ultrasonographic evaluation did not show any change in pancreatic structure and volume. In conclusion, this study suggests that mild pancreatic damage may occur during viral hepatitis.     

A unique case of breast carcinoma producing pancreatic-type isoamylase

A 71-yr-old woman with a widely metastatic lipid-rich variant of breast cancer was found to have striking hyperamylasemia (85-fold normal). By isoelectric focusing, agarose gel electrophoresis, and a wheat protein inhibitor assay, the predominant serum amylase appeared to be identical to pancreatic isoamylase. Serum trypsin, serum lipase, and an abdominal computed tomography scan were normal, excluding the possibility of pancreatitis. Furthermore, both the primary breast tumor and skin metastases that developed 10 yr later stained immunohistochemically for amylase. Thus, breast carcinoma must be added to the list of tumors causing ectopic hyperamylasemia, and this case shows that nonpancreatic malignancies may produce pancreatic-type hyperamylasemia.     

Age and sex-dependent alterations of serum amylase and isoamylase levels in normal human adults

Total amylase and salivary-and pancreatic-type isoamylase levels were assayed in sera from 606 apparently healthy adults of different sex and age groups. There were significant differences in both total amylase and isoamylase levels, depending on age and sex, one of the characteristics being that levels of these three enzymes were significantly higher in the elderly group in both men and women than in other age groups. Another feature was that all of these enzyme levels were significantly greater in women in the third and fourth decade than in men. Age and sex differences should be taken into consideration in the evaluation of mild hyperamylasemia.     

Effect of Pancreatic Stimulation on Serum and Urine Amylase Isoenzymes in Man

Alpha amylase of pancreatic origin is cleared by the kidney more rapidly than the salivary isoamylase. To determine whether alterations in the ratio of pancreatic to salivary amylase in sera caused alterations in over all renal clearance, the clearance of amylase was measured before and after the exocrine pancreas was stimulated with a prolonged intravenous infusion of secretin plus cholecystokinin. Serum and urine samples collected prior to and following stimulation were analyzed for amylase activity and creatinine concentration. Amylase isoenzymes were separated using isoelectric focusing. Over all renal clearance of amylase and of the separated amylase isoenzymes were calculated as a percentage of the clearance of creatinine. The hormone infusion was associated with an increase in serum and urine amylase activities, this increase being mainly accounted for by pancreatic amylase. The renal clearance of the salivary and pancreatic isoamylases was not altered by the hormone infusion but the over all amylase clearance by the kidney rose from 2.31 +/- 0.74 to 3.42 +/- 1.46% of creatinine clearance. In some cases the renal clearance of amylase following stimulation entered the range considered diagnostic for acute pancreatitis.     

Aging changes of pancreatic isoamylases and the appearance of "old amylase" in the serum of patients with pancreatic pseudocysts

Human serum and pancreatic secretions contain at least three isoamylases of pancreatic origin. As determined by gel electrophoresis and saccharogenic amylase assay, P1, P2, and P3 designate isoenzymes with slow, intermediate, and rapid electrophoretic mobilities, respectively. The P1 isoamylase normally accounts fo 80-90% of total amylase activity, P2 for 10-20%, and P3 for 0-4% in both serum and pancreatic juice. When pancreatic amylase is incubated at 37 degrees C, P1 decreases, and P2 and P3 increase within hours. Whereas P2/P1 is always < 0.25 in fresh pancreatic juice, normal serum, acute pancreatitis serum, chronic pancreatitis serum, or pancreatic cancer serum, the ratio was elevated in 11 of 12 pseudocyst contents (mean P2/P1 = 0.51) and in 14 of 16 sera from patients with proven pseudocysts (mean P2/P1 = 0.43) (P < 0.001). After pseudocysts were surgically drained, the proportions of the pancreatic isoamylases in serum reverted to normal. The precise, characteristic, and predictable changes in electrophoretic mobility, presumably a result of specific spontaneous chemical alterations of the isoenzyme molecules, allow identification of "old amylase" in the serum of patients with pancreatic pseudocysts. This finding may be a useful adjunct for diagnosis, but whether the amount of "old amylase" can be used to estimate the age of a pseudocyst is not yet known.