Amylase Clearance Research Articles

Makroamilazemija kao razlog hiperamilazemije u klinički nejasnim stanjima - prikaz slučaja

Macroamylasemia is a rare condition characterized by elevated serum amylase levels due to the existence of macromolecules - complex amylase that cannot be secreted normally by the kidneys due to their size. It is a benign condition that is usually free of marked clinical symptoms and signs. The prevalence of macroamylasemia in the population is between 1 and 2%. The main clinical significance of macromylasemia is that it is often a diagnostic problem because it requires differentiation of this condition from other causes of hyperamylasemia. Therefore, it is rational to avoid unnecessary diagnostics and treatment, which burden both the patient and the health system. Macromalasemia as a diagnosis should be considered in any patient with elevated serum amylase activity in whom serum lipase and urine amylase levels are normal. Laboratory confirmation of the diagnosis of amylasemia rests on tests: electrophoresis or polyethylene glycol precipitation test. This paper presents a patient who has been routinely treated clinically due to non-specific gastrointestinal problems. During laboratory treatment, the patient's serum was found to have elevated amylase values. Additional diagnostics did not identify organic pancreatic diseases or other diseases that may be related to hyperamylasemia, and by calculating the ratio of renal amylase clearance to creatinine clearance of less than 1%, we conclude that it is most likely a macroamylasemia. Specific treatment was not required. The patient comes for a check-up once every 6 months.

Macroamylasemia; a Dilemma in Morbidity.

To the Editor, A one-year-old boy was referred for randomly detected hyperamylasemia. Physical examination was completely normal, including growth parameters. Medical history and family history were unremarkable. In laboratory, the repeated serum amylases were found elevated with the values of 280 IU/mL, 290 IU/mL and 310 IU/mL (N: 10–90 IU/mL) together with a lower urine amylase level of 10.5 IU/mL (N: 25–500 IU/mL). The complete blood count, acute-phase reactants, liver–renal–thyroid functional tests, serum lipase and immunoglobulins were normal. Antibodies against endomysium, gliadin, thyroglobulin, thyroid peroxidase, nucleus and dsDNA were negative. Abdominal ultrasonography and computed tomography (CT) scans were normal. According to these normal findings, hyperamylasemia due to macroamylasemia (MA) was found in patient. An informed consent form to research was obtained from parents. At analysis for MA, a precipitated activity was 87% at polyethylene glycol (PEG) precipitation (MA>60%) (1). Macroenzymes are complexes of serum enzymes commonly with immunoglobulins or lipoproteins that have a higher moleculer weight. Macroamylase is the most common macroenzyme, in which pancreatic or salivary amylase binds to plasma proteins and cannot be excreted by renal glomeruli. Macroamylasemia may be seen either isolated or associated with a variety of disorders, such as IgA deficiency, celiac disease, lymphoma, carcinoma, systemic lupus erithematosus, rheumatoid arthritis, liver disease and various autoimmune diseases (2–4). The pathogenesis of macroenzyme formation in these diseases is estimated by ‘antigen-driven’ theory: a self-antigen is altered or released from a sequestered site in the body and cross-reacts with an antibody initially formed against a foreign antigen. A second theory is the ‘dysregulation of immune tolerance, which is likely to occur in autoimmune disorders (4). The cross-reactivity to either gluten-related or other antigens resulting in autoantibody formation against pancreatic serum amylase at intestinal level is considered (5). The elevated serum amylase, low 24-hour urine amylase and reduced ratio of amylase clearance / creatinine clearance are important at diagnosis. It has been shown that PEG precipitation is also a precise method in the detection of MA by purifying protein from macromolecules more easily and quickly (1). Due to the problem at urine collection, PEG precipitation was preferred in this case. It was considered as isolated MA due to the absence of associated disorders. In conclusion, MA is generally a benign condition, but associated potential disorders should be considered at follow-up. The case highlights the dilemma of additional invasive explorations and unnecessary treatments by differentiating MA from other situations associated to hyperamylasemia.

Hyperamylasaemia: don’t forget undiagnosed carcinoma

The case report in this issue by Akinosoglou et al. describes increased serum amylase activity in a patient with a small oat cell tumour of the lung. Alpha amylase is a relatively small 45 kDa enzyme that breaks down glycogen and starch to maltose and dextrin. Amylase is a glycoside hydrolase which acts upon a-1,4-glycosidic bonds. It is located principally in the pancreas and salivary glands but is also found in other organs, including the gonads, fallopian tubes and intestinal tract. Total amylase activity in human plasma usually comprises pancreatic (P) and salivary (S) isoenzymes. There are many causes of hyperamylasaemia. This is an essential laboratory practical point as although raised serum amylase activity is associated with acute pancreatitis, it is not specific for this condition. Lankisch et al. report that increased serum amylase activity occurs in about 8% of hospitalized patients with non-pancreatic diseases. Jones et al. observed that patients with non-diagnostic hyperamylasaemia presenting with abdominal pain should be thoroughly investigated: in 50% cases, gallstones were found. There are also important ethno-racial differences in mean serum amylase activity. Tsianos et al. reported that serum amylase activity was highest in West Indians, intermediate in Asians and lowest in Caucasians. This poses the question of whether laboratories should report ethno-racial reference ranges for serum amylase. Table 1 shows some of the possible causes of hyperamylasaemia. Alpha amylase is filtered by the renal glomeruli and excreted in the urine, hence its elevation in the plasma in certain renal disorders. In macroamylasaemia, amylase is bound to immunoglobulins and can be diagnosed by a low ratio of amylase clearance to creatinine clearance. Electrophoretic and chromatography techniques can also be used to determine macroamylasaemia. Macroamylasaemia is associated with certain autoimmune disorders including coeliac disease, ulcerative colitis and rheumatoid arthritis as well as lymphoma and human immunodeficiency virus (HIV) infection and after administration of hydroxyethyl starch. Hyperamylasaemia can also be associated with malignant disease. Serum amylase activity may be increased in some patients with multiple myeloma. A common feature of the myeloma cell lines associated with hyperamylasaemia was a translocation of chromosome 1, which harbours the gene for amylase. Shigemura et al. reported that in multiple myeloma with hyperamylasaemia, a majority of cases were due to salivary-type hyperamylasaemia (sialyl salivary-type amylase). In patients with amylase-producing multiple myeloma, the onset of hyperamylasaemia was associated with a rapid disease progression associated with extensive bone destruction and increased mortality. It has been suggested that serum amylase activity may be a prognostic index of multiple myeloma disease progression. Ovarian carcinomas are also associated with hyperamylasaemia as are some lung tumours. Amylase-producing tumours of the lung are rare and may comprise in total only 1–3% of all lung carcinomas and, in these cases, the salivary amylase isotype is generally found. As Akinosoglou et al. point out, small cell lung carcinoma originates from Kulchitsky cells, which are of neural crest origin and able to secrete various polypeptide hormones and enzymes such as amylase. However, this hyperamylasaemia may also be due to an increase in the amylase content within

The amylase-to-creatinine clearance ratio--a non-specific response to acute illness?

It was felt that the apparent specificity of the amylase-to-creatine clearance ratio (ACCR) in several previous studies of pancreatitis might reflect a failure to utilize adequately ill control subjects. The ACCR and the renal clearances of beta 2-microglobulin (B2-m), similarly related to creatinine (BCCR) as well as the urinary concentration of albumin, were compared in 27 patients with acute pancreatitis, 8 with a perforated peptic ulcer and 7 with mild biliary colic, during the first 5 days in hospital. Acute pancreatitis was graded as mild (6), moderate (14) or severe (7), using a combination of clinical data, diagnostic peritoneal lavage and multiple criteria. Further assessment of the severity of the acute illness was obtained from measurement of C-reactive protein (C-RP). Lowest C-RP levels were found in the patients with mild pancreatitis and biliary colic, and highest levels in the patients with severe pancreatitis and perforated ulcer (P less than 0.002). Similarly, ACCR and BCCR levels were significantly lower in the two mild groups than in the two severe ones (P less than 0.01 and less than 0.002 respectively), although plasma amylase was raised only in patients with pancreatitis and plasma B2-m was similar in all groups. Electrophoresis of urine showed dense bands of tubuloprotein in patients from both severe groups. Urine albumin was higher in severe pancreatitis than in perforated ulcer (P less than 0.1), perhaps indicating a more specific glomerular lesion in pancreatitis. Thus a rise in amylase clearance appeared to be related to the severity of the acute illness, and may be a component of a non-specific tubuloproteinuria. In this study patients with a perforated peptic ulcer had increases in ACCR similar to those seen in patients with severe pancreatitis, and we are therefore doubtful whether ACCR has any role in the clinical diagnosis of pancreatic disease.

Effect of electric charge on the transperitoneal transport of plasma proteins during CAPD

Controversy exists as to whether electric charges of plasma proteins influence their transport across the peritoneal membrane during CAPD. Fixed negative charges in the peritoneal membrane are diminished during peritonitis in rats. Peritoneal clearances of 10 proteins and their isoforms were used to establish the relationship between peritoneal clearance and molecular weight. The observed protein clearances were compared with the predicted clearances based on molecular weight. Clearances of proteins with different charge but identical size were compared. Stable patients and peritonitis patients were compared. Results. Only the peritoneal clearance of lipase, LDH 4/5 and IgG3 were significantly different from the predicted values (P<=0.05). The peritoneal clearance of slightly anionic beta2 microglobulin (1072 microl/min) and cationic lysozyme (572 microl/min) showed no evidence for charge selectivity; neither did the peritoneal clearance of slightly anionic transferrin (86 microl/min) and highly anionic albumin (99 microl/min). The peritoneal clearance of IgG1, IgG2 and IgG4 were identical (32, 31 and 31 microl/min), despite their different charge. The peritoneal clearance of cationic LDH 4/5 was 137 microl/min and higher than the peritoneal clearance of neutral LDH 3 (97 microl/min, P=0.01) and LDH 1 (59 microl/min, P=0. 02). These results suggested charge selectivity; however in five additional patients during peritonitis the peritoneal clearance of LDH 4/5 increased to 10 times the peritoneal clearance of LDH 1. Local LDH isoenzyme release from the cells present in the dialysate was shown to be responsible in stable and peritonitis patients. Likewise, the higher peritoneal clearance of neutral pancreatic amylase (234 microl/min) compared to anionic salivary amylase (142 microl/min, P=0.03) could probably be attributed to local release of the former from the pancreas, as the peritoneal clearance of lipase (highly anionic) was higher than predicted and the difference remained during peritonitis. The peritoneal membrane constitutes a size- but probably not a charge-selective barrier for the transport of macromolecules between blood and dialysate during stable CAPD.

Renal isoamylase clearance as a measure of altered renal charge selectivity in patients with diabetes mellitus.

Microalbuminuria is well established as a marker for early renal damage in diabetic patients. Differences in charge selectivity in glomerular protein filtration may also be an early marker of renal damage. We investigated the possible usefulness of the renal clearances of pancreatic and salivary amylases, and the ratio of the two, as markers of early renal damage in 55 diabetic subjects and 21 healthy controls. Diabetic patients with established albuminuria and microalbuminuria had increased clearance of salivary amylase and a trend toward lower pancreatic/salivary amylase clearance ratios compared to healthy controls and diabetic subjects without albuminuria, but the overlap with controls and diabetics without albuminuria was too large for the test to be useful.

Rate of clearance of ethanol from the blood of intoxicated patients in the emergency department

One hundred and three patients presenting to the Mt. Sinai Medical Center emergency department (ED), who appeared on clinical grounds to be acutely intoxicated, were studied to determine the rate of clearance of ethanol from blood. The mean presenting serum ethanol level was 299 mg/dL. The rate of clearance was found to be 20.43 mg/dI/h with a standard deviation of 6.86 mg/dL/h. No correlation was found between rate of ethanol clearance and serum levels of amylase, alkaline phosphatase, glutamate-oxaloacetate or glutamate-pyruvate transaminase, lactic dehydrogenase, or total bilirubin. Similarly, no correlation was found between rate of clearance and race, sex, age, or time of day. We conclude that although the average patient presenting to the emergency department will clear ethanol at about 20 mg/dL/h, a standard deviation of 6 mg/dL/h means that only 83% of these patients will have clearance rates between 8 and 32 mg/dL/h, and that if accurate estimates are necessary, serial determinations of two or more levels are needed.

Electrophoretic characterization of posttranslational modifications of human parotid salivary α‐amylase

Human salivary alpha-amylase displays multiple bands upon native polyacrylamide gel electrophoresis. In fresh saliva, due to posttranslational modifications, a pattern of 5-6 isozymes is observed. The isozymes are designated 1-6, in the order of increasing anodal mobility. As a result of the development of a rapid and sensitive electrophoresis system, with markedly higher resolution than previously reported, we concluded that a previously proposed model (Karn et al., Biochem. Genet. 1973, 10, 341-350) is inadequate to explain the origin of the various bands. We propose an alternative model that fits in with our new and previously made observations. According to this model, band 2 is the primary gene product and band 1 is its glycosylated counterpart--with only one neutral oligosaccharide present on each molecule. Band 3 originates from band 1 by the transialidase-catalyzed incorporation of sialic acid into the biantennary chain. Bands 4 and 6 originate from bands 2 and 4, respectively, by deamidation; band 5 is the deamidation product of amylase with an acidic oligosaccharide (band 3). Only a minor part of band 3 consists of the deamidation product of band 1. Peptide Asn-Gly-Ser (residues 427-429) is the most probable candidate for glycosylation; literature data suggests that deamidation occurs in the stretch Glu-Asn-Gly-Lys-Asp (residues 364-368) and Asn-Gly-Asn-Cys (residues 474-477). Both glycosylation and deamidation might play a role in the clearance of amylase from the systemic circulation. The electrophoresis system described is a powerful tool to determine amylase isozyme distributions in health and disease, especially for the screening of alterations seen in ectopically produced amylase.

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.

Pancreatic-type hyperamylasemia in end-stage renal disease

Pancreatic-type isoamylase (P-type) and salivary-type isoamylase (S-type) activities were determined by the wheat protein inhibitor method in 29 patients with end-stage renal disease and in 38 healthy volunteers. Serum levels of total amylase (322 +/- 43 units/liter) and P-type (212 +/- 39 units/liter) in ESRD were significantly higher than those of controls (total: 142 +/- 7 units/liter, P less than 0.01; P-type: 52 +/- 4 units/liter, P less than 0.01). There was no significant difference between S-type activities in ESRD (110 +/- 16 units/liter) and in controls (90 +/- 6 units/liter). The ratios of amylase clearance to creatinine clearance (Cam/Ccr) and S-type clearance to creatinine clearance (Cs-amy/Ccr) rose significantly in ESRD(Cam/Ccr: 5.7 +/- 0.6%; Cs-amy/Ccr: 4.3 +/- 0.55%) compared to controls (Cam/Ccr: 3.2 +/- 0.24%, P less than 0.01; Cs-amy/Ccr: 2.1 +/- 0.17%, P less than 0.01). The ratio of P-type clearance to creatinine clearance (Cp-amy/Ccr) revealed no significant difference between ESRD (5.5 +/- 0.54%) and controls (5.6 +/- 0.42%). The renal excretion of P-type appeared to be more impaired than that of S-type in ESRD.

Relationship of Urinary Amylase Activity and Proteinuria in the Dog

-I In normal dogs and in some dogs with marked hyperamylasemia there is very little urinary amylase a c t i ~ i t y . ' . ~ . ~ . ~ . ~ ~ This is surprising since the molecular weight of purified canine pancreatic amylase determined using a denaturing technique is about 54,000 daltons) which is below the threshold of the glomerular filter. It has been hypothesized that canine amylase does pass the glomerular filter but is totally reabsorbed from the filtrate or is ina~tivated.~ Recently, it has been shown that these mechanisms probably do not play an important role in amylase clearance and that canine amylase probably does not pass the intact glomerular filter.6 It appears that unique characteristics of the canine glomerular filter and amylase might account for the lack of appearance of urinary amylase activity. It has been reported that native human amylase appears as a polymer.8 Since the polymerization phenomenon may be important in understanding the clearance of canine amylase, an estimate of the molecular weight of canine serum amylase and the influence of this weight on glomerular filtration were determined. Four pools of canine sera, each composed of ten canine sera chosen randomly from routine laboratory accessions at the University of Guelph Veterinary Teaching Hospital, were made. Random urine samples were obtained from 13 normoamylasemic 1-year-old Samoyed dogs in a colony bred to propagate the sex-linked trait for hereditary glomerulopathy.' Urine samples were obtained from 16 normoamylasemic, non-hematuric, non-hemoglobinuric dogs with proteinuria. Total amylase activities were determined using a p-nitrophenylmaltohexaoside substrate (Coulter Electronics of Canada, Ltd., Burlington, Ontario, Canada). Urinary protein concentrations were determined by a Ponceau S (Aldrich Chemical Co., Milwaukee, WI) dye binding method and purified human albumin standards (Dade Diagnostics, Inc., Aguada, Puerto R i ~ o ) . ~ Urinary specific gravities were determined refractometrically. The four pools of canine sera were each diluted 300 times in a buffer containing 0.086 M NaC1, 7 mM KCl, 2 mM MgCl,, 7 mM CaCI,, and 0.02 M tris(hydroxymethy1)-aminomethane-HC1 (pH 7.2). Aliquots of diluted pooled sera were concentrated to the original volume of serum using 100 and 300 kilodalton molecular weight cut-off ultrafilters (Amicon Canada Ltd., Oakville, Ontario, Canada). Prior to use, 10 ml of a 0.15 M NaCl solution containing l.Oo/o bovine serum albumin was passed through each ultrafilter to prevent non-specific adherence. The ultrafiltration was done at 4 C and 10 Ib/sq. in. About 20% of albumin is retained by both sizes of ultrafilters (Amicon Canada Ltd., Oakville, Ontario, Canada). Amylase activities in the retentates were determined and results expressed as the percent amylase activity retained by each filter. The relations between urinary protein concentration, specific gravity, and amylase activity were determined by linear --

Renal Disposition of Amylase, Lipase, and Lysozyme in the Dog

Normal adult dogs were given intravenously lysine hydrochloride to abolish renal tubular reabsorption. The treatment caused tubular proteinuria. Once forced diuresis was established, fractional clearances for amylase, lipase, and lysozyme increased five-, 18-, and 857-fold over the baseline values, respectively. There was relatively little tubular reabsorption of amylase, and urinary amylase activity remained low. A renal arteriovenous difference in amylase activity was not present. Urinary amylase activity could not be reactivated by the addition of serum or treatment with dithiothreitol. Urinary inhibitors of amylase activity were not detected. Immunoreactive urinary amylase did not exceed kinetically measured urinary amylase. Therefore, the presence of irreversibly inactivated amylase did not explain the low fractional clearance of amylase. A small amount of serum macroamylase was present, but macroamylasemia did not account for canine amylase failing to pass the glomerular filter. It appears that the renal loss of amylase in the dog is not an important excretory route.

Renal clearance of pancreatic and salivary amylase relative to creatinine clearance in patients with renal disease and proteinuria.

To study the charge-selective properties of the glomerular filter in renal disease, we measured the fractional clearance, relative to creatinine clearance (ECC), of the amylase isoenzymes pancreatic amylase and salivary amylase, which have identical size but different charge. In 63 healthy subjects the mean (and SD) fractional excretion of pancreatic amylase, 4.07% (1.24%), was fourfold that of salivary amylase: 1.02% (0.54%). For 29 patients with renal disease and proteinuria, the mean fractional excretion of pancreatic amylase was significantly lower, 3.31% (1.94%), and that of salivary amylase significantly higher, 2.06% (1.41%), than in controls. In these patients, fractional excretions of both these isoenzymes were negatively correlated with urinary excretion of beta 2-microglobulin and ECC. Evidently, differences in clearances of pancreatic and salivary amylase are a consequence of differences in charge-related glomerular filtration. The relative increase of salivary amylase clearance in patients with renal disease and proteinuria is most probably caused by a loss of the charge-selective properties of the glomerular basement membrane.

Experience with OKT3 in Vascularized Pancreas Transplantation

With refinements in technical aspects of whole organ pancreas transplantation, allograft rejection is currently the major cause of graft failure. The monoclonal antibody OKT3 has emerged as a highly effective antirejection therapy in renal and hepatic allograft recipients, but its efficacy in pancreas transplantation remains to be determined. During a 12-month period, 28 vascularized whole organ pancreas transplants were performed with pancreatico-cystostomy. Sixteen episodes of allograft rejection were treated with monoclonal antibody OKT3. Indications for OKT3 use included steroid- or antilymphocyte globulin (ALG)-resistant allograft rejection in isolated pancreas (n = 8) or simultaneous kidney-pancreas (n = 8) transplants. A total of 34 rejection episodes occurred in the 16 patients (mean, 2.1; range, one to five). The diagnosis of rejection was based on clinical criteria, a reduction in urinary amylase clearance, radionuclide scanning, hyperglycemia, or associated renal allograft dysfunction in combined engraftments. Postoperative immunosuppression consisted of cyclosporine, prednisone, azathioprine, and prophylactic ALG. OKT3 was administered for a full 14-day course concomitant with low-dose steroids, azathioprine, and cyclosporine. The mean age of the patient population was 32.1 years (range 24 to 39) with a mean duration of insulin-dependent diabetes mellitus (IDDM) of 20.9 years. Monoclonal antibody therapy was instituted in two clinical settings: early rejection (within 3 months of transplant, n = 10); and late rejection (after 3 months, n = 6). OKT3 successfully reversed allograft rejection in ten (62.5%) cases, including six early (60%) and four late (66.7%) episodes. In isolated pancreas transplants, OKT3 therapy reversed pancreas allograft rejection in only two patients (25%).(ABSTRACT TRUNCATED AT 250 WORDS)

Macroamylasemia with a markedly increased amylase clearance ratio in a patient with renal cell carcinoma.

We report hyperamylasemia, macroamylasemia, and a markedly increased amylase clearance/creatinine clearance ratio in a patient with renal cell carcinoma. Serum amylase activity was characterized as macroamylase by gel exclusion chromatography. Electrophoretic separation revealed an atypical band of amylase, migrating anodal to the S2 control fraction. Electrophoresis of urine revealed the presence of both S1 and S2 fractions, but not the atypical band found in serum. Quantification of the salivary- and pancreatic-type amylase fractions showed amylase in urine to be 100% salivary. Immunofixation disclosed the macroamylase to consist of an immune complex between amylase and IgA-lambda antibody. Binding-capacity studies showed that the serum immunoglobulin was present in excess and could bind 46% and 49% additional S-type amylase activity derived from saliva and the patient's urine, respectively. The amylase clearance/creatinine clearance ratio was markedly supranormal (0.134), unexpected in a patient with macroamylasemia. A biopsy specimen of the renal cell tumor was found to contain significant salivary-type amylase activity. These results suggest production of amylase by tumor tissue in the renal carcinoma and secretion of S-type amylase into the patient's urine. Evidently, macroamylase should be confirmed by gel exclusion chromatography.

臨床分野におけるα‐アミラーゼ分別測定の現況

Amylase isoenzyme analysis in serum is widely used in clinical medicine for the differential diagnosis of hyperamylasemia. The major clinical application of amylase isozyme determination at the present time is the differentiation of salivary from pancreatic hyperamylasemia . In the present study, we evaluated the usefulness of the established means of amylase isozyme analysis; electrophoresis, the amylase to creatinine clearance ratio (ACCR) and the amylase inhibitor test. Isozyme analysis by electrophoresis has not been widely used in clinical laboratories because it is too technically complex and time-consuming for ready clinical use. In spite of these short comings, electrophoresis is a useful and sensitive tool for the identification of minor components of amylase isozyme observed in the serum of patients with acute pancreatitis, the variant amylase isozyme pattern seen in patients with lung cancer as well as in normal persons, macro amylasemia, and aged amylase in cases of pancreatic pseudocysts or pleural effusion . The ratio of the renal clearance of amylase to that of creatinine (ACCR) is a simple, rapid, readily available and reliable means of assessing the presence or absence of acute pancreatitis. In addition to in acute pancreatitis, recent studies have revealed a nonspecific increase in ACCR in normoamylasemic conditions such as burns, diabetic acidosis and Bence-Jones proteinemia, and in postoperative patients. In patients with conditions other than pancreatitis, however, hyperamylasemia and a high ACCR are likely to be unrelated phenomena. A cause of hyperamylasemia is not a cause of an increased ACCR and vice versa. At present, therefore, acute pancreatitis appears to be the only condition that causes elevation of both the serum amylase level and the ACCR. The assay method involving wheat inhibitor, which is a relatively specific inhibitor of salivary amylase, is a simple and accurate means of differentiating salivary from pancreatic hyperamylasemia. Except for very high or very low pancreatic amylase levels, the results obtained with the inhibitor assay show good correlation with the results as to amylase isozyme distribution determined by electrophoresis. Thus, the inhibitor assay can be reliably used to determine if a patient's hyperamylasemia or the aged amylase in biological fluids is of pancreatic or salivary origin. The inhibitor assay, however, leads to clinical confusion in patients with macroamylasemia or with variant amylase isoenzymes. Although all three techniques mentioned above can differentiate salivary from pancreatic hyperamylasemia, salivary amylases are known not to be specific to the salivary gland. At present, there is no method for determining the origin of the amylase. In addition to the knowledge that hyperamylasemia is of nonpancreatic origin, knowledge that hyperamylasemia is related to some specific organ may greatly improve the diagnostic accuracy.

Temperature Dependence of Macroamylase Binding

The presence of macroamylasemia can be suspected when a patient has low renal clearance of amylase relative to creatinine (Cam/Ccr). However, the Cam/Ccr is seldom reduced to the extent expected given the fraction of amylase that appears to be bound in the macroamylase complex by gel filtration studies at 3 degrees C. Therefore, we tested the possibility that the binding of amylase in macroamylase complexes was temperature dependent and that binding at physiological temperatures might be less than at 3 degrees C. Gel filtration of five macroamylase-containing sera was carried out at temperatures of 3 degrees, 25 degrees, 37 degrees, 43 degrees, and 49 degrees C. Binding of amylase in the complex decreased as the temperature increased with virtually all amylase bound at 3 degrees C and virtually no amylase bound at 49 degrees C. The fraction of the amylase that bound at 37 degrees C was approximately what would have been predicted from the Cam/Ccr of each of the five subjects. We conclude that amylase binding in macroamylase complexes is extremely temperature sensitive and appreciable changes in binding may occur over the physiological temperature range of 37-41 degrees C.

Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages.

Salivary-type amylase normally comprises about 60% of the amylase activity in human serum, but only a small fraction is a glycosylated isoenzyme (amylase A). In contrast, 1/3 of amylase in human saliva is glycosylated. Since glycosylation can affect circulatory clearance, we studied the clearance of amylase A in rats and its uptake by rat alveolar macrophages. Following intravenous injection, 125I-labeled amylase A disappeared rapidly from plasma (t 1/2 = 9 min) and accumulated in the liver. Simultaneous injection of mannose-albumin slowed its clearance to a rate comparable to that of 125I-labeled nonglycosylated salivary amylase (t 1/2 = 45 min). In contrast, galactose-albumin had no effect. Electron microscope autoradiography of the liver following injection of 125I-labeled amylase A revealed a localization of grains over the hepatic endothelial cells. In vitro studies indicated that amylase A is taken up by alveolar macrophages via receptor-mediated pinocytosis. Uptake was linear over time, saturable, and inhibited by mannan and mannose-albumin, but not by galactose-albumin. We conclude that amylase A, which is a naturally occurring human glycoprotein with at most three terminal L-fucose residues per molecule, is recognized in rats by a mannose receptor located on hepatic endothelial cells. We speculate that this receptor, by rapidly clearing circulating amylase A, may be responsible for the low level of amylase A in human serum.

Identification of unique macroamylase with abnormally low affinity for macromolecular substrate by short-chain amylose assay methods

An abnormally large sized serum amylase was identified by Wilding et al [l]. Binding of normal amylase to some high molecular weight substances was reported to be the cause of macromolecular amylase, designated as ‘macroamylase’ [2]. This macroamylase can be identified by its large molecular size [2], low ratio of amylase clearance to creatinine clearance [3] and/or unique electrophoretic pattern [4]. Binding of amylase to serum components may lead to differences in apparent amylase activity between various methods. In this communication, we describe an immunoglobulin A-linked macroamylase, whose activity varied with the assay used.

Foregut mucosal defects: An etiology of hyperamylasemia

To evaluate a preliminary correlation of hyperamylasemia to upper gastrointestinal bleeding, total serum amylase and serum isoamylase profiles were determined in 50 patients with upper gastrointestinal bleeding. Etiologies of the bleeding were determined in 46 patients including gastritis or duodenitis in 25, gastric ulcers in 12, duodenal ulcers in 3, Mallory-Weiss tears in 3, gastric carcinoma in 2, and esophageal varices in 1. Gastritis or duodenitis was seen incidentally in 14 more patients. Hyperamylasemia was seen in 38 patients, most commonly being due to a rise of both nonpancreatic and pancreatic isoamylases (18 patients). In 13 patients it was due to an elevation of nonpancreatic amylase alone, and in 7 patients secondary to elevated pancreatic isoamylase alone. Acute pancreatitis raises only the pancreatic component and cannot explain the hyperamylasemia in most of these patients. Hyperamylasemia did not correlate to etiology of the bleeding; gastritis or duodenitis present in the majority of these patients appears to be the unifying factor. Since both nonpancreatic and pancreatic amylases are present in the duodenum and the stomach with pyloric reflux, reabsorption of intraluminal amylase across damaged mucosa is postulated as a mechanism to explain the observed isoamylase patterns. The possibility of decreased amylase clearance as an explanation is unlikely. An alternative central nervous system mechanism might be invoked. It is concluded that hyperamylasemia is a complex event which the use of isoamylase analysis is beginning to elucidate. The hyperamylasemia seen commonly in patients presenting with upper gastrointestinal bleeding does not imply the presence of acute pancreatitis.