Direct spectrophotometric determination of alpha-amylase activity in salive, with p-nitrophenyl alpha-maltoside as substrate.

We describe a simple, direct kinetic method for determination of salivary alpha-amylase (1,4, alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1). The assay makes use of a well-defined substrate, p-nitrophenyl alpha-maltoside, which is hydrolyzed by alpha-amylase to a chromogenic product, p-nitrophenol. Activity is determined by directly monitoring the increase in absorbance of the reaction mixture. Amylase activity can be defined in international (IUB) units of micromoles of product/min per liter of saliva. For 22 healthy subjects, the mean +/- SD of amylase activity in mixed saliva was 2.77 +/- 1.12 U/liter. Activity and instrumental response were linearly related over the entire range tested (0.224 to 11.90 U/liter). The within-run precision (CV) over this range was better than 3% for all but the lowest activities. Values obtained with this assay correlate well with those obtained with a modified Nelson-Somogyi saccharogenic method (r = 0.979). The precision and simplicity of this assay suggest that it is the method choice for determining amylase activity in human saliva.     

Fluorescence depolarization assay for quantifying alpha-amylase in serum and urine

We have developed a new method for quantifying alpha-amylase (EC 3.2.1.1) in serum and urine by fluorescence depolarization. Amylase in the sample catalyzes the hydrolysis of the substrate, a fluorescein-labeled amylose. This results in decreased fluorescence polarization, owing to the increased rate of rotation of the amylose fragment relative to the intact substrate. The TDx amylase assay is calibrated with six human-serum-based pancreatic amylase calibrators. Amylase activities are determined by interpolation from the calibration curve, which is stored in the TDx analyzer's memory. Results correlate well with those by the Du Pont aca assay and the Beckman "DRI-STAT" assay. Endogenous glucose does not interfere. CVs are less than 6%, and the reagents are stable in liquid form.     

New method for the determination of pancreatic amylase evaluated.

We have carried out a multicenter evaluation of a new reagent carrier for Reflotron, specific for pancreatic amylase where the salivary isoenzyme is inhibited by two specific monoclonal antibodies. This new procedure combines easy handling with low imprecision (median CV less than 3%) in control material, serum, heparinized blood, and plasma) and close correlation (r = 0.991 to 0.999) with established manual and automated methods. The same close correlation was found with values obtained from either venous or capillary finger-stick blood. Salivary amylase up to 54 kU/L (37 degrees C) was inhibited to about 97%. Endogenous interference by hemoglobin, bilirubin, triglycerides, cholesterol, or hematocrit was found to be negligible within a wide range of interferent concentrations. Out of a panel of 28 commonly used drugs it was shown that only two (ascorbic acid and paracetamol), and then only at toxic concentrations, caused a deviation in amylase activity of greater than 10%. From the results of this study we conclude that this new method is suitable for highly precise and accurate measurements of pancreatic amylase in emergency and routine laboratories.     

Creatine kinase determination: a European evaluation of the creatine kinase determination in serum, plasma and whole blood with the Reflotron system.

We evaluated a new dry-reagent carrier system for the determination of creatine kinase (EC 2.7.3.2) activity, Reflotron CK, with special attention to analytical performance with whole blood. We found a good within series imprecision. The median coefficient of variation was 3.1% for Reflotron CK (blood, serum and plasma) and 0.9% for the automatic analysers (serum and plasma only). The between-days imprecision with Reflotron CK (median CV: less than or equal to 3%) was similar to that for the comparison method on different analysers. Fresh samples of human blood, plasma and serum were examined by Reflotron CK and by a N-acetylcysteine activated creatine kinase method in six different clinical laboratories and in the Evaluation Department of Boehringer Mannheim GmbH. The correlation between these methods was excellent (r greater than or equal to 0.99), the median systematic deviation (bias) for all samples being smaller than -5%. Haematocrits between 0.25 and 0.50, haemolysis up to 6 g/l haemoglobin, and icteric samples with bilirubin concentrations up to 0.2 g/l showed no interference. No drug in therapeutic concentration was found to affect the Reflotron CK results; ascorbic acid, calcium dobesilate and sulphamethoxazole lowered the values only when present in high concentrations. Reflotron CK may be considered as a suitable alternative for decentralized testing sites, especially in situations where creatine kinase results are needed quickly.     

An automated method for determination of serum and urine alpha-amylase.

An automated method for the determination of alpha-amylase activities of serum and urine using the Phadebas Amylase Test is described. The procedure has been adapted for the System Olli 3000 analyser using amylase tablets whose weight is half of that of normal tablets. The results are calculated by the computer with the aid of a standard curve calculated theoretically. This automated procedure is fast, as many as 120 analyses per hour can be carried out. Intra-assay precision of 2.0 and 2.2% (CV) is obtained from serum samples containing 301 and 181 u/l of alpha-amylase, respectively (n = 20), and inter-assay precision is 4.4 and 3.3% with mean values of 337 and 196 u/l, respectively (n = 20). When the automated procedure is compared with the manual procedure on 85 sera with alpha-amylase activities below 1000 u/l, a good correlation r = 0.992, and a regression equation y = 1.01x-6 are found. In the case of serum and urine samples, which contain high enzyme activities, the automated method gives slightly higher results than the manual method.     

Determination of alpha-amylase in biological fluids using a new substrate (beta-2-chloro-4-nitrophenyl-maltopentaoside)

A novel substrate, beta-2-chloro-4-nitrophenylmaltopentaoside (beta CNPG5), was used for the enzyme-coupled determination of alpha-amylase in biological fluids. It was hydrolyzed specifically by alpha-amylase to about 90% producing beta-2-chloro-4-nitrophenylmaltoside (beta CNPG2) and maltotriose. Under the assay conditions, the absorption of 2-chloro-4-nitrophenol (CNP) generated by the secondary reaction of alpha-glucosidase and beta-glucosidase as auxiliary enzymes is about twice the absorption of 4-nitrophenol (PNP), which is the end product currently measured in some alpha-amylase assay methods. The sensitivity of the assay using beta CNPG5 was thus much higher than that using 4-nitrophenyl-maltopentaoside (PNPG5) as substrate. The absorption of CNP did not fluctuate with temperature or with pH between 6.8 and 7.2, which are the conditions normally used for determination of amylase activity in biological fluids.     

Cholesterol screening: comparative evaluation of on-site and laboratory-based measurements

We measured cholesterol in capillary blood samples from 9683 volunteers over a four-day on-site community screening program, using the "Reflotron" desk-top analyzer (Boehringer-Mannheim Diagnostics, Indianapolis, IN). We also measured cholesterol in venous blood samples from 3% of those screened (a) with the Reflotron at the screening sites, (b) in a qualified hospital clinical laboratory, and (c) in a Centers for Disease Control standardized lipoprotein research laboratory. The sensitivity (and specificity) of the Reflotron measurements, with use of the lipoprotein laboratory measurements as the point of reference, was 0.95 (0.73) in capillary blood samples and 0.88 (0.93) in venous blood samples, compared with 0.99 (0.87) in the hospital clinical laboratory. The Reflotron measurements correlated less well with the lipoprotein laboratory values in both venous blood (r = 0.91) and capillary blood (r = 0.89) samples than did the clinical laboratory values (r greater than 0.99). Furthermore, the capillary blood measurements averaged 7% higher than venous measurements when both kinds of samples were analyzed in the Reflotron.     

Development of a direct assay for alpha-amylase

We describe a direct colorimetric assay for alpha-amylase, with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. Both human pancreatic and salivary amylase split this substrate without the use of helper enzymes, yielding free 2-chloro-4-nitrophenol, which is monitored at 405 nm. The performance of this reagent compares well with that of Pantrak Amylase (Behring Diagnostics) for both serum and urine samples. The reagent is very stable in dry powder form and is stable for one month at 2 to 8 degrees C after reconstitution. Because of the rapid color development and linear kinetics (less than 30 s), the assay is easily automated. Results can be obtained in less than 5 min.     

Methods compared for determining total amylase activity and amylase isoenzymes in serum

We evaluated two kinetic methods for determining total amylase activity and isoenzyme composition in serum. Stability studies of reagents for measuring total activity indicate that reagents containing 4-nitrophenyl-alpha-glucosides or enzyme-linked reagents can be stored only for seven days at 4 degrees C. Methods based on 4-nitrophenyl-alpha-glucoside substrates cannot be used if the reagent absorbance at 405 nm exceeds 2. However, in the alpha-amylase EPS method (Boehringer Mannheim) an ethylidene-protected 4-nitrophenyl-alpha-D-maltoheptaoside substrate is stable for up to 28 days after reconstitution. Further studies indicated that the Amylase-DS (Beckman) and the alpha-Amylase EPS standard curves are linear to at least six times the upper limit of the reference interval. Within-batch imprecision (CV less than 1.1%) and between-batch imprecision (CV less than 3.3%) for these two methods are comparable with those for other kinetic methods, and there is excellent correlation (r2 = 0.983) between the two methods. The reference interval, determined by use of samples from 90 healthy blood donors, is 31 to 141 U/L for the amylase-DS method, 22 to 92 U/L for the alpha-Amylase EPS method. We also used these two methods to measure amylase isoenzymes after inhibiting the salivary isoenzyme with either a lectin or a monoclonal antibody. We found the monoclonal antibody method more specific than the lectin inhibition method for determining the isoenzymes.     

Lipase and pancreatic amylase versus total amylase as biomarkers of pancreatitis: an analytical investigation

OBJECTIVE: To evaluate the biomarkers of pancreatitis Colorimetric Lipase, Total Amylase and Pancreatic Amylase (immunoinhibition) assays on the Roche COBAS INTEGRA 700. RESULTS: Pancreatic and Total Amylase assays and Colorimetric Lipase showed excellent imprecision of 1.6 to 2.3% and linearity (slope = 0.94-0.99, y-intercepts-1 to +3 U/L, r = 0.999) over the range of 17 to 900, 35 to 880, and 21 to 150 U/L, respectively. There was an excellent correlation between Pancreatic and Total Amylase: Pancreatic Amylase = 0.99 (+/- 0.02) x Total Amylase-36(+/- 8) (n = 106, r = 0.97, p < 1 x 10(-5), y intercept p < 1 x 10(-5)). Colorimetric Lipase showed some correlation to Total and Pancreatic Amylase results: Colorimetric Lipase = 1.54 (+/- 0.16) x Total Amylase-81(+/- 37) (n = 100, r = 0.70, p < 1 x 10(-6), y intercept p = 0.03), and Colorimetric Lipase = 1.78 (+/- 0.15) x Pancreatic Amylase-50(+/- 29) (n = 99, r = 0.78, p < 1 x 10(-6), y intercept p = 0.09). CONCLUSION: We recommend running the more specific Pancreatic Amylase as biomarker of pancreatitis on the Roche COBAS INTEGRA.     

Measuring carboxypeptidase A activity with a centrifugal analyzer: analytical and clinical considerations

This adaptation of a commercially available kit for automated measurement of carboxypeptidase A (CPA; EC 3.4.17.1) activity in serum with the Cobas Bio centrifugal analyzer extends the linear range to an activity concentration of 82 U/L. Results obtained by the described method correlated closely (r = 0.98) with those by the manual kit method. The reference interval for 150 apparently normal individuals was 0.12-0.91 U/L. Total CVs of the method ranged from 4.0% to 13.1%. Bilirubin and glucose decreased the CPA activity in serum by as much as 98% and 26%, respectively. Substantial CPA activity was found in pancreatic tissue, with little activity in intestinal tissue. CPA activity was not as widely distributed in extra-pancreatic tissues as were amylase and lipase activities. Peak activities of CPA, amylase, and lipase in the sera of patients with acute pancreatitis were significantly correlated (r = 0.45 to 0.78, P less than 0.05-0.01). The optimized diagnostic efficiency of CPA for acute pancreatitis was 0.85 at a cutoff value of 5 U/L. Amylase and lipase exhibited similar optimized efficiencies, and parallel testing did not significantly improve diagnostic accuracy. We conclude that automated analysis for CPA activity, even in the absence of interferences, does not add to the diagnostic information provided by the widely available assays for amylase and lipase activity.     

Evaluation of a radioimmunoassay of urinary cortisol without extraction

We evaluated the Kallestad "Quanticoat" cortisol RIA for direct (no extraction) measurement of urinary free cortisol, which requires no solvent extraction. An analytical-recovery study showed a linear regression of y (measured) = 0.65x (added) + 37.5 micrograms/L (Sy.x = 21.4 micrograms/L, r = 0.978, n = 48). Recovery appeared to vary with the urine used and with the concentrations of cortisol added. Within- and between-run CVs were less than or equal to 4.1% and less than or equal to 3.8%, respectively. Cross reactivities were low, except for prednisolone (20.5%). This no-extraction method gave higher values for urinary free cortisol than did either an RIA method involving extraction or an HPLC method. A comparison study with the HPLC (x) and with the method involving extraction (x') gave the following Deming-debiased regression equations: y = 1.60x + 68.8 (Sy.x = 34.4, n = 29) and y = 1.33x' + 0.69 (Sy.x = 40.3, n = 66), respectively. We conclude that the no-extraction method may give misleading results for patients' diagnosis or management if this cross reactivity is not taken into account.     

Incidence and source of hyperamylasemia after cardiac surgery

We investigated the incidence and possible mechanisms of postoperative hyperamylasemia in 101 patients after cardiac surgery. Amylase (EC 3.2.1.1) activities in serum were increased in 36% of patients after bypass surgery, 59% of patients after valve replacement, and in 69% of patients after combined bypass and valve replacement. Lipase (EC 3.1.1.3) activity was increased in 30% of all patients. We found enzymatic evidence for pancreatitis in six patients. Thirty-six patients showed increased salivary (S-type) amylase activity, with a positive correlation (r = 0.55, P less than 0.001) between the severity of pleural effusions and the peak S-type amylase activity. Hyperamylasemia after cardiac surgery is apparently often related to absorption of S-type amylase from pleural fluid and (or) from aspirated salivary secretions. Monitoring patients for postsurgical pancreatitis necessitates assay of amylase isoenzymes to distinguish abnormalities resulting in release of pancreatic (P-type) amylase from those involving release of S-type amylase.     

Reflotron cholesterol measurement evaluated as a screening technique

We evaluated the analytical performance of Boehringer Mannheim Diagnostics' "Reflotron" analyzer for the measurement of cholesterol. Coefficients of variation (CVs) for whole-blood cholesterol were: within-day 2.0% and 2.2% at 1680 and 2670 mg/L, respectively; between-day 1.8% and 2.4% (n = 9 and 8). Results were similar for serum and heparinized or EDTA-treated single-donor plasma (CV 1.4% to 2.6%). CVs of results for two reconstituted commercial quality-control materials were 3.4% and 4.6%. Heparin and hematocrit were evaluated as interferents, and critical limits for interference were identified for bilirubin, hemoglobin, and triglyceride in blood and plasma or serum. When sample collection and analysis were controlled by trained personnel, results with the Reflotron (y) compared well with those by the Ektachem procedure (x) for both blood and serum samples: r = 0.950, y = 0.944x + 130 mg/L; and r = 0.955, y = 0.93x + 43.5 mg/L, respectively. The same comparability was observed when the analysis was performed by briefly trained high-school students: r = 0.980, y = 0.949x + 23 mg/L. Performance decreased when both collection and analysis were performed by laymen: r = 0.880, y = 0.870x + 186 mg/L.     

Total and pancreatic amylase measured with 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltoside

BACKGROUND:: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex. METHODS:: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into beta-D-galactopyranosylmaltose and 2-chloro-4-nitrophenol, which was measured at 405 nm. RESULTS:: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and beta-D-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6-1.6% and 0.5-2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8-3.7% and 0.6-4.1%, respectively. T-AMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those determined by the 2-chloro-4-nitrophenyl 4-O-beta-D-galactopyranosyl-beta-maltotetraoside method or the modified IFCC method. CONCLUSIONS:: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures.     

Isopropylidine maltoheptosyl fructofuranoside, doubly blocked substrate for determination of endoamylase activity.

We synthesized o-(4,6-o-isopropylidene-alpha-D-glucopyranosyl)-(1----4)- [o-alpha-D-glucopyranosyl-(1----4])5-o-alpha-D-glucopyranosyl-(1----2)- alpha-D-fructofuranoside (IPG7F) and developed an assay for determining the activity of amylase in human serum and urine by using this substrate. Glucoamylase, alpha-glucosidase, and mannitol dehydrogenase are used as coupling enzymes. The coupled reactions are monitored by continuously measuring the oxidation rate of NADH. In this procedure, various substances in the test specimens do not interfere with the detection of amylase activity. Exactly one molecule of NADH is oxidized by one attack of amylase on the substrate, although four products can be produced in the reaction. The within-assay coefficient of variation (CV) ranged from 1.0% to 4.1% and the between-assay CV ranged from 2.6% to 5.3%. The results of our new assay correlate well with those of the amylase assay involving p-nitrophenol maltoheptaoside as substrate (r = 0.978) and with those of the amylase assay involving maltopentaose (r = 0.987).     

Revised calibration of the Reflotron cholesterol assay evaluated

We evaluated the Boehringer Mannheim (B.M.) Reflotron Total Cholesterol "dry-chemistry" method after its recalibration in 1987. Reports in the literature up to 1986-1987 of a negative bias (up to -10%) in the method prompted a revision of the factory-set calibration of the Reflotron. For this, B.M. prepared a new set of calibrators with 12 different concentrations of cholesterol. We checked in two ways whether accuracy had been achieved: (a) The values assigned to the calibrators by B.M. were checked with the manual Abell-Kendall Reference Method (MAK) performed in an official Reference Center. These were shown to be correct. (b) Concurrently, a direct comparison was made by analyzing 200 fresh samples of human serum. Reflotron cholesterol values obtained for these samples proved to be accurate, meeting the current World Health Organization/Centers for Disease Control criterion of maximum bias less than or equal to 5%. Orthogonal regression analysis yielded the following correlation: Reflotron = 0.985 MAK + 0.238 mmol/L (y = ax + b). Reflotron mean = 6.26 mmol/L; MAK mean = 6.09 mmol/L. SDa = 0.015 mmol/L; SDb = 0.120 mmol/L, and r = 0.989.     

Determination of amylase activity and amylase isoenzymes in serum and urine using a solid phase blue starch substrate.

The alpha-amylase of serum and urine was determined in 40 healthy people using the modified "Phadebas Amylase Test". The original method was modified by decreasing the incubation volume to one millilitre and by adding to all urine speciemens a small amount of albumin in saline. The normal values obtained are slightly higher than those obtained by the original method. The amylase isoenzymes were likewise determined from the serum and urine of the same 40 healthy people. For separation were used electrophoresis on cellulose acetate and Phadebas tablets as substrate. These urine and serum isoamylases were investigated and compared. The distributions obtained deviate somewhat from ones reported earlier. The clinical usefulness of isoamylase is briefly discussed.     

Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.     

A novel automated assay for malate dehydrogenase isoenzymes

We have developed a new automated method for the determination of malate dehydrogenase (MDH) isoenzymes in serum employing guanidine hydrochloride. Our proposed method showed good reproducibility; within-run precision coefficient of variations (CVs) were less than 2.5 (mean 13.6–42.9 U/L) for total MDH (T-MDH) and less than 6.7% (mean 6.3–23.6 U/L) for mitochondrial MDH (m-MDH) (n = 10). The upper detection limit of the proposed method exhibited good linearity up to 1,000 U/L for both T-MDH and m-MDH. In the proposed m-MDH reagent, the presence of up to 2,000 U/L of cytosolic MDH(c-MDH) activity had no effect on the outcome of m-MDH assay. Results of our proposed method (y) correlated well with those of the electrophoretic method (x) giving the regression equation: y = 1.46 x + 6.87 (N = 30); r = 0.99. Normal concentrations of various anticoagulants and bilirubin did not affect the assay results. Both ascorbic acid and glucose exhibited a slight positive interference with the proposed assay. Clinically, we found that m-MDH activity in serum had greater diagnostic predictive value than T-MDH activity for judging successful outcome of reperfusion therapy; the prognosis was poor when the m-MDH/T-MDH ratio was greater than 20%. © 1996 Wiley-Liss, Inc.