ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 9, No. 5 Copyright 1979, Institute for Clinical Science, Inc. Rapid Enzymatic Determination of Amylase in Serum and Urine Using a Centrifugal Analyzer* STEVEN P. CROUSE, PH.D.,f ROBERT E. CROSS, Ph.D.* NANCY C. PARKER,! and JO H N SAVORY, Ph.D. t Division o f Pathology, Abington Memorial Hospital, Abington, PA 19001 The Department o f Hospital Laboratories, North Carolina Memorial Hospital, Departments o f Pathology, Medicine, and Biochemistry, University o f North Carolina, Chapel Hill, JVC 27514 Division o f Pathology, University o f Virginia Medical Center, Charlottesville, VA 22901 ABSTRACT A convenient, totally enzymatic procedure for amylase assay using a centrifugal analyzer is described. The reaction scheme is examined using both starch and m altotetraose as substrates. Data for the reaction kinetics, linearity and sensitivity are presented. The proposed m ethod exhibits linear reaction kinetics betw een three and five m inutes after the initiation of the reaction and is linear to 1200 U per liter of amylase. The determ ination is rapid, (five m inutes analysis time), convenient, (only one reagent), and precise, (1.2 percent within-day C.V. and 4.6 percent day-to-day C.V.), at a level of 62 U per liter. The proposed m ethod compares favorably with chrom ogenic procedures; (r = 0.979, n = 84). Principle Alpha-am ylase (EC 3.2.1.1) is quantitated using an enzym e-coupled reaction sequence first described by Pierre.6 The hydrolysis of substrate by amylase is followed by m easuring the production of n icotinam ide-adenine din u cleo tid e, re- * Send reprint requests to Dr. Steven P. Crouse, Director of Clinical Chemistry, Division of Pathology, Abington Memorial Hospital, Abington, PA 19001. ducedfrom (NADH) kinetically at 340 nm and 37 C in a system utilizing the coupling enzymes maltophosphorylase, phosphoglucom utase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The reaction sequence is outlined in table I. Because the coupling enzymes do not utilize glucose in the coupling reactions, interference from endogenous glucose present in the sample is non-existent. 420 0091-7370/79/0900-0420 $00.90 Institute for Clinical Science, Inc.
AUTOM ATED AMYLASE ASSAY 421 TABLE I R eaction Sequence fo r th e Enzym e-coupled Assay fo r Amylase Amylase Maltotetraose ------------------------- 2 Maltose (EC 3.2.1.1) MP Maltose + P. ------------------------- ^ Glucose + Glucose-l-phosphate 1 (EC 2.4.1.8) Glucose-l-phosphate PGM -----------------------Glucose-6-phosphate (EC 2.7.5.1) Glucose-6-phosphate + NAD G-6-PDH ------------------------ ^ 6-Phosphogluconate + NADH (EC 1.1.1.49) 6-PGDH 6-Phosphogluconate + NAD -------------------- (EC 1.1.1.44) Reagents The reagents for the enzyme-coupled procedure were obtained commercially.* C o u p led enzym e reagent system s contain in g starch and m altotetraose substrates are used in the evaluation. Each of the reagents system s is reconstituted using 80 percent of the recom m ended amount of distilled water diluent to account for the flush volume required during the autom atic p ipetting stage. The reagent for the dye-coupled system (Amylochrome) was obtained com m ercially f and is used exactly as described in the product insert. Standard Solutions For the enzym e-coupled procedure, no activity standardization is feasible. H ow ever, to insure optimal results, it is importa n t th at centrifugal analyzers be calibrated as to cuvet tem perature and path length. T em perature calibration is performed using the m ethod described by Bowie e ta l,1and path length calibration is accom plished using th e procedure rec * Beckman Microbics, Carlsbad, CA 92001. t Roche Diagnostics, Hoffman-LaRoche, Inc., Nutiey, NJ 07110. om m ended by the analyzer m anufacturer incorporating p-nitrophenol as the dye material. The manual dye-coupled procedure is standardized using the protocol recom m ended by the m anufacturer. For this standardization, varying concentrations of triazine dye solution are m easured for their absorbance at 625 nm and a standard curve is generated. The w ater bath tem perature for the m anual procedure is m onitored using a National Bureau of Standards calibrated therm om eter. Apparatus A centrifugal analyzer $ is used for the enzyme-coupled determ ination. Sample and diluent are delivered using the Rotofil II dispensing system. R eagent is delivered m anually using m icroliter pipets. F or th e dye-coupled procedure, reagents are dispensed using a reagent delivery system. 1 The sam ple is d eliv ered using an E ppendorf microliter pipet. The reaction is m aintained at 37 C using a t Rotochem II, American Instruments, Co., Silver Springs, MD 20910. E ppendorf, Brinkman Instrum ents, Inc., Westbury, NY 11590. 1Dispensette, Brinkman Instruments.
422 CROUSE, CROSS, PARKER AND SAVORY thermostatted water bath.! The absorbance of the final reaction mixture is determ ined using a grating spectrophotom eter.** Procedure For the analysis of the kinetics of the enzym e-coupled reaction, coupled enzyme reagent systems containing either the starch or the maltotetraose as substrate are reconstituted using the am ount of diluent corresponding to 80 percent of the am ount required by the m anufacturer. After mixing for 15 m inutes by slow inversion, the transfer disc of the centrifugal analyzer is loaded with sample, 0.025 ml, flush, 0.100 ml, and reagent, 0.400 ml. An absorbance print routine is loaded into the system computer, the transfer disc placed into th e instrum ent and the reaction initiated. Absorbance readings are taken at one m inute intervals. Both the starch and m altotetraose substrates are analyzed d u rin g th e sam e batch to e lim in a te betw een-batch variation. For the linearity study, both the starch and m alto tetrao se system s are used. Saline dilutions of an elevated serum sample are assayed by each m ethod using a Kinetic Rate I program containing subroutines for substrate exhaustion and absorbance change errors. Because of the differing kinetics of the two substrates indicated by the kinetic rate experim ents, 11 Lab-Line Magna-Stir, Scientific Products, McGaw Park, IL 60085. ** Model 25, Beckman Instruments, Inc., Fullerton, CA 92634. T A B L E II Program Parameters fo r th e M altotetraose Enzyme-coupled Procedure fo r Amylase Program KR I Sample volume 0.025 ml Flush volume 0.100 ml Run temperature 37 C Factor 3376 Lag phase 180 s Sampling interval 15 s Filter position 1 (340 nm) two separate sets of com puter parameters are used for the linearity studies. When the starch substrate is used, a lag phase of five m inutes and reading phase of two m inutes is incorporated. T he concentration factor, based on the volume fraction of serum and m olar absorptivity of NADH, is calculated to be 1688 for this assay system. The maltotetraose system, on the other hand, requires only a three m inute lag phase and two m inute reading interval. The factor calculated for this system is 3376. The computer param eters for the maltotetraose system used for routine determ inations are shown in table II. T he protocol for the dye-coupled procedure is outlined in the reagent insert. This protocol was followed explicitly with no modifications. T he m altotetraose (y-axis) and dyecoupled procedure (x-axis) are compared using the same samples. Analysis is perform ed on norm al and abnorm al specim ens by each m ethodology and the results com p ared by lin e a r reg ressio n analysis. The precision of the maltotetraose assay was determ ined by replicate analysis, both within-day and day-to-day, o f an abnormal control specim en. Results and D iscussion The data for the kinetic study are illustrated in figure 1. Linear kinetics data, using samples having elevated activities, are obtained after a three m inute lag phase for the defined substrate, maltotetraose and, after five minutes, for the starch substrate. In both cases, a reading interval of two m inutes after the initial lag phase is found to exhibit linearity. Also illustrated in figure 1 is the striking change in sensitivity w hen the two substrates are com pared. This characteristic is a result of two factors. From an examination of the reaction principle, it is evident that two moles of maltose are produced for each mole of
AUTOM ATED AMYLASE ASSAY 423 m altotetraose cleaved by the action of the amylase. Conversely, only one mole of m altose is produced by each catalytic attack of amylase on the starch. It has also b e e n show n by M en so n 5 th at alphaamylase exhibits a higher affinity for small oligosaccharides than for large, long chain carbohydrate moities. These two factors lead to an increase in sensitivity in assay system s u tiliz in g m alto tetrao se as substrate. T he linearity of the m ethod is graphically illustrated in figure 2. Analysis of the data shows that the assay system utilizing the maltotetraose substrate is linear to approximately 1200 U per liter of activity. For the starch substrate, the reaction is non-linear by dilutional analysis throughout the dynamic range studied. This fact is a result of the attack of amylase on the starch m olecule. Initially, the reaction m ixture contains only starch, but as the amylase cleaves the parent starch m olecule, small oligosaccharides are produced w hich serve as better substrates in the reaction. H ence, the apparent activity of the enzyme is increased. The rate of conversion of starch to oligosaccharide is dependent on the enzyme activity contained in the reaction mixture. As a result, the apparent activity is increased in samples with high amylase activities and decreased in samples with low activity when these two activity regions are compared to samples exhibiting m idrange activities. The apparent increase in activity of elevated samples found when the maltotetraose is used as substrate can be the result of several factors. It is quite possible that the maltotetraose contains som e starch fragm ents w hich cause increases in apparent activity as before, or the mechanism of action of amylase on the su b strate is very com plex, in clu d in g transglycosylation, condensation, and hydrolysis of the defined substrate. A mechanism of this complexity has been described for bacterial amylase by Matsuno et al.4 FIG U RE 1. Kinetic analysis o f the enzym e- coupled procedure for amylase. A and B. A serum sample exhibiting 340 U per liter of activity when using maltotetraose as the substrate. C and D. A serum sample having 30 U per liter activity using maltotetraose as substrate. The comparison of the enzym e-coupled procedure (y), and the dye-starch procedure (x) is graphically illustrated in figure 3. By analyzing 84 normal and pathological specim ens in duplicate, a linear regression equation of y = 0.874x 24 is obtained. The m ethod correlation is r = 0.979, and this level of correlation agrees with that shown earlier by Crouse,3 using a different enzym e-coupled reaction and the dye- starch method. The precision data are summarized in table III. W ithin-run precision of 14 rep licates of 1.2 percent at a level of 160 U per liter and day-to-day precision on 62 suc- FlGURE 2. The linearity of the enzyme-coupled procedure using both substrates. A serum sample having 2000 U per liter (maltotetraose) was diluted and each dilution was assayed using both substrates. Note the non-linearity of the dilutions when starch is used as a substrate.
424 CROUSE, CROSS, PARKER AND SAVORY TABLE I I I P recisio n o f the Automated Assay W i t h i n - R u n Day-to-Day N 14 N 62 X ± SD, U/liter 159-163 X ± SD, U/liter 156-172 CV, percent 1.2 CV, percent 4.6 cessive replicate analysis of 4.6 percent dem onstrate the reproducibility of the assay system. The data presented here demonstrate the utility of the enzym e-coupled assay and its applicability to the study undertaken. The m ethod is precise, has a broad range of application (to 1200 U per liter of activity), is not interfered w ith by en dogenous glucose and uses kinetic m ethodology which eliminates blanking and end-point interferences. The ability to automate the analysis of amylase is of prime importance to the clinical laboratory. The m ethod proposed here meets all requirements of an assay system in the modern laboratory. F ig u r e 3. The comparison o f the enzym e- coupled procedure with a dye-coupled procedure. The linear regression equation was determined to be y = 0.874x - 24, r = 0.979 and n = 84. Normal Range T he norm al range for th e assay of amylase used in this study is 20 to 110 U per liter. How ever, it is important that users define th eir ow n reference intervals. Resum e of C linical Interpretation Amylase is of prim e interest in the diagnosis of acute pancreatitis.2 However, it has been well docum ented that other diso rd ers m im ic the sym ptom atology of acute pancreatitis.8 Elevation of amylase may be due to acute pancreatitis, mumps, renal disease, trauma and a wide variety of abdominal disorders such as cholecystitis.7 References 1. Bow ie, L., E ster, F., Bo l in, J., and Gochm an, N.: Development of an aqueous temperature indicating technique and its application to clinical laboratory instrumentation. Clin. Chem. 22:449-455, 1976. 2. Brooks, F.: Testing pancreatic function. New Eng. J. Med. 286:300-303, 1972. 3. Cr o u se, S. P., C r o ss, R. E., and Savory, J.: The adaptation of an enzyme-coupled amylase procedure to the centrifugal analyzer. Clin. Chem. 22:1196, 1976. 4. Ma t su n o, R., S uganum a, T., F ujimori, T., N akanishi, K., Hiromi, K., and Kam iku b o, T.: Rate equation for am ylase catalized hydrolysis, transglycosylation, and condensation for linear oligosaccharides and amylase. J. Biochem. 83:385-394, 1978. 5. Me n so n, R., A dam s, T., N arayam sw amy, V., Co l l in g s, P., and D a v id so n, D.: A stoichiometric approach for determinations o f serum amylase. Clin. Chem. 22:1164, 1976. 6. Pierre, K., Tu n g, K., and N adji, H.: A new enzymatic kinetic method for determination of amylase. Clin. Chem. 22:1219, 1976. 7. Sa lt, W. and Sc h en k d er, S.: Amylase its clinical significance: A review of the literature. Medicine 55:269-289, 1976. 8. SlNGH, G.: Clinical significance and experimental studies on amylase: Review. Indian Med. Sci. 20:185-193, 1966.