Larsen: -amylase determination using maltopentaose äs Substrate 45

J. Clin. Chem. Clin. Biochem,Vol. 21, 1983, pp.45-52

-Amylase Determination Using Maltopentaose äs Substrate

By /£. Larsen

Department of Clinical Chemistry Sunderborg Sygehus, Stinderborg, Denmark

(Received January 15/July 5, 1982)

Summary: The rationale of choosing a NADP-coupled continuous method, with the Substrate maltopentaose,äs a method for the determination of -amylase (EC 3.2. L1) activity is investigated. The method presented isinvestigated with respect to all reaction parameters, including possible influence of protein, and shows zeroorder reaction kinetics after a 5—6 minute lag phase. The blank reaction from maltopentaose Substrate isconstant and is 13% of the upper limit of the reference interval for serum. The course of the blank reactioncan be used to check that the maltopentaose is of adequate purity for use in the assay. Km for maltopentaose is0.48 mmol/1. There is no interference from endogenous glucose when the total NADP turnover is less than0.25 mmol/1. Data for sensitivity, linearity and long term precision over an eighteen month period are given,together with reference intervals for serum and for urine. The method is recommended for consideration äs areference method.

Bestimmung von a-Amylase mit Maltopentaose als Substrat

Zusammenfassung: Die rationelle Grundlage für die Wahl einer NADP-gekoppelten kontinuierlichen Me-thode mit dem Substrat Maltopentaose als Methode für die Bestimmung von -Amylase (EC 3.2.1.1) wurdeuntersucht.Die beschriebene Methode wurde im Hinblick auf alle Reaktionsparameter, einschließlich eines möglichenEinflusses von Protein untersucht und zeigt nach einer 5 bis 6 minütigen lag-Phase eine Kinetik nullter Ord-nung.Die Blindreaktion des Maltopentaosesubstrats ist konstant und beträgt 13% der oberen Referenzwertgrenzefür Serum. Der Verlauf der Blindreaktion kann als Grundlage für die geforderte Reinheit der angewandtenMaltopentaose dienen.Km für Maltopentaose ist 0,48 mmol/1. Es besteht keine Interferenz der endogenen Glucose bei einem totalenNADP-Umsatz von weniger als 0,25 mmol/1.Es liegen Daten vor über die Sensitivität, Linearität und Langzeitpräzision über eine 18 monatige Periode undReferenzwerte für Serum und Urin.Die Methode wird für die Beurteilung als Referenzinethode empfohlen.

IntroductionMethods for the determination of -amylase (1.4-a-D-glucan glycanohydrolase) in biological fluids in-clude starch-iodine (1), starch with measurement ofreducing groups (2) and fecently dyerlabelled starch(3) methods. The newer NAD(P)H-coupled, contin-uous methods (4-8) have been developed for use ina variety of mechanized procedures.

No single method is generaily accepted äs a refer-ence or recommended method. The evaluation of amethod is therefore difficult, äs correlation arid re-gression studies are only of restricted value and inparticular do not solve the main problem: The lackof standardization, when methods are used overlonger periods, with different Substrates and differ-ent Substrate batches.

034Ö-076X/83/0021 -0045$02.00© by Walter de Gruyter & Co. · Berlin New York

46 Larsen: -amylase determination using maltopentaose äs Substrate

The starch-reducing-group method (2) is generallyused äs a basis for nominal -amylase values in sev-eral commercial reference sera. However, calibra-tion of other methods with such a reference serum isan arbitrary and unsatisfactory solution.

It is desirable to develop methods with well docu-mented characteristics that fulfil the requirementsfor method standardization. The NADH methodsreviewed by Lorenz (9) and Kaufman et al. (10) arepotentially interesting in this connection. Thesemethods measure glucose formed by coupled en-zyme processes from the maitose formed by the hy-drolytic action of -amylase on different oligo- orpolyglucan Substrates.

Substrates can be divided into polysaccharides, suchäs starch, that are chemically poorly defined, andwell defined 1,4-pligosaccharides, such äs fnaltote-traose and maltopentaose. On the basis of Robyt &French's work (11), maltopentaose may be consi-dered to be an ideal Substrate, because active sites inporcine pancreatic amylase — which shows similari-ties to human pancreatic amylase — consist of 'fiveglucose' binding sites with the catalytic groupsplaced at the second binding site, so that hydrolysisof maltopentaose is dominated by the products mai-tose and maltotriose.

Maltopentaose is used — following preliminary re-moval of endogenous glucose - in the Du Pontmethod (12, 13), but basic and detailed method stu-dies for this and other commercially developedNADH methods are lacking.

Therefore, the prerequisite for agreement on meth-od standardization based on the molar absorbance ofNADH is not fulfilled.

The present study concerns a maltopentaose methodusing NADP and without removal of endogenousglucose:

Maltopentaose

Maltose+.Maltotriose

5 Glucose+5 ATP

5 Glucose-6-phosphate-h5 NADP+

a-Amylase

EC 3.2.1.1

a-Glucosidase

EC 3.2.1.20

Hexokinase

EC 2.7.1.l

G-6-P dehydrogenaseEC 1:1.1.49

Maltose+Maltotriose

5 Glucose

5 Glucose-6-phosphateH-5 ADP

5 6-Phospho-gluconateH-5 NADPH+5 H+

Optimal reaction conditions, requirements for thepurity of maltopentaose, and long-term reprodud-bility have been determined, in order to evaluate theoverall potential of the method äs a candidate refer-ence method.

Preliminary to the study, maltopentaose (not com-mercially available - US patent 4039383) and otherglucose homologues (Gi—Gn) with 1.4 glucosidebonds were produced by partial arid hydrolysis ofamylose and colunm chromatography of the hydro-lysate using polyacrylamide gel (14), colüinn eluatesbeing identified with thin layer chromatography andquantified using a spectrophotometric phenol^sul-phuric acid test.

Materials and MethodsApparatus

LKB 8600 Reaction Rate Analyzer 37 °C with plotter and 340 nminterference filter.

Reagents

Amylose, Merck 4561Maltose, Merck 5912Maltotriose, Sigma grade IIHPTLC 'Fertigplatten', Kieselgel 60, MerckG-6-P-DH (> 75 kU/1) / HK (> 125 kU/1), mixture in 50% gly-cerol, from Glucoquant®, Boehiinger Mannheim Biochemicals.NADP-disodium salt 98% and ATP-diso,dium salt, BoehfingerMannheim Biochemicals.

Maltopentaose:

a) Donated by Lic. Techn. Bent Stig Enevoldsen, Carlsberg Re^search Centre, Copenhagen, Denmark;

b) Donated by Noda Institute for Scientific Research, Japan;

c) Own production (14), together with maltohexaose and othermaltosyl oligosaccharides.

The purity of different maltopentaoses is described in the section*Experimental Results'.

Phosphate buffer reagent pH 6.8 ± 0.05 (20^25 ÖC), contaiiüngthe following (mmol/l):Phosphate 70, sodium chloride 51, calcium chlonde10.6, magnesi-

'um chloride 4, NADP 2.7 and ATP 1.5 (stäble for at least 5months at -20 °C and at least l month at +4°C).

-Glucosidase solution (with ammonium sulphate 0.6 mol/1): a-Glucosidase 250 kU/1 (Boehringer Mannheim Biochemicals) iscentrifuged and 4/5 of the Volume of supernatant containing am-monium sulphate 3.2 mol/1 is removed. The enzyme concentrateis diluted with 4/5 volume of redistillied water (Stäble at leastl month at 4 0C).

Albumin dilution reagent, containing:Human serum albumin, Kabi® 2 g/l and sterile sodium chloridesolution 9 g/l.Other chemicals: all Merck analytical grade;

J. Clin. Chem. Clin. Biochem. / Vol. 21,1983 / No. l

Larsen: α-amylase determination using maltopentaose s Substrate 47

Specimens

Serum and urineSaliva, centrifuged, sterile filtered and diluted with sterile albumindilution reagent.

Serum pools, enriched with α-amylase from sterile filtered saliva,s well s serum pools with mainly pancreatic α-amylase from pa-

tients with acute pancreatitis.

All serum and saliva samples were stored at -20 °C up to a maxi-mum of 18 months, with no measurable change in α-amylase ac-tivity. Urine samples were mixed, centrifuged and analysed on theday of sampling.

Thin layer chromatography

Glucose (Gi) and other maltosyl oligosaccharides including mal-topentaose (Gs) and up to maltoundecaose (Gn) can be identifiedby asccending thin layer chromatography on Merck HPTLC Kie-selgel 60 using 2-propanol: acetone: lactic aeid, 24-2+1 (by vol.)·,and naphthoresorcin - sulphuric acid (Merck) s the location rea-gent.

Proposed α-amylase assay procedure (see also tab. 1)

Phosphate buffer reagent ΙΟΟΟμΙG-6-P-DH/HK mixture 10 ulct-Glucosidase solution 80 ulSerum, urine or water (substrate blank) 20 μΐMix and incubate at 37 °C (min. 5 min, max. 4 hours)Start reagent: Maltopentaose 24 mmol/1 100 μΐ

Following a lag phase of 6 min, ΔΑ is measured at 37 °C and340 nm with one minute s assay interval.

The substrate blank ΔΑ = 0.007 (constant) is deducted. Calcula-tion of α-amylase activity in the sample:

χ 10* = (AA min-1) x 1945[U/1]

Tab. 1. Assay conditions in proposed α-amylase assay.

Molar lineic absorbance of NADPH (340 nm):

ε = 622 m2/mol

The sample is diluted when the total absorbance at the end of thelag phase is higher than A = 1.6.

Experimental Res ltsThe proposed assay procedufe for determination ofα-amylase described above gives the reaction condi-tions described in table 1. These conditions arebased on a series of optimisation studies for the indi-vidual reaction parameters described below (α-amy-lase from a suitable dilution of saliva was used inthese investigations, but all the results were also sub-stantiated using human serum pools With mainlypancreatic α-amylase). Reaction conditions in thefigures shown and in the tables are, with the excep-tion of the parameter uftder investigation, the sames those shown in table 1.

Phosphate (Na/K)Sodium ChlorideCalcium ChlorideMagnesium ChlorideAmmonium sulphateATPNADPMaltopentaosea-GlucosidaseHexokinaseGlucose-6-phosphate dehydrogenaseTemperaturepHSerum volume fraction

58 mmol/142 mmol/1

0.5 mmol/13 mmol/1

40 mmol/1l .2 mmol/12.2 mmol/12.0 mmol/1

16 kU/l(25°C)

2*0.6 kU/1 (25 °C)37 °C6.60.0165

Buffer, pH and ion concentrationComparisons of acetate, tris and phosphate buffersshow that phosphate is the most suitable, with an ac-ceptable buffer capacity of 58 mmol/1 at pH 6.6 inthe feaction mixture. Increasing the buffer concen-tration results in significant anion Inhibition at con-centrations above 100 mmol/1. The optimal pH forthe reaction mixture is 6.6 ± 0.2 (fig. 1).

0.120

0.100_

0.080

0.060

6.2pH

6.6 7.0

Fig. 1. Dependence of α-amylase reaction rate on pH (37 °C) inthe reaction mixture. Assay conditions s given in tab. 1.Enzyme: Dilutions of saliva with albumin diluent.

ChlorideIncreasing the concentration from 30 to 100 mmol/1increases the reaction rate to twice that of the ratewithout Chloride. Higher concentrations of chloridecause Inhibition.

CalciumCalcium at 0.5 mmol/1 activates slightly, and higherconcentrations have a slight inhibitory effect on thereaction rate.

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48 Larscn: -amylase determination using maltopentaosc äs Substrate

MagnesiumMagnesium is known to be necessary for the glucosereaction system and was not investigated in the pres-ent study.

SulphateSulphate ions from the -glucosidase preparation1)and added sodium sulphate inhibit the reaction ässhown in figure 2. Sulphate concentration in the pro-posed assay conditions is 40 mmol/1 (tab. 1). Inter-polation to zero sulphate concentration gives a maxi-mal 4% greater reaction rate in the total reactionsystem. Inhibition is found to be about five times äsgreat in the glucose indicator reaction, which is dem-onstrated by addition of glucose without -amylase.The lag phase for the two indicator reactions aiid forthe total -amylase reaction System is increased athigher sulphate ion concentrations.

0.200

0.180

| 0.160l30. HO

^0.120

0.100

200 0)0SO,2' Immol/l)

600 800

Fig. 2. Inhibition of -amylase reaction rate with sulphate ion,sulphate final concentrations originating from a-glucosi-dase preparation (see tab. 1) and from added sodium sul-phate. Assay conditions äs given in tab. 1. Enzyme: Dilu^tions of saliva with albumin diluent.

about 16'kU/l chosen in the proposed assay (tab. 1)gives a lag phase of 5-6 min, which is found to beconstant and equal for different sources of -amy-lase (see 'specimens').

Hexokinase and glucose-6-phosphate de^hydrogenase

These auxiliary enzymes afe pfeseiit in the sameconeeiitrations äs described for Glucoquant® for thedetermination of glucose. Halviüg or doubling theconcentrations of both enzymes has no significant ef-fect.

ATP and NADP

ATP 1.2 mmol/1 and NADP 2.2 miiiol/l were ehosenafter consideration of the results shown in figure 3.With a maximum turnöVer of ATP and NADP of0.25 mmol/1 in the reaction mixture, correspondiiigto a maximum absorption of A = 1.5, the ATP andNADP concentrations will still remain in the optimalranges of concentration.

0.200

0.150

10,100

0.050H

a-Glucosidase

-Amylase reaction rates were investigated at cata-lytic concentrations of -glucosidase between 2 and32 kU/1 at constant sulphate concentration and con-stant pH. Between about 4 and 32 kU/1, the maxim-um reaction rate is constant, but the lag phase variesbetween about 15 min at the lowest and 4-5 min atthe highest concentration. A proportionally increas-ing maltopentaose blank reaction is also obtainedwith increasing catalytic concentration of -glucosi-dase, in äs much äs maltopentaose is also slowly hy-drolysed by -glucosidase. The concentration of

Lyophilized -glucosidase became commercially available af-ter the termination of this study.

0 1 2 3 4 . 5 6 7ATP or NADP mmol/ l

Fig. 3. Dependence of -amylase reaction rate on ATP ( —€>)and ori NADP (O—O). Assay conditions äs given intab. 1. Enzyme: Dilutions of saliva with albumin diluent.

Maltopentaose Substrate

Maltopentaose is used in the three gf ades a, b and c.ä) is piirified by paper chromatography*

b) has the following specifications: wäter 0·,017, car ·̂bohydrate 0.983, and an oligoiner distributioncöfttaining maltopentaose 0.980 together withmaltotetraose 0.002 and G6-s »Qligömers 0.018.

J. Cliii. Chem. Clin. Bioehem. / Vol. 21,1983 / No. l

Larsen: α-amylase determination using maltopentaose s Substrate 49

c) has been compared with a) and b) using thin lay-er chromatography, which showed that all threegrades were of the same purity.

a-Amylase activities measured using the threegrades of maltopentaose give similar results·.

Following a suitable period of reaction at 37 °C, mix-tures of 2 mmol/1 maltopentaose in α-amylase con-taining serum or in dilutions of serum with phos-phate buffer pH 6.8 were analysed by thin layerchromatography. In every case there were two reac-tion products, maitose and maltotriose, and less than0.10 unhydrolysed maltopentaose.Examination of the blank reaction of the total α-am-ylase reaction System, in which the sample contain-ing α-amylase is replaced by distilled water (fig. 4),gives the best indication of the content of lower olig-omers in the maltopentaose; the rate of reaction isgreatest for glucose, and this reaction is seen in thefirst minute of the blank reaction; it is less for mai-tose and maltotriose, which react in the first 3—4minutes, and least for maltotetraose, which gives the

0.200

0.150

0.100

0.050

6 8t Cmin]

10 12

Fig. 4. Relationship between α-amylase reaction rate on time ofmeasurement for sample (•^- ) and for reagent blank(O—O). Assay conditions s given in tab. 1. Enzyme: Di-lutions of saliva with albumin diluent.

curved part of the reaction course in the last part ofthe lag phase. Finally, the blank reaction of malto-pentaose is seen s a constant low reaction rate withAAmin"1 == 0.007. The results of experiments withlow concentrations of glucose, maltpse, maltotriose,maltotetraose and maltohexaose s Substrates (for a-glucosidase) in the α-amylase reaction System, con-firm the Interpretation of the course of the blank

reaction described above. Maltohexaose and higherglucose homologues (from our own preparation ofmaltopentaose) give no measurable blank reaction,and do not give a higher α-amylase reaction rate.

Km for maltopentaoseThe Km for maltopentaose using a Lineweaver-Burkplot (fig. 5) is 0.48 mmol/1. Two mmol/I is chosen(tab. 1), because the greatest α-amylase activitymeasurable with a linear response, 850 U/l in thesample, will convert about 0.05 mmol/1 in the reac-tion lasting 7 minutes, i.e. a maximum of 0.02 of theSubstrate. Maltopentaose concentrations higher than4 mmol/1 give a maximum reaction rate (blank sub-tracted) about 17% higher, but the blank reactiondue to maltopentaose is increased by a factor of 2.

0.200

0.160

0.120

0.080

0.040.ι ' l * ' ' ' ι \ ι

4 6 8 10Maltopentaose l mmol/l]

12

Fig. 5. Substrate curve and determination of Km for α-amylasewith maltopentaose s a Substrate by a Lineweaver-Burkplot. Assay conditions s given in tab. 1. Enzyme: Dilu-tions of saliva with albumin diluent.

Endogenous glucose and the serum blankreactionGlucose from serum or urine will react completely inthe α-amylase reaction System during the lag phase;Δ A min""1 after 5 min at 37 °C, and without malto-pentaose in the reaction System is 0.000 ± 0.002. Avariety of serum samples, including very icteric andlipaemic samples, also give serum blank values thatare not significantly different from zero. Using thecomplete reaction System, α-amylase activity is con-stant after addition of various glucose concentrationsup to 12-14 mmol/1 in the sample (tab. 2). Varia-tions of glucose content in the samples and thus in-itial variations in NADP/NADPH ratios do not ef-fect the α-amylase activities measured for glucose

J. Clin. Chem. Clin. Biochem. / Vol. 21,1983 / No. l

50 Larsen: -amylase determination using maltopentaose äs Substrate

concentrations up to twice the upper reference levelfor normal values. Higher glucose concentrations(l 6-20 mmol/1) give a slight negative interference.

Tab. 2. Effect of variable glucose concentration pn measured a-amylase activity.

Glucose(mmol/1 0 2 4 6 8 12 16 20in sample)a-amylase(U/l) 149 147 144 143 141 143 137 129(x,n = 4)

Assay conditions äs given in table 1. Enzyme: Constant concen-tration of saliva, diluted with albumin diluent.

Lineari ty - the effect of proteinKnown dilutions qf high catalytic activity serum withup to at least 850 U/l -amylase activity (corre-sponding to the usable ränge of measurement withthe apparatus concerned) give a linear response, i.e.zero order kinetic, and the activity-absorbance linegoes through the origin. Variation of the proteincontent by diluting high -amylase serum with sodi-um chloride solution 9 g/l, or low -amylase serumhas no influence either on the reaction rates mea-sured per liter serum or on the ränge of linearity.Glucose concentrations in the sera used were about4 mmol/1.

Dilution 1:2 of 39 normal urines (see fig. 7), used todetermine the reference ränge, gives correspondingresults when the dilution is performed with sodiumchloride 9 g/l (x = 288 U/l), or with diluent contain-ing albumin 4 g/l and sodium chloride 9 g/l(x = 291 U/l); statistical comparison using Wilcox-on's matched-pairs test for the two sets of resultsgives p = 0.10. On the other hand, saliva only retainsits -amylase activity when diluted with albumin dil-uent, and increasing dilution with sodium chloride9 g/l results in a total loss of activity in comparisonwith activities over 100 U/l in corresponding albu-min-containing dilutions.

Method data

Data describing the method from the above studiesand from precision and reference interval determi-nation are collected in table 3. The results for blankvalues are given äs means and ranges for betweeii-

run determinations carried out over a period of 18months. Between-run precision data are from a peri-od of one year, including several changes of reagentbatches. In addition, other precision data over a 18months period using a variety of samples, äs de-scribed in 'specimens', show coefficienfts of Variationof 3-5% for -amylase activities 50-400 U/L

Tab. 3. Assay data for the proposed -amylase assay.

Lag phase 5 - 6minMeasurement interval l minSerum blank 0± 4 U/lMaltopentaose Substrate blank 14 ± 4 U/lLinearity 0 - 850 U/l

(with NADP turnover less than 0.25 mmolA)Serum reference interval (N = 173) . 30 - 112 U/lUrine reference interval (N = 39) <600 U/l

Precision3)Within -run, mean 174 U/lBetween-run, mean 179 U/lBetween-run, mean 24 U/l

SD = 4.2 U/l CV = 2.4%SD = 7.9 U/l CV = 4.4%SD = 2.3 U/l CV = 9.5%

a) Serum pool, n = 19 for each determination.Between-run values are calculated from means of triplicates as-sayed during a one-year period.

The reference interval for serum was determmed us-ing a group of 173 outpatients over 18 years old, andwith no known pancreatic or gästro-intestmäl dis-eases. The histogram in figureo shows a slightlyright-skewed distribution. The 95.% interval calcu-lated from log values is 30-112 U/l.

30

; zo

10

24 48 72 96a- Amylase (serum) tu/ll

120 144

Fig. 6, Histogram of the serum -amylase activity in 173 outpa-tients with no recognized disease of the pancreas — gastro-intestinal Systems. Assay conditions äs given in tab. 1.

J. Glin. Chem. Clin. Biochem. /Vol. 21,1983 /No. l

Larsen: -amylase determination using maltopentaose äs Substrate 51

For urine, the upper reference interval limit of600 U/I is based on a group of 39 healthy hospitalemployees over 18 years old (fig. 7). The.urine sam-ples were taken at random and analysed on the sameday äs sampling.

5erS4gr$3

200 400 600-Amylose (urine) [U/ l ]

800 1000

Fig. 7. Histogram of the urine -amylase aetivity in 39 healthypersons from the hospital staff. Assay conditions äs givenin tab. 1.

BiscussionA theoretical argument for chosing maltopentaose äsa Substrate is given in the study by Robyt & French(6), mentioned above. The present study substan-tiates their findings that only maitose and maltotri-ose are formed by the action of human -ainylase onmaltopentaose.The chosen reaction conditions give an apparentlyzero order reaction after a 5—6 minute lag phase. Bysetting a maximum limit of As4o =1.6 for the ab-sorbance at the end of the lag phase, a linear re-sponse can be ensured for all samples, whatever theircombined a-amylase-glucpse coiitent and inclüdingthe äbsorbance of the reagents and samples them-selves.The course of the Substrate blank cän be used äs abasis of the requirements of purity for the malto-pentaose used. Among the lower linear 1,4 glucosehomologues (Gi^G4), maltotetraose could give ahigh and variable ('kinetic') blank value, dependingon its concentration, the -glucosidase concentratipnand the chosen period of measurement.In the study of linearity, the proteins of serum orurine samples did not irifhience the results for a-amylase aetivity. Urine samples used to determine areference interval show no significant differences

with and without albumin addition, but experimentsalso show that saliva completely loses its aetivitywhen diluted with water or sodium chloride (9 g/l),and retains it when there is protein in the diluent.

The method data in table 3 show that the presentmethod has a high sensitivity and an excellent long-tenn reproducibility, even with several changes ofreagent batch.

An overall comparison with the Du Pont aca -amy-lase procedure is made difficult by the lack of pub-lished Information about reagent composition, espe-cially buffer type, added activators and purity ofmaltopentaose. From the manufacturer's assay man-ual it can be seen that the chosen concentrations ofthe specified reactants do not differ essentially fromthe present method (tab. 1), differences being thepreliminary removal of endogenous glucose, the useof NAD, and a total reaction time of 4.3 min.

The main differences between the characteristics ofthe present method and the aca method are that theaca method is limited by measurement of a non-Iine-ar rate change in äbsorbance (13), and has a variableSubstrate blank of 100-120 U/l (9); both featurescan be explained by contamination of maltopentaosewith maltotetraose. Furthermore, albumin activationof urinary -amylase with the aca method (13, 15)contradicts the results of the present study, but thenature of possible protein interference is not clari-fied and needs further study. Finally, the originallyreported aca reference interval shows an upper limitof 75 U/l (12) compared with 112 U/l in the presentmethod, but it should be emphasized that the maxi-mum obtainable aetivity using an assay System with ahigher maltopentaose concentration of 4 mmol/1 andwith a sülphate^free -glucosidase preparation, isexpected to give an upper limit of reference intervalof about 140 U/l.

ConclusionIn summary, it is concluded that maltopentaose,theoretically a well chosen and chemically well de-fined Substrate for -amylase in the reagent Systemdeveloped in the present study, is also suitable inpractice, both for mechanized and for manual Sys-tems.The method conforms to the majority of the require-ments that can be made of a prospective referencemethod, especially concerning the important possi-bility of a reliable calibration based on the molarlineic äbsorbance of NADPH.

J. Clin. Chem. CHn. Biochem. / Vol. 21, 1983 / No. l

52 Larsen: -amylase determination using maltopentaose äs Substrate

Acknowledgement

This work was supported by the Medical Research Fund for theHospitals in Ringkobing, Ribe and South Jutland Counties. Ithank Lic. Techn. Bent Stig Enevoldsen, Carlsberg Research Cen-

tre, Copenhagen for supplying reference materials and for itsvaluable assistance -in the preparation of maltopentaose, and theNoda Institute for Scientific Research, Japan, for making avail-able maltopentaose. I am also very grateful for the interest andskilful work of the technical assistant Rita Bork.

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Mr. K. Larsen, M. Pharm. Sc.Dept. of Clinical ChemistrySonderborg SygehusP.O. Box 160DK-6400 Senderborg

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