Scholer and Hohenwallner: Evaluation of -amylase assay with p-nitrophenylheptaoside äs Substrate 677

J. Clin. Chem. Clin. Biochem.Vol. 22, 1984, pp. 677-684

Evaluation of a Colorimetric Test for the Determination of ct-Amylasewith jp-Nitrophenylheptaoside äs Substrate

By A. Scholer

Kantosspital, Basle, Switzerland and

W. Hohenwallner

Allg. öffentl. Krankenhaus der Barmherzigen Schwestern, Linz, Austria

Participants:Dr. D. H. den Blaauwen

Dr. J. H. Jansen-Ziekenhuis, Emmeloord, The NetherlandsProf. Dr. A. Burlina, Dr. P. Rizzotti

Istituti Ospitalieri B. Trento, Verona, ItalyG. Clayes

Kath. Universiteit, Leuven, BelgiumProf. Dr. J. P. Colombo, Dr. E. Peheim, P. Degiampietro

Inselspital, Berne, SwitzerlandDr. H. J. Gibitz

Landeskrankenanstalten, Salzburg, AustriaDr. C. van der Heiden

Wilhelmina Kjnderziekenhuis, Utrecht, The NetherlandsPriv.-Doz. Dr. E. Henkel

Krankenhaus Oststadt, Hannover, GermanyDr. W. Hohenwallner

Allg. öffentl. Krankenhaus der Barmherzigen Schwestern, Linz,Austria

Prof. Dr. O. Lalegerie, A. VassaultHöpital Necker Enfants Malades, Paris, France

Prof. Dr. K. LorentzInstitut für Klinische Chemie, Med. Hochschule, Lübeck,Germany

Dr. E. MagidBispebjerg Hospital, Copenhagen, Denmark

Dr. K. MayrAllg. Krankenhaus, Wels, Austria

Prof. Dr. P. Maiais, Prof. G. FerardCentre de Traumatologie et d'Orthopedie de Strasbourg,Illkirch-Graffenstaden, France

Dr. A. Noma, Dr. H. OkabeTokyo Metropolitan Geriatrie Hospital, Tokyo, Japan

Dr. H. Pilgerstorfer, E. RuprechtsbergerMed. Diagn. Laboratorium, Linz, Austria

Prof. Dr. W. Rick, Dr. E. SchnaithZentralinstitut für Klinische Chemie undLaboratoriumsdiagnostik, Düsseldorf, Germany

Dr. S. RosalkiRoyal Free Hospital, London, United Kingdom

A. ScholerKantonsspital, Basle, Switzerland

Prof. Dr. N. Tieiz, Dr. F. ShueyUniversity of Kentucky, Lexington, USA

Dr. 7. WaldenströmSahlgrenska Sjukhuset, Göteborg, Sweden

Prof. Dr. L. Weiss, Dr. G. HoffmannKrankenhaus München-Harlaching, Munich, Germany

G. Staber, C. MöllerBoehringer Mannheim GmbH, Tutzing, Germany

(Received August 16, 1983/May 25, 1984)

Summary: A continuous method for the determination of a-amylase activity with a defined Substrate has beenpublished by Rauscher et al. in Berichte der österreichischen Gesellschaft für Klinische Chemie 4, 150(1981). It offers the followiiig advantages: good practicability, easy adaptation to analysers and short incuba-tion time.

Tweüty-one laboratories took part in a multicentric study on this method, working with different Instrumentsand manual procedures at three temperatures. Additionally, all laboratories participated in a laboratory im-provement program, in order to discriminate systematic from accidental errors. Data on imprecision, linearity,interferences and comparability of the method have been presented at a workshop.

J. Clin. Chem. Clin. Biochem. / Vol. 22,1984 / No. 10

678 Scholer and Hohenwallncr: Evaluation of ct-amylasc assay with p-nitrophenylhcptaoside äs Substrate

The resulting coefficients of Variation for imprecision in series äs well äs from day to day were 0.6—6.3% and1.3-7.7%, respectively. No interferences from bilirubin, glucose, maitose and 37 selected drugs were found.The results from the described method correlate well with those from an U V-test (substrate: maltoheptaose);compared with other methods, however, the expected systematic deviations were observed. Reference valuesfor 25, 30 and 37 °C are indicated in this report.

• l

Evaluierung eines kontinuierlichen Farbtests zur Bestimmung von a-Amylase mit p-Nitrophenyl-maltohepta-osid als Substrat.Zusammenfassung: Rauscher et al. beschrieben in Berichte der Österreichischen Gesellschaft für KlinischeChemie 4, 150 (1981) eine kontinuierliche Methode zur Bestimmung der -Amylase-Aktivität mit einemdefinierten Substrat. Diese Methode bietet als Vorteile: gute Praktikabilität, Automatisierbarkeit und kurzeInkubationszeit.Einundzwanzig Laboratorien nahmen an einer multizentrischen Studie über diese Methode teil, wobei ver-schiedene Automaten und manuelle Arbeitsverfahren bei drei Temperaturen verwendet wurden. Um syste-matische von zufälligen Fehlern zu diskriminieren, nahmen die Laboratorien zusätzlich an einem Ringversuchteil. Ergebnisse über Ungenauigkeit, Linearität, Störungen und Vergleichbarkeit der Methode wurden aufeinem Workshop vorgestellt.Die erhaltenen Variationskoeffizienten für die Ungenauigkeit in der Serie sowie von Tag zu Tag lagen zwi-schen 0,6—6,3% bzw. 1,7—7,7%. Störungen durch Bilirubin, Glucose, Maltose und 37 ausgewählte Pharma-ka wurden nicht festgestellt. Die Resultate der beschriebenen Methode korrelieren gut mit einem UV-Test(Substrat: Maltoheptaose); im Vergleich mit anderen Methoden traten hingegen die erwarteten Abweichun-gen auf. Referenzwerte für 25, 30 und 37 °C werden angegeben.

Introduction

According to the German Society for Clinical Chem-istry (1), about 200 methods for measuring ct-amy-lase, based on nine different principles, have beendeveloped during the last 100 years. The main re-quirements, postulated also by other national com-mittees, are the following:

- continuous measurement- defined Substrate of constant quality- sufficient specificity for endo-cc-amylase- defined reaction mechanism

From practical considerations, the method shouldsatisfy further conditions, such äs

— short incubation time- adaptability to all types of analysers- no interference by endogenic glucose- formation of a chromogenic reaction product (2).

For the new method, some of these points have beenpublished separately (3, 4).

The following report presents the results obtainedduring the evaluation of the continuous colour test,"a-Amylase PNP". Twenty-one laboratories in Eu-rope, USA and Japan took part in this study. Tho-rough examinations of the methodology were madeat 25, 30 and 37 °C, according to manual work sheets

äs well äs on the following analysers: ACP 5040,ABA-100®, Abbott VP, CentrifiChem® 400, Co-bas® Bio, ENI Gemsaec, Hitachi 705, LKB 2086, ILmultistat® III. Reference values in serum and urinewere established for the three tempefatüres.

Materials and MethodsBoehringer Mannheim's test kit for automatic analysers, "a-Amylase1) PNP", contains phosphate buffer solution pH 7.1 andlyophilisates of a-D-glucosidase2) (30 U/l) and p-nitrophenyl-a-0-maltoheptaoside (5 mmol/1), all in separate bottles.

To improve stability, particularly fof samples with extreme pH-values, the buffer concentration was meanwhile increased from 50mmol/1 to 100 mmol/1. Comparison of tests at 25 and 37 °Cshowed tolerable deviations up to 4%. For the comparison ofmethods, the following additional reagent kits were used: amylo-clastic and chromogenic tests, one reductometric and. two ÜV-methods (substrates: maltoheptaose and maltotetraose) andanother kinetic colour test.

The measurements were made on the following analysers accord-ing to the instructions of the reagent manufacturers:

- Abbott 100® and Abbott VP (Abbott Scientific ProduetionsDivision, S. Pasadena Ca. 91030, USA)

- CentrifiChem® System 400 (Union Carbide Inc., Tarrytown,N.Y. 10591^ USA)

- I Gemsaec Centrifugal Analyzer (Electro Nucleonics, Fair-field, N.J. 07006, USA)

') EC 3.2.1.12) EC 3.2.1.20

J. Clin. Chem. Clin. Biochem. / Vol. 22,1984 /No. 10

Scholcr and Hohenwallner: Evaluation of u-amylase assay with /?-nitrophenylheptaoside äs Substrate 679

- Cobas® Bio (Hoffmann-La Röche & Cie AG, CH-4002 Basle,Switzcrland)

- IL multistat® 111 (Instrumentation Laboratory Inc., Lcxington,Mass. 02173, USA)

- Hitachi 705 (Bochringer Mannheim GmbH, D-6800 Mann-heim 31. Germany)

- Clinicon AB 2086 Mark II (Boehringer Mannheim GmbH, D-6800 Mannheim 31, Germany)

- Greiner GSA (Greincr Electronics AG, CH-4900 Langenthai,Switzerland)

- ACP 5040 (Eppendorf Gerätebau, Nethcler und Hinz, D-2000 Hamburg, Germany)

The study was rcalized with fresh and frozen human sera, urinesamplcs, in one casc with pancreas fistula fluid, and the followingcontrol sera:Monitrol and EnzatrolMerz & Dade, Dept. American Hospital

Supply GmbH Deutschland, D-8000 Mu-nich 50, Germany

Kontrollogen L Behring-Werke AG, D-35SO Marburg,Germany

Validatc

Hylarid

Seronorm

Versatol

Gödecke AG, D-1000 Berlin, Germany

Div. Travenol Laboratories S.A., B-7860Lessines, BclgiumNyegaard & Co., AS-Olso, Norway

General Diagnostics, Div. of Warner-Lambert Company, Morris Plains, N.J.07950, USA

Results

Imprec is ionThe imprecision of the method in series äs well äsfrom day to day was measured in human sera of low,medium and high amylase catalytic activity on thedifferent analysers and for three temperatures. Thesamples were selected by the particular laboratories.Examples are shown in table l.

Tab. 1. Precision of the colorimetric lest -Amylasc PNP at 25, 30 and 37 °C.

Instruction sheets for Precision within runn = 30Catalytic Temperatureconcentration

ACP 5040 Start withSubstrate

® 100

Abbott VP

Centrifichem®400

Cobas® Bio

ENI Gemsaec®

Manual procedurestaft with sample

Hitachi 705

LKB 2086

Manual procedureStart with SubstrateMultistat® III

X

(U/l)

41101426

84152684101227

115052

126450

60121656

9619666765

13949110222758542692214765152

127485

25 °CCV (%)

2.84.00.6_

-~

——-2.81.84.2

——-

——-3.63.62.5

——-_

-

—

———

30 °CCV(%)

--4.82.32.2

——-

—-—1.51.00.8

——-

-

0.70.50.5_

-3.91.33.53.91.4

37 °CCV(%)

--

—--1.70.61.3--——-—2.22.01.7

-

——-3.52.1—

—-—

Precision between runn = lOduplicatesCalalytic TemperatureconcentrationX

(U/l)

48106607108165745101230

115353

121428

65121655108192651

6714550010322756841689715564756

127494

25 °CCV(%)

5.44.52.3_

—-

——-2.11.31.8—-———-3.63.02.6-----—

--—

30 °CCV(%)

_—-7.76.64.6

——-

—-—2.51.80.9---

-

3.91.82.2

4.05.07.73.01.8

37 °CCV (%)

_—-_

-- '

2.22.62.0------5.93.62.4

-

---3.04.3

-

J. Clin. Chem. Clin. Biochem. / Vol. 22, 1984 / No. 10

680 Scholer and Hohenwallner: Evaluation of α-amylase assay with p-nitrophenylheptaoside s Substrate

B e h a v i o u r of reac t ion and l i n e a r i t y

Figure l shows the reaction curves of four differentsera: one control serum, one icteric, one lipaernicand one serum with increased amylase catalytic ac-tivity.The linearity of the continuous measurement was ex-amined with stepwise dilution of human sera andurine samples with 154 mmol/1 (9 g/l) NaCl solution(fig. 2).For all adaptations on analysers, the linearity up tthe indicated dilution limit or higher is given s afunction of the sample to reagent ratio (fig. 2).

2.50

2.00

ε 1.50

1.00

0.50

0.00 6 8 10 12 14t [min]

Fig. 1. Absorbance/time diagram of α-amylase PNP from differ-ent samples at 25 °C.

.Φ 30° C/^-Abbott VP

30° CHitachi 705

25°C-Atentrifi-

chem®400ι ι ι ι ι ι ι ι L

"0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00Fraction of serum/urine samples

Fig. 2. Dilution series for serum ( ) and urine ( ) samples(triple determinations) at 25, 30 and 37 °C.

of sample Material and reagents

Amylase-containing sera can be stored frozen or atrefrigerator temperatures without any problem,whereas the urine samples show a decrease in cata-lytic activity. The stability of the'Yeagents was testedby storing them for thfee weeks at +35 ΡΟ, Solutionsmade from these reagents were again stored for fivedays at +25 °C and for two weeks at +2 to +8 C.The amylase activities found in two control samplesof pathological and normal r nge were within tolera-ble limits.

Interference

As one important eornponent of this ev luation, thereliability of the "a-Amylase PNP" test was checkedby extensive ititerference studies.

The following possible interferiiig substances wereexamined:Bilirubin, haemoglobin, glucose, anticoagulants,maitose, 37 drugs, lipaernic sera, turbidity, hydroxy-ethyl starch. A serum of high bilirubin concentration(225 μηιοΐ/ΐ) and low amylase activity was mixed inseveral Steps with a serum of high amylase activityand low bilirubin concentration. A linear functionwas found for the amylase activity (fig. 3). Thedashed line shows the correspondirig bilirubin con-tent.The influence of haemoglobin was tested by dilutinga haemolytic with a non-haemolytic serum sample ofthe same blood donor. Figure-4 shows that samplescontaining up to 0.15 mmol/1 haemoglobin may beused for analysis without problems.

E

1300

1100

900

700

500

300

100

\

Ί30

110

90 §o

70 !,!

Fig.

1 2 3 4Serum [fi(]

3. Relationship between aliquot size (μΐ) of a high a-amylaseserum added to high bilirubin serum and the resulting a-amylase catalytic activity concfehtration) (closed circles)and bilirubin concentration (closed triangles).

J. Clin. Chem. Cjin. Biochem. / Vol. 22,1984 / No. 10

Scholcr und Hohenwallncr: Evaluation of u-amylasc assay with /j-nitrophenylhcptaosidc s Substrate 681

1.07

1.03

0.99

0.95

0.91

0.87

0.83

0.79

η?ς

-

ι • ί ,[ h n

-

-

ι ι ι ι ι

' l 1ι.

ι

-·

ι ι ι| 0.04 0.09 0.13 0.17 0.22 0.26 0.30 0.35 0.39 0.43l Hoemoglobin I m m o l / l )Non-

hoemolyticFig. 4. Influence of hacmoglobin (A/r 68000) on α-amylase cata-

lytie activity conccmration. Results given s fraction ±2 SD of the non-hacmolytic sample.

16

U

12

£10o»

1 8U.

6

4

2

-

-

χk

Medi n1

^

498 524 550 576 602 628 654 680 706α-Amylose measured ( U / l ]

- Abbott VP- ΑΒΑ® 100- ACP 5040- Manual procedure- ENI GEMSAEC®

frt\- Cobas ΒΙΟ®- Hitachi 705- Multistat I

•

e

•

e

1

•

••

1

*

A normal serum (approx. 70 U/I) was mixed withincreasing quantities of glucose up to 56 mmol/1. Nointerference was recognized. A maitose concentra-tion of 6.2 mmol/1 also had no influence on the testSystem. On the other hand, decreased values wererecorded in citrate, fluoride, EDTA, and oxalateplasmas, since these anticoagulants complex calcium,which is an active component of the enzyme's activecentre. For this reason, only serum, urine and hepar-in plasmas can be used. None of the other drugs ex-amined in vitro (5) interfered at therapeutic concen-trations. For extremely turbid sera, a predilution isrecommended. Interference by hydroxyethyl starchwas shown in vitro s well s in vivo. The in vitroinfluence is most probably based on a removal of theenzyme via absorption by hydroxyethyl starch. In vi-vo, an increase of α-amylase in serum appears afterinfusions with this plasma substitute, while on theother hand, the excretion of the enzyme in urine iscorrespondingly reduced.

Accuracy

Laboraiory improvemenl pro gram

The comparabiiity of the results from the particularInstruments with those obtained by the original man-ual working instruction was tested by measurementof two contf l sera of unknown amyla.se catalytie aotivity on five days at 25, 30 and 37 °C.

The results of this comparison program are plottedin histograms (e.g. 30 °C see fig. 5).

Fig. 5. Determination of α-amylase catalytie activiiy conccntra-tion in an unknown controi sample by 8 laboralories on 5days, double determinations. 30 °C.N = 80; χ = 584.9 U/l; median = 585.5 U/l; SD = 55U/l; CV = 9.5%.

The good results of the study are documented by afairly Symmetrie distribution. The deviation of themean value from the assigned value is ±5%. Infor-mation about the distribution of the values in the dif-ferent laboratories is given in the data-structureanalysis (6).

This diagram analyses the distribution of the valuesof the particular laboratories. The position of everylaboratory's median is marked by a point. Figure 5shows e.g. that the classes 654-680 U/I and 680-706 U/l contain only those values from multistat®III. The median is in the class 680-706 U/l.

The systematic differences showed by figure 5 point-ed to the need for the correction of the worksheets ofmultistat® III and Hitachi 705. The Hitachi 705should be calibrated with a calibration serum, be-cause calibration with p-nitrophenol results in valuesthat are too low. After the adaptation of the manualinstructions to the multistat® III the recovery in de-pendence on the sample volume was found to be dif-ferent. At 25 and 30 °C the most favourable ratiobetween sample and total volume was 1 4 - 2 1 forserum and urine. Smaller sample volumes showed atendency to higher values.

J. Clin. Chem. Clin. Bjpchem. / Vol. 22,1984 / No. 10

682 Schoier and Hohcnwallner: Evaluation of α-amylase assay with p-nitrophenylheptaoside s Substrate

Qudlity controlA further item of the laboratory improvement pro-gram was the examination of the aptitude of severalcommercially available control sera for the continu-ous colour test at different temperatures and accord-ing to different instructions. Figure 6 shows an ex-ample, using Boehringer Mannheim control sera at37 °C (Precinorm® U and Precipath® E).

The recovery of the assigned values for Precinorm®U (407 U/l) and Precipath® E (798 U/l) was be-tween 90 and 115%. For both samples the lowestvalues resulted from the Cobas® Bio and the highestones from ACP 5040.

0)

£ 1.30cu

•l 1.20(Λ° 1.10§ i.oo1 0.90t' 0.80(Λ-E 0.70f 0.60

a-

'_ i ' '

3-

-

ACP 5040RA®

f ;

mn

F Jt J

Abbott VPENI GEMSAE

fL

c®

i[

Cobas BIO®

b

{ _ ί •ACP 50,40ΑΒΑ® 100

Abbott VPENI GEMSAEC®

Cobas BIO®LKB 2086 LKB 2086

Fig. 6. a-Amylase in Precinorm® U (a) and Precipath® E (b) ondifferent Instruments at 37 °C: fraction of assigned value.Method: α-Amylase PNP.

Comparison of the methods

The comparability of the results, using the newmethod on different Instruments, can be judged bythe values of the multicentric study. The comparabil-ity with other methods, however, was investigated bythe comparison of the following methods: proce-dures measuring α-amylase catalytic activity by thedecrease of the iodine-starch colour, chromogenic,one reductometric, two UV methods (Substrates:maltoheptaose and maltotetraose) and another colo-rimetric test (substrate: a mixture of maltopentaoseand maltoheptaose). Pathological and normal r ngehuman sera s wei s heparin plasma and urine wereused s samp.es. After the determination on analys-ers, the results may be evaluated by the means of aComputing . ctor (based on the ε-v lue of p-nitro-phenol especially determined for this System) or of a

control serum based on human serum. When devia-tions occur, due to systematie influences, calibrationwith a control serum is recommended.

The following examples show the correlation of thenew "α-Amylase PNP" colour tost on different in-struments and vs. the compared methods (fig. 7—10).

1000

Ξ 800

600

400

200

0 200 400 600 800 1000 Ϊ200""α-Amylase (reductometric method [U/l (25*01

Fig. 7. Reductometric method, 25 °C (x) vs. o>Amylase PNP onACP 5040, 25 °C (y). n = 48 serum samples.Regression line: y = -16.3 + 0.53xCorrelation coefficient: r = 0.995

95 165 235 305 375 445" 515 585 655α-Amylase (ehromogenic, manual)[U-/ l (37°C)]

Fig. 8. Chromogenic colorimetric test (manual), 37 °C (x) vs, a-Amylase PNP on Cobas® Bio, 30 °C (y). n = 70 serumsamples.Regression line: y = -=8.17 -K»0.49xCorrelation coefficient: r = 0.994

J. Clin.-Chem. Clin. Biochem. / Vol. 22, 1984 / No. 10

Scholer and Hohenwallner: Evaluation of α-amylase assay with />-nitrophcnylheptaoside s Substrate 683

The correlations between the four methods are ex-cellent. According to the respective lest conditions(selection of the Substrate), all Computing faetorsshowed larger or smaller, theoretically expectablesystematic differences.

540

420

g 360

300•Eg 240

l 180>%

? 120d

60

Y = X

0 60 120 180 240 300 360 420 480 540α-AmylQse (continuous chromogenic) [U/l (30eC)l

Fig. 9. Continuous chromogenic colorimetric test, 30 °C (x) vs. a-Amylase PNP on Hitachi 705, 30 °C (y). Substrate: a mix-ture of maltopentaose and maltohexaose. n = 80 heparinplasma samples.Regression line: y = ^5.07 + 2.44xCorrelation coefficient: r = 0.989

540

480

420

.S 300

L.

0-

240

180

Ο-

Y = X

Χ

sS \. \ 1 1 I I 1 1

60 120 180 240 300 360 420 480 540'a- Amylase (UV, -maltoheptoose) IU/K30^°G)]

Fig. 10. UV-test, substrate: maltoheptaose, 30 °C (x) vs. ct-Amy-lase PNP on Hitachi 705, 30 °C (y). n = 80 heparin plas-ma samples.Regression line: y - -3.52 + 0.99xCorrelation coefficient: r = 0.997

For completion, the results at 37 °C are also present-ed (fig. 11-12). The Abbott VP results were ob-tained with human sera and urine samples vs. anUV-test (substrate: maltotetraose) and an amylo-clastic method on an LKB 2086. The Abbott VP wascalibrated with a Precinorm® U control serum. Agood correlation was found for the whole measuringr nge, s well s for the LKB examinations.

500LJoC-.ΓΟ

300

ib 200

100

100 200 300 400a-Amylase (UV maltotetraose) [U/l (37°C)1

500

Fig. 11. UV-test, substrate: maltotetraose, 37 °C (x) vs. a-Amy-lase PNP on Abbott VP, 37 °C (y). n = 65 urine (O) andserum (O) samples.Regression line: y = -1.294 + 1.64xCorrelation coefficient: r = 0.983

630

„ 560<_>e

B 490\-=>S 420GO

S

2 350c

^ 280Q_

| 210>v

1 HO

70

Y = X

•0 70 140 210 280 350 420 490 560 630α-AmylQse (amyloclastic, Greiner G S A D t U / l (37°0)

Fig. 12. Amyloclastic methodon Greiner GSA 11,37 °C(x)vs. a-Amylase PNP on LKB 2086, 37 °C (y). n = 59 serumsamples.Regression line: y = -10.2 + 0.95xCorrelation coefficient: r = 0.976

J. Clin. Chem. Clin. §iochem. / Vol. 22,1984 / No. 10

684 Scholcr and Hohcnwullncr: Evaluation of <t~amylase assay with /7-nitrophenyIhcptaoside äs Substrate

Tab. 2. Reference intcrvals of -amylase in scrum and urinc with PNP-maltohcptaoside at 25, 30 and 37 °C.

Samplc-matcrial Collcctive 25 °C 30 °C

24 hur ine 1 )

ambulalorium and staffof the Basle Kantonsspital3)Staff of theBasle Kantonsspital

37 °C

SerumHe pari n plasmaUrincportion

9 -i n9 H

H (5

H 6

H d

Selected blood-donors2 )Selected blood donors2)Selected patients of an

194

91

34-121-32-557

U/l

U/l

197

138

39-158-57-703

U/l

U/l

83(3860

69- 2 10 U/l63- 228 U/l92 -l 239 U /l

57 34-335 U/24 h 57 57-505 U/24 h 52 68-1021 U/24 h

') To be applicd only with reservation (see "Stability of sample materiell and reagents'\ p. 680):) Normal values requested for: aspartate aminotransferase, alanine amiunotransferase, total bilirubin, -glutamyltransferase, aikalinc

phosphatase, total protein, albumin, glucose, lipase and -amylase from a routine method.3) Normal values requested for all other urine parameters and -amylase from a routine method

R e f e r e n c e v a l u e s

In addition to the evaluation of the "a-AmylasePNP" colorimetric test, reference values for serum,heparin plasma and urine had to be determined fordifferent temperatures. Some participants have al-ready presented provisional values since the end ofthe test period. The final results have been publishedby W. Hohenwallner & A. Scholcr (7) and others (8).The following limits are recommended (tab. 2).

lzi rf'Er/7 et al. also used the above-mentionedmethod for the determination of reference values forserum, spontaneous urine and 12 hours-urine at37 °C (9). For serum (97.5 percentile: 225 U/l) andspontaneous urine (97.5 percentile: 1222 U/l), theresults demonstrate an excellent agreement with thedefined reference values.

Discussion

Unimportant scattering in series and good reproduc-ibility of the measurements from day to day confirmthe practicability of the tka-Amylase PNP" method

with every experienced worksheet. After a lag phaseof 3 min at 25 °C and of 2 min at 37 °C, the selectedreaction conditioris showed zero order reaction ki-netics.As the response for serum is linear up to the tenfoldupper reference Hmit, only few sera have to be dilut-ed.In view of its excellent comparability with other sim-ilarly based methods (e.g. continuous measurementsof -arnylase with maltotetraose or a mixture of p-nitrophenylpentaoside//7-nitrophenylhexaoside äsSubstrates), the new method offers a real alternative.In comparing these methods, particular accountshould be taken of the lade of interference by secon-dary reactions, the stability of the reagents and thechoice of a defined Substrate for the new "a-Arriy-läse PNP" method.

AcknowledgementsAll the participants are thanked for their helpful cooperation inthis extensive multiccntric study and for much uscful Information.

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504.2. Rauscher, E., von Bülovv, S. & Hagele, E. O. (1982) Fresenius

Z. Anal. Giern. 31L 454.3. Hagele, E. O., Schaich, E., Rauscher, E., Lehmann, P., Bürk,

H. & Wahlefeld, A. W. (1982) Gin. Chem. 28, 2201-2205*4. Rauscher, E., Neumann, U., Schaich, E., von Bülow, S. &

Wahlefeld, A. W. (1984) Gin. Chem. in print.5. Staber, G., Busch, E. W. & Koller, P. U. (1982) Med. Lab 35

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6. Markowetz, D. (1982) Med. Lab. 35, 53-66.7. Hohcnvvallner, W., Hagele, E. O., Schüler, A. & Staber, G.

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