Parviainen, Koivula and Jokela: Automated serum amyläse and isoamylase assay 41

J. Clin. ChenT. Clin. B loche m.Vol. 22, 1984, pp.41-45

Serum Amyläse and Isoamylase Assayon the Hitachi 705 Automatic Clinical Chemical Analyzer

By Markku T. Parviainen, Timo Koivula and Hanna Jokela

Department of Clinical Chemistry, Tampere University Central Hospital, Tampere, Finland

(Received May 26/October 31, 1983)

Summary: The automated continuous -amylase assay using p-nitrophenyl-a-D-maltoheptaoside (Boehrin-ger Mannheim) äs Substrate on the Hitachi 705 clinical cheniical analyzer, was compared with the Phadebas®Kinetic Amyläse Assay (Pharmacia Diagnostics) on the Hitachi 705. The two methods showed good correla-tion.

The precision varied from 1.0 to 2.5% (CV) within-day and from 1.1 to 5.6% (CV) day-to-day. The sub-strate, /?-nitrophenyl-a-D-maltoheptaoside, was also applied to an automated isoamylase assay. The amyläseinhibitor from wheat was used to determine the ratio of pancreatic and salivary amyläse activities of serum.About 80% of salivary type amyläse was inhibited up to an activity level of 1000 U/l, while Inhibition ofpancreatic type amyläse activity was only 10—15%. Ratios of pancreatic to salivary amyläse from 0.1 to 10can be evaluated in serum with wheat inhibitor. The precision of the isoamylase determination by the Boeh-ringer amyläse method was acceptable.

Bestimmung von Amyläse und Isoamylase im Serum mit dem Hitachi 705Zusammenfassung: Die automatisierte kontinuierliche Bestimmung von a-Amyläse mit p-Nitrophenyl-a-D-maltoheptaosid (Boehringer Mannheim) als Substrat mit dem Hitachi 705 würde mit dem Phadebas® KineticAmyläse Assay (Pharmacia Diagnostics) am Hitachi 705 verglichen. Beide Methoden zeigen gute Korrela-tion.

Die Präzision lag in der Serie zwischen 1,0 und 2,5%, von Tag zu Tag zwischen 1,1 und 5,6%. Das Substrat p-Nitrophenyl-a-D-maltoheptaosid wurde auch zur automatisierten Bestimmung von Isoamylase eingesetzt.Der Amylasehemmstoff aus Weizen wurde zur Bestimmung des Verhältnisses von Pankreas-/Speichel-Amy-läse im Serum verwendet. Speichelamylase bis zu einer katalytischen Konzentration von l kU/1 wurde zu etwa80% gehemmt, Pankreas-Amylase nur zu 10 bis 15%. Quotienten von Pankreas- und Speichel-Amylase imSerum von 0,1—10 können mit dem Inhibitor aus Weizen untersucht werden. Die Präzision der Bestimmungvon Isoamylase mit der Methode von Boehringer Mannheim war annehmbar.

IntroductionContinuous amyläse assays have recently been intro-duced by several manufacturers. Unfortunately thevarious methods are difficult to compare with eachother because of differences in the Substrates used.At least four different Substrates are in common use:p-nitrophenyl-a-D-maltoheptaoside (BoehringerMannheim), maltotetraose (General Diagnostics), a

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

mixture of oligosaccharides containing a knownamount of polymers with 4 to 10 glucose residues(Merz-Dade) and a carboxymethylated starch (Phar-macia Diagnostics). Differences in the specificity ofamyläse to these Substrates make various standardi-zation procedures necessary. So far no recom-mended standardization procedure is available foramyläse.

42 Parviainen, Koivula and Jokela: Automated serum amylase and isoamylase assay

The assay for amylase isoenzymes has been used toseparate the various amylase isoenzymes, whichmainly originale in the pancreas and salivary glands.Electrophoretic (1—3), electrofocusing and Chroma-tographie (4) techniques, and Inhibition tests (3, 5,6) have been used in this assay. The inhibitor proteinfrom wheat (Triticum aestivum) exhibits 100-foldspecifity for human salivary (S) s compared to hu-man pancreatic (P) amylase (5), which makes it suit-able for inhibitor assay of amylase isoenzymes. ARIA method has also been introduced for salivaryisoamylase (7).The aim of this study was to evaluate the applicationof two continuous amylase tests and of an automatedisoamylase assay on the Hitachi 705 automatic clini-cal chemical analyzer.

Tab. 1. An outline of the α-amylase assays on the Hitachi 705Automatic Analyzer.

A. Reagent preparation

Reagent 1.

Reagent 2.

1.Boehringercontinuousamylase

Dissolve thereagent vial 2in 45 ml ofsolution I.Dissolve oneSubstrate vialin 5 ml ofsolution 1.

2.Phadebas®KineticAmylase

Redistilledwater

Dissolve thecontents ofone reagentvial in 20.5 mlof water

3.Phadebas®KineticIsoAmylase

Dissolve oneinhibitor vialin 15 ml pfwaterAs in 2.

B. Chemistry parameters

Materials and Methodsα-Amylase assay kitsContinuous assay of amylase was performed with the CBR Pro-gram α-amylase Farbtest kit from Boehringer Mannheim GmbH,Mannheim, F.R.G. and the Phadebas® Kinetic Amylase Kit fromPharmacia Diagnostics, Uppsala, Sweden, who also supplied thePhadebas® Kinetic IsoAmylase Kit.

TemperatureAssay code

Sample (ul)Reagent 1 (μΐ)Reagent 2 (μΐ)Wavelength 1 (nm)Wavelength 2 (nm)

37 °C2(Kinetic)-2501

1040040

660415

37 °CAsinl.

5250500376340

37 °CAsinl.

5250500376340

Automated amylase assay using the Hitachi 705 clini-cal chemical analyzer

Continuous amylase and isoamylase assays with various kits wereperformed using a Hitachi 705 (Clinicon, Mannheim, F.R.G.) sshown in table 1. The table shows essentially the reagent prepara-tion and the chemistry parameters needed for the automated as-say.

OL-Amylase Standards

Human-based Phadebas® IsoAmylase Test Serum Standards (P/S0.1—8) and Humylase H, M and L control serums containinghigh, medium and low levels of amylase, and high and low P/Sratios were all from Pharmacia Diagnostics. Isoamylase Standardswere also prepared in our laboratory from a pooled sample ofsaliva (total amylase activity concentration 300 kU/1) and from asample with high pancreatic amylase (total amylase 30 kU/1) ob-tained from a pancreas drain. These samples were diluted in phos-phate buffer, 50 mmol/1, pH 6.9 and assayed for exact amylaseactivity, and were mixed to yield P/S ratios 0.1—10.

Amylase Inhibitors

Amylase inhibitor from wheat seeds (Triticum aestivum) was ob-tained from Sigma Chemical Co., St. Louis, MO 63178, contain-ing 2700 inhibiting units per mg of protein. Inhibitor stock solu-tion was prepared by dissolving the contents of one inhibitor vial(5 mg protein corresponding to 13500 inhibiting units) in 1.0 mlof tris buffer, 20 mmol/1 containing NaCl 10 mmol/1, pH 8.0.This solution was diluted 1/100 in the phosphate buffer, and in>mediately before use it was further diluted 1/4 in the same buffer.

The inhibitor Solutions were stored frozen. Additionally, inhibitorfrom the Phadebas® IsoAmylase Test Kit was used and the lyo-philized wheat inhibitor redissolved ceordingto the manufactur-er's recommendations. The inhibitor solution was diluted 1/3 inthe phosphate buffer before use.

Isoamylase assay with Inhib i t ion

150 μΐ of Standards, controls or samples werepipetted in duplicateinto glass tubes. 150 ul of the phosphate buffer was pipetted intoone tube of the duplicates and 150 μΐ of the inhibitor workingsolution into the other. The tubes were mixed and analysed fortotal amylase activity after at least 5 min at room temperature.The fraction remaining uninhibited was calculated. Then the stan^dard curve was constructed in which the uninhibited fraction isrelated to the P/S ratio in the Standard. The P/S ratio in samplesand controls was read frorh this curve. After correcting for thedilution (x2) in controls and samples, the P and S isoenzyme ac-tivity concentrations were calculated (5):

P (Pancreatic) amylase (U/l) = Total amylase x / P/S VU + P/S/

S (Salivary) amylase (U/l) = Total amylase -- P

In addition the isoamylase assay with the Phadebas® KineticIsoAmylase method was also performed on the Hitachi 705. Theinhibited and uninhibited reactions were performed automaticallyin the analyser, which calculated the activities directly. The Stan-dard curve relating uninhibited fraction to the P/S ratio was con-structed and the results calculated s above.

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

Parviainen, Koivula and Jokela: Automated serum amylase and isoamylase assay 43

ResultsTotal continuous amylase assay on the Hi-tachi 705The routine amylase assay in our laboratory is theBoehringer continuous method for the Hitachi 705.This was compared with the Phadebas® Kinetic Am-ylase Test in the same analyser.The day-to-day and within-day coefficients of Varia-tion of the two methods are presented in table 2. TheBoehringer amylase assay is linear at least to 3500U/l, whereas the Phadebas® kinetic method in theHitachi 705 was not linear above 750 U/l.

An excellent correlation was found between themethods compared, s will be seen in figure 1. Thesame assay level in the two methods was achieved bystandardizing both with human serum. However, theresults from various commercially available serumsdiffered considerably from one method to the other.Normally there was no need for daily standardiza-tion of the Boehringer amylase method.

Isoamylase assayThe routine Boehringer continuous total amylase as-say was used combined with preliminary incubationof 5 min with the inhibitor at room temperature toseparate the pancreatic (P) isoenzyme from salivary(S) isoenzyme. The incubation time proved to belong enough together with about 10 min processingtime of the Instrument before starting the measure-ment. Figure 2 shows the Standard curves for thevarious methods relating the uninhibited amylasefraction to the respective P/S ratio in the Standard.

£5, 10oo-L. x-s..£.§

*.

ΙΛ L_Λ je

500-

500 1000Amylase (Phadebas Kinetic

Amylase Kit) [U /1 )

Fig. 1. The Boehringer continuous amylase method on a Hitachi705, compared to Phadebas® Kinetic Amylase Kit methodin 28 patient serum samples.y = 1.105x - 19.013, r = 0.998, n = 28

Tab. 2. The within-day and day-to-day coefficients of Variation inthe total amylase assays with Hitachi 705 E.

A. Boehringer continuous method

Total amylaseactivityconcentration(U/l)

130520

2120

Coefficients of Variation

Within-day

2.5 (n = 8)1.0 (n =12)1.1 (n = 12)

Day-to-day

3.4 (n =16)1.1 (n =12)1.2 (n =13)

B. Phadebas® kinetic method

Total amylaseactivityconcentration(U/l)

Coefficients of Variation

Within-day Day-to-day

185370445

Variationnot determined

<»

5.6 (n = 5)2.2 (n = 10)4.8 (n = 8)

0 0.1 1 10 ooPancreatic amylase

Salivary amylase

Fig. 2. Isoamylase Standard curves relating the fraction of totalamylase remaining uninhibited to the respective P/S ratio.The curves were c nstructed by mixing pancreatic and sa-livary amylases in P/S ratios in a r nge of 0.1 — 10, andanalysing the mixtures for amylase activity in the presenceand absence of inhibitor. In the lower two curves eachpoint is the mean of 7 analyses. The bars indicate ±2 SD.(A) Phadebas® Kinetic IsoAmylase Kit (direct assay),(O) Boehringer continuous amylase preincubated for 5min with Phadebas® inhibitor,(Φ) Boehringer continuous amylase preincubated for 5min with Sigma inhibitor.

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

44 Parviainen, Koivula and Jokela: Automated serum amylase and isoamylase assay

The various commercially available inhibitors exhi-bited a marked Variation in the Inhibition with theBoehringer continuous method äs illustrated in fig-ure 2. This is probably due to different quality andpurity of each commercial inhibitor. Recalibration ofthe assay is essential whenever the inhibitor batch ischanged. The Phadebas® Kinetic Isoamylase Assayon Hitachi 705 yielded somewhat lower Inhibition ofthe S activity äs compared to the Boehringer assaycombined with preliminary Inhibition. Apparentlythe 5 min incubation time with inhibitor in Hitachi705 is not enough for a complete Inhibition,The inhibitor from Sigma was adopted for our rou-tine amylase assay because of its commercial availa-bility. In figure 3 the linearity of the assay is shownfor the P and S amylase isoenzymes. The curves areconstructed by analyzing total amylase and isoamy-lase from a serial dilution of a patient serum withhigh total amylase activity concentration (3500 U/l)and low P/S ratio. The assay for total amylase activi-ty is linear at least to 3500 U/1, but the total activitymust be diluted below 1000 U/l for the isoamylaseassay.

4000-

3000-

•CDOH- 2000-

< 1000-

1000 2000 3000 4000Amylase calculated [ U / l ]

Fig. 3. Linearity of the amylase and isoamylase assay methodswith Boehringer continuous amylase and Sigma inhibitor.A patient serum containing elevated total and S amylaseactivity was diluted and assayed for total amylase and Pand S isoamylases.(O) total activity, (O) S activity, ( ) activity concentra-tion.

In table 3 the coefficients of Variation are summa-rized for the within-day and day-to-day analyses ofthe P and S isoamylases. The coefficients were deter-mined by assaying both patient serum samples con-taining normal or elevated total amylase, and high,normal and low P/S ratios and with Phadebas® hu-man based Standards. The Phadebas® Standards gavesomewhat higher Variation coefficients.

Tab. 3. The within-day and day-to-day coefficients of Variation inthe isoamylase assays at various P/S rations at normal orslightly elevated serum amylase concentration.

A. Boehringer continuous methodHuman-based serum samples made in our laboratory.

Ratio P/S Totalamylaseactivity

(U/l)

Coefficients of VariationWithin-day Day-to-day(n = 8) (n = 8)P type S type P type S type

6.51.50.2

390340280

7.96.75.1

9.88.63.4

5.76.6

12.0

20.510.72.1

B. Boehringer continuous methodPharmacia isoamylase Standards (n = 20)

8.01.00.1

204180380

10.614.715.5

18.510.36.7

Variationnot determined

C. Phadebas® Kinetic Isoamylase AssayPharmacia isoamylase controls (n = 8)

8.00.7

450370

Variationnot determined

9.28.6

13.86.4

Isoamylase assays from seiüm samplesTotal serum amylase, and P and S isoamylase activi-ties were determined from apparently healthy nor-mal subjects (34 of both sexes, tab. 4), ages 32.2 ±10.3 years (rneän ± SD), ränge 16—58 years. Thetotal amylase activities were not statistically differentin the sexes. Females however had significantly (p<0.001) higher P isoamylase activity äs compared tomales.

Tab. 4. Amylase and isoamylase activity concentrations in nor-mal subjects (means ± SD).

Subjects n Mean amylase activity concentration(U/l) ± SDTotal Pancreatic (P) Salivary (S)

Normald 34 157.8 ±43.1 76.9 ± 29.7 81.l ±41.39 34 179.6 ±59.5 111.4 ±43.4a 70.3 ± 37.9

Statistical significance of difference between rnaks and femalesaccording to the Two Sample t-test: a p <0.001.

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

Parviainen, Koivula and Jokela: Automated serum amylase and isoamylase assay 45

Discussion

Automated total amylase assays have been intro-duced in routine laboratories, using the continuousmode of analysis with commercially available kits(6). One difficulty is, however, the variety of Sub-strates used in the assay kits pending internationalrecommendations.In this work we have tested the continuous amylaseassays from Boehringer Mannheim and PharmaciaDiagnostics on a Hitachi 705 analyzer. The methodscorrelated well with each other, but the Boehringermethod seemed to be more readily suited for routineuse in the Hitachi 705 analyzer by reason of its widerlinearity. The methods also showed excellent preci-sion when compared to each other.

The isoamylase assays in this work are based on Inhi-bition with inhibitor from wheat (5). The methodswere easily applied to the Hitachi 705 analyzer. Theisoamylase assays with inhibitor are thus rapid andeasy to perform äs compared to other isoamylasemethods, e.g. electrophoresis (1—3) or radioimmu-noassay (7). Based on appropriate absorbancechanges, at leäst the Phadebas® Kinetic IsoAmylasetest is also readily applicable to routine manual pho-tometric measurement either at 37 PC or at 30 °C.

The precision characteristics of our isoamylase assayare comparable to those given by O'Donnell et al.(5) for their Inhibition methods of by Rammeloo et

al. (3) for their electrophoresis and Inhibition meth-ods. The isoamylase analyses from serum samples innormals showed a statistically very significant differ-ence (p <0.001) in P isoamylase between femalesand males; the latter showed lower levels, whichagrees with the findings of O'Donnell et al. (5).

The clinical value of serum isoamylase measure-ments is still a matter of controversy. The usefulnessof the electrophoretic method has been criticizedmainly because of the laborious methodology to-gether with a somewhat questionable value in clini-cal decision-making. The present isoamylase testmakes possible a fast and easy evaluation of pan-creatic and salivary type isoamylase activities. Pre-vious studies have shown that results of the inhibitortests correlate with the electrophoretic method (3).Although a considerable bias between the methodsmay exist, further studies are however needed to as-sess whether this correlation is valid in all clinical si-tuations. These studies are now in progress in ourlaboratory^and will be published later.

AckiiowledgementsPhadebas® Kinetic Amylase and Kinetic IsoAmylase kits weregenerously provided by Star Oy, Tampere, Finland.

References1. Ojala, K. & Harmoinen, A. (1975) Scand. J. Clin. Lab. Invest.

35, 163-169.2. Skude, G. (1975) Scand. J. CÜn. Lab. Invest. 35, 41-47.3. Rammeloo, T., Haard, P. M. M. & Beunis, M. H. (1982) Clin.

Chem. 28, 145-149.4. Takeuchi, T. (1979) Clin. Chem. 25, 1406-1410.

5. O'Donnelli M. D., FitzGerald, O. & McGeeney, K. F. (1977)Clin. Chem. 23, 560-566.

6. Huang, W. Y. & Tietz, N. W. (1982) Clin. Chem. 28, 1525-1527.

7. Boehm-Truitt, M., Harrison, E., Wolf, R. O. & Notkins, A. L.(1978) Anal. Biochem. 85, 476-487.

Markku T. Parviainen, Ph. D.Department of Clinical Chemistry,Tampere University Central Hospital,SF-33520 Tampere 52

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