P o s t e r A

has to be introduced into the formula for the calculation of the zooo- activity of c~-amylase relating to the turnover of the substrate.

The assay conditions were chosen as follows: HEPES buffer 100 mmol/1, pH 7.1, sodium chloride 50 retool/l, magnesium 900 chloride 10 mmol/1, substrate 5 retool/l, e-glucosidase 30 U/ml. Volume fraction of the sample ~ = 0.02 to 0.04 depending on 000 the handling, used instruments and measuring temperature.

The absorption coefficients of 4-nitrophenol at 25 ~ 30 ~ and 37~ were determined in a ring trial. The values found were 7~ 700 9.7 respectively 9.8 respectively 10.1 (1 �9 m o l - 1 , mm-1). It was concluded that the average value of ~ = 9.9 (1 �9 mol - 1 . ram- 1) should be used for all measuring temperatures. ~ 6oo

The reagent mixture containing all ingredients may be used T

3 days at 25~ or 10 days at 4~ An increase in absorption is caused by traces of contaminants of glucoamylase in the auxili- ~ s00- ary enzyme ct-glucosidase. Therefore, the quality of this enzyme 2 has to be improved to increase the stability of the reagent ~ 40o mixture. If the working solutions of c~-glucosidase and of the substrate are stored separately at 4~ they may be used for 2 r weeks, aoo

The activity of a-amylase in serum, heparin-, EDTA-plasma or urine may be determined either at 25, 30 or 37~ But the 2oo reference values for this method have to be determined yet.

The lag phase of the assay is less than 3 min at 25 ~ C and 30~ and less than 2min at 37~ For manual handling a 100 measuring time of 3 min is recommended. The method may be adapted to all automated instruments but preincubation of the assay during the lag phase is necessary. 0

Dilution of the sample is recommended when the increase of absorption is higher than AA/min 0.25 by manual handling. Using automated equipment the limit for dilution may be twice as high.

No difference of the activity of c~-amylase is found starting the reaction either by sample or by the substrate.

No influence was observed by lipemia, hemoglobin < 35 btmol/1, bilirubin < 170 btmol/1, glucose < 55 mmol/1, py- ruvate < 10 mmol/1 or ascorbic acid < 3 mmol/1 of sample.

The method described here correlates well with the method using 4-nitrophenylmaltoheptaoside as substrate [1]. The cor- relation coefficient r was found higher than 0.99 at all measuring temperatures. The results for the comparison of both methods by measuring the activity of 44 human sera using the instrument Hitachi 705 (37 ~ C) are given in Fig. 1.

• x

l l l l ~ l l [ l l ~ l l l l l

200 400 600 800 1000 I200 1400 1600 1fi00 2000

X: D T - P N P I U / L ]

S tand ,Pr lnc ,Comp.Ana[ .= Y = -1 .56 + .428 t X Re~res~lon of X to Y = Y - - 1 . 95 + ,428 �9 X Relilresston of Y to X = Y = -1 .17 + ,427 �9 X Coeff,of Correlation = .898

NL~ber of Sera : 44

Fig. 1. Comparison of methods

References

1. Rauscher E, Neumann U, Schaich E, vBiilow S, Wahlefeld AW (1985) Clin Chem 31 : 1 4 - 1 9

Fresen ius Z Ana l C h e m (1986) 3 2 4 : 3 0 4 - 3 0 5 �9 Spr inger -Ver lag 1986

A 57 Quality control material for the determination of the alpha-amylase (EC 3.2.1.1) Results of an experimental round robin

E. Henkel 1, G. R6hle 2, and A. Louderback 3

1 Institut fiir Klinische Chemic, Medizinische Hochschule Hannover, Podbielskistr. 380, D-3000 Hannover 51, Federal Republic of Germany

2 Institut fiir Klinische Biochemie, Universitfit Bonn, Federal Republic of Germany

3 CCC Laboratories, Temple City, California, USA

Qualit~itskontrolimaterial fiir die Bestimmung yon ~-Amylase (EC 3.2.1.1). Ergebnisse eines Ringversuchs

Introduction

Quality control material for the determination of a-amylase (EC 3.2.1.1) has some well-known limitations. The main problems are:

1. Alpha-amylase from spiked control sera has a different affinity to the substrates with short chain length, especially when enzyme from hog pancreas is added.

2. Some lyophilized control materials are turbid after re- constitution.

3. The stability of the control material varies. 4. The variety of methods makes it difficult to define a

reference method. To find a suitable control material for the different methods

currently used we started a round robin with six different control sera.

Material and methods

The following control sera were included in this study: S 1 Monitrol II Lot Nr. 68 (human S-amylase spiked); S 2 Wellcome unassayed (bovine serum); S 3 Beckman Benchmark (porcine amylase spiked); S 4 Beckman Decision Level 1 (porcine amylase spiked); S 5 Liquid Level A (Serum + EG) (human P-amylase spiked); S 6 Liquid Level B (Serum + EG) (human P-amylase spiked); S 7 Liquid Level in BSA (human S-amylase); S 8 Liquid Level in BSA (human P-amylase).

305

Pes ers A

Table 1. Results of amylase activity after transformation

Meth. 3 Meth. 4 Meth. 5 Meth. 6 Meth. 7 Meth. 8 Phadeb. G 7 PNPG 5 + G 6 PNPG 7 PNPG 7 G 4

S 1 442 U/1 517 U/1 485 U/1 483 U/1 512 U/1 440 U/1 S 2 136 U/1 326 U/1 436 U/1 286 U/1 314 U/1 102 U/1 S 3 135 U/1 113 U/I 168 U/1 83 U/1 93 U/1 95 U/1 S 4 306 U/1 570 U/1 655 U/1 586 U/I 587 U/1 164 U/I S 5 779 U/1 631 U/1 513 U/1 521 U/1 606 U/1 475 U/1 S 6 780 U/1 678 U/1 542 U/I 531 U/1 613 U/1 494 U/1 S 7 327 U/I 306 U/1 311 U/1 323 U/1 323 U/1 350 U/I S 8 325 U/1 262 U/1 249 U/1 248 U/1 297 U/1 202 U/I

Factor 1.00 2.63 4.70 3.00 2.45 7.83

Serum 1 and 2 were lyophilized materials, serum 3, 4, 5, and 6 were based on 33% ethylenglycol (EG) and a serum matrix. Control material 7 and 8 were made up in a stabilized liquid bovine serum albumine (BSA) matrix with highly purified S- and P-amylase added.

Eight different methods currently used in the laboratories in Germany, Danmark, Sweden and Norway were included in this study. These methods were performed manually and with different automated analytical instruments. The measuring temperatures were 25~ and 37 ~ All tests were performed twice a day over a period of two days. So the precision within runs and from day to day under routine conditions can be calculated.

The following methods were used: 1. Amylochrome (Roche) 37~ 2. DY-Amyl (G6decke) 37~ 3. PHADEBAS (Pharmacia) 37 ~ 4. Maltoheptaose (G 7) (Boehringer Mannheim) 25~ 5. Testomar Amylase-PNP (PNPG 5 + G 6) (Behringwerke)

25~ 6. Alpha-Amylase-PNP (PNPG 7) (AHS Miinchen) 25~ 7. Alpha-Amylase-PNP (PNPG 7) (Boehringer Mannheim)

25~ 8. Maltotetraose (G 4) (Beckman Miinchen) 25 ~ C.

Results and discussion

All methods show good results for precision and accuracy in the range from 1.04 to 17.17% CV. The liquid materials reveal better results for precision and accuracy as for these controls reconstitution is not necessary. As shown in earlier studies [1] the stability of the liquid control material is excellent under different storage conditions.

The main problem at this time is not the control material itself. The problem is the variety of methods using different substrates and also the different affinity of the isoenzymes to these substrates. The differences in affinity are depending on the total activity of the amylase and the ratio of the P- and S- type amylase in the sample and additionally on the different modes of calculation.

To make the results comparable a transformation of the data is helpful. Therefore we used the activity determined in one of our laboratories for the serum 8 as a standard to recalculate the results of the other laboratories. The Phadebas procedure used, also the other methods applied by our laboratory, revealed the factor for the transformation.

In a study recently published by the Scandinavian Group [2] a "standard" with a specified activity was measured in each laboratory and used for the calculation of the results. So in- terlaboratory differences are eliminated.

A summary of our results is listed in Table 1.

The results of method 1 and 2 were eliminated due to the few number of laboratories performing this methods. Th e results returned by a small group of participants showed some major discrepancies due to different calculation procedures or different measuring temperatures in contrast to the specifications given in the questionnaire returned to the organizers of the round

robin. These differences could not be cleared; therefore the data were not removed from this study and so the results show still some scatter after transformation of the data.

Condu~on

Control material based on human serum and only spiked with human salivary and/or pancreatic amylase is recommended for quality control of alpha amylase determination with the recently developed methods.

If a transformation or factor is derived from measurings by only one laboratory some scattering of the results cannot be avoided due to the laboratory specific differences, e.g. tempera- ture, calculation factor considering the reaction products or using only the lineic molar absorbance. All these differences can be easily eliminated by relating to a "standard" with a known activity measured in each laboratory and used for the calculation of the results in relation to the activity of this "standard".

The procedure of calculating enzyme activities with a stan- dard or a calibrator has to be discussed thoroughly and is only recommended for a study like this.

The results achieved with liquid control material show better precision and reproducibility. As known from earlier studies, the stability of the control material made up with ethylenglycol has better stability under different storage conditions and is always ready for use in the analytical instruments as reconstitu- tion is not necessary [3]. This is the main advantage for use in the emergency laboratory and additionally due to the stability, the use of liquid control material is more economic.

References

1. Henkel E (1981) Workshop Quality control in enzymology, Int Congress Clin Chemistry, Vienna

2. Gerhardt W, Waldenstrom J, H6rder M, Magid E, Stromme JH, Theodorsen L, H/irk6nen M, Ic6n A (1985) Scand J Clin Lab Invest 45:397 -404

3. Henkel E, Henkel R, Louderback A (/983) Experiences with different control sera monitoring the determination of c~- amylase. 2nd African mediterranean and near-east congress for clinical chemistry. Nov. 20.-24. 1983 Cairo Egypt

Fresenius Z Anai Chem (1986) 324: 305-306 �9 Springer-Verlag 1986

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