NATL INST OF STAND & TECH NBS

PUBLICATIONS A111D7 257SD2

NBS SPECIAL PUBLICATION 260-83

U.S. DEPARTMENT OF COMMERCE/National Bureau of Standards

QC

100

U57

260-33

1983

C.2

Standard Reference Materials.

The Measurement of the

Catalytic (Activity)

Concentration of Seven

Enzymes in NBS Human

Serum SRM 909
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards' was established by an act of Congress on March 3, 1901. The Bureau's overall goal is to strengthen and advance the Nation's science and technology and facilitate their effective application for public benefit. To this end, the Bureau conducts

research and provides: (1) a basis for the Nation's physical measurement system, (2) scientific and technological services for industry and government, (3) a technical basis for equity in trade, and (4) technical services to promote public safety. The Bureau's technical work is per- formed by the National Measurement Laboratory, the National Engineering Laboratory, and

the Institute for Computer Sciences and Technology. THE NATIONAL MEASUREMENT LABORATORY provides the national system ot physical and chemical and materials measurement; coordinates the system with measurement

systems of other nations and furnishes essential services leading to accurate and uniform

physical and chemical measurement throughout the Nation's scientific community, industry, and commerce; conducts materials research leading to improved methods of measurement,

standards, and data on the properties of materials needed by industry, commerce, educational

institutions, and Government; provides advisory and research services to other Government

agencies; develops, produces, and distributes Standard Reference Materials; and provides

calibration services. The Laboratory consists of the following centers: Absolute Physical Quantities2 — Radiation Research — Chemical Physics —

Analytical Chemistry — Materials Science THE NATIONAL ENGINEERING LABORATORY provides technology and technical ser- vices to the public and private sectors to address national needs and to solve national problems; conducts research in engineering and applied science in support of these efforts; builds and maintains competence in the necessary disciplines required to carry out this research and technical service; develops engineering data and measurement capabilities; provides engineering measurement traceability services; develops test methods and proposes

engineering standards and code changes; develops and proposes new engineering practices; and develops and improves mechanisms to transfer results of its research to the ultimate user. The Laboratory consists of the following centers:

Applied Mathematics — Electronics and Electrical Engineering2 — Manufacturing

Engineering — Building Technology — Fire Research — Chemical Engineering2 THE INSTITUTE FOR COMPUTER SCIENCES AND TECHNOLOGY conducts

research and provides scientific and technical services to aid Federal agencies in the selection, acquisition, application, and use of computer technology to improve effectiveness and economy in Government operations in accordance with Public Law 89-306 (40 U.S.C. 759), relevant Executive Orders, and other directives; carries out this mission by managing the Federal Information Processing Standards Program, developing Federal ADP standards

guidelines, and managing Federal participation in ADP voluntary standardization activities; provides scientific and technological advisory services and assistance to Federal agencies; and

provides the technical foundation for computer-related policies of the Federal Government. The Institute consists of the following centers: Programming Science and Technology — Computer Systems Engineering.

'Headquarters and Laboratories at Gaithersburg, MD, unless otherwise noted; mailing address Washington, DC 20234. 'Some divisions within the center are located at Boulder, CO 80303.
Standard Reference Materials: AUG 1 1983

1 The Measurement of the Catalytic (Activity)

Concentration of Seven Enzymes

in NBS Human Serum SRM 909

fipioaji omJl

George N. Bowers, Jr.

Hartford Hospital

Hartford, CT 06115 Robert Alvarez, J. Paul Cali* Keith R. Eberhardt, Dennis J. Reeder,

Robert Schaffer, George A. Uriano

National Bureau of Standards Washington, DC 20234

Robert Elser, 1 Lilian M. Ewen,^

Robert B. McComb,3 Robert Rej, 4

and Leslie M. Shaw,5

1 York Hospital, York, PA 1 7405

2Royal Columbian Hospital, British Columbia, Canada V3L 3W7

3Hartford Hospital, Hartford, CT 061 15

4New York State Department of Health, Albany, NY 12201

5Hospital of the University of Pennsylvania, Philadelphia, PA 19104

* Present Address: 16405 Kipling Road, Rockville, MD 20855

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director Issued June 1 983
Library of Congress Catalog Card Number: 83-600543

National Bureau of Standards Special Publication 260-83

Natl. Bur. Stand. (U.S.) Spec. Publ. 260-83, 108 pages (June 1983)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE WASHINGTON: 1983

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402

Price $4.50

(Add 25 percent for other than U.S. mailing)
PREFACE

Standard Reference Materials (SRM's) as defined by the

National Bureau of Standards are we 1

1 -c h ar ac ter i zed materials,

produced in quantity and certified for one or more physical or chemical properties. They are used to ensure the accuracy and compatibility of measurements throughout the Nation. SRM's are widely used as primary measurement standards in many diverse

fields in science, industry, and technology, both within the

United States and throughout the world. They are also used

extensively in the fields of environmental and clinical analysis.

In many applications, traceability of quality control and measurement processes to the national measurement system is carried out through the mechanism and use of SRM's. For many of the Nation's scientists and technologists it is therefore of more

than passing interest to know the details of the measurements

made at NBS in arriving at the certified values or supplemental

data for the SRM's produced. An NBS series of monographs, of which this publication is a member, called the NBS Special

Publication - 260 Series , is reserved for this purpose.

This 260 Series is dedicated to the dissemination of information on different phases of the preparation, measurement,

certification and use of NBS-SRM's. In general, much more detail

will be found in these monographs than is generally allowed, or desirable, in scientific journal articles or on the SRM certificate. This enables the user to assess the validity and accuracy of the measurement processes employed, to judge the

statistical analysis, and to learn details of techniques and

method-s utilized for work entailing the greatest care and

accuracy. These monographs also should provide sufficient

additional information not found on the SRM certificate so that

new applications in diverse fields not foreseen at the time the

SRM was originally issued will be sought and found.

The scope of the NBS Clinical SRM program has recently been

extended to include the development of complex "matrix"

type reference materials such as the ant i -convu 1 sant drugs in serum (SRM 900). More recently with SRM 909, a lyophilized human

serum, the concentrations of a number of c 1 i n i c a 1

1 y- i mpor t ant analytes were certified by definitive methods. Analytes include

total calcium, chloride, cholesterol, glucose, lithium,

magnesium, potassium, and uric acid. (See Appendix 1, The

Certificate of Analysis for SRM 909.) The catalytic (activity)

concentrations of seven enzymes also have now been determined by

the reference method type protocols included in this publication.

NBS policy generally precludes certifying a property of any

SRM unless one of its laboratories and NBS staff have actively

and fully participated in the analytical work. For determination

of the enzyme activities in SRM 909, NBS only coordinated the

efforts of a voluntary group of 28 cooperating scientists from 18 laboratories. The list of cooperating scientists and laboratories, the team leaders, and the names of other participating

personnel and their institutional affiliation is given in the

Introduction.

i i i
Prior to the start of the experimental work for this study,

several general features of its organization and design were discussed and agreed upon. Among these were:

1) The scientific aspects of the work would be under the

general direction of G. N. Bowers, Jr. and a team of enzyme

measurement experts working with him.

2) Team leaders would be recognized for the specific enzyme

methods assigned to them.

3) As much as possible, the reference method type protocols to

be used would follow the recommendations of the Expert Panel

on Enzymes of the International Federation of Clinical

Chemistry (EPE/IFCC). Team leaders would write measurement

protocols for each of the enzymes after discussion and

agreement with G. N. Bowers, Jr.

4) Because the control of temperature is critical in enzyme

analysis, it was agreed that a uniform reaction temperature

would be used by all laboratories. The set-point would be defined by the melting point of gallium, 29.77 °C, and the

temperature would be controlled within narrow limits.

Accordingly, each team was supplied with an NBS Gallium

Melting Point standard (SRM 1968).

5) The design of the testing schedules and statistical analysis

of the resultant data would be performed by NBS under the

direction of K. R. Eberhardt. Unless the preliminary data

were to prove unsatisfactory, three rounds were to constitute the work program for evaluation of each enzyme

method. Rounds 1 and 2 were to be considered preliminary - in the nature of a "training" exercise - and only Round 3 data would be used for reporting final results. The number

of participating laboratories for the specific enzyme

analysis was to range from a minimum of 3 to not more than 7.

6) Citation of the enzyme data as it would appear on the SRM

909 Certificate would be solely an NBS responsibility.

However, a full report of the interlaboratory tests would be made, through an NBS publication - this publication being

the resu 1 1

.

7) The organization of schedules, sample distribution, data

collection, and similar duties were initially the

responsibility of J. P. Cali. After July 1981, R. Alvarez

assumed this responsibility.

The catalytic (activity) concentration values given in this

publication are consensus values, obtained from data submitted by

the cooperating laboratories. They are provided for information

only ; they are not NBS certified values . Full recognition should be given to G. N. Bowers, Jr. for

coordinating the scientific activities, to J. P. Cali for

planning and organizational contributions, and to R. Schaffer for

special editorial contributions. Special recognition is also

i v
given to the 28 scientists who dedicated their time and talents

to providing the analytical data that resulted in a successful

project.

Inquiries concerning the technical content of this document

should be directed to the authors. Other questions concerned

with the availability, delivery, price, etc. of SRM 909, will

receive prompt attention from:

Office of Standard Reference Materials

National Bureau of Standards

Washington, DC 20234

George A. Uriano

Chief

Office of Standard Reference Materials

National Measurement Laboratory

National Bureau of Standards

v
OTHER NBS PUBLICATIONS IN THIS SERIES

Catalog of NBS Standard Reference Materials (1981-83 edition), R. W. Seward, ed., NBS

Spec. Publ. 260 (November 19X1). Michaelis, R. E., and Wyman, L. L. Standard

Reference Materials: Preparation of White Cast

Iron Spectrochemical Standards. NBS Misc. Publ. 260-1 (June 1964). CO M 74- 1 1 06 I * * Michaelis, R. E., Wyman, L. L., and Elitsch, R., Standard Reference Materials: Preparation of NBS Copper-Base Spectrochemical Standards. NBS Misc. Publ. 260-2 (October 1964). COM74-

1 1063**

Michaelis, R. E., Yakowitz, H., and Moore, G. A., Standard Reference Materials: Metallographic

Characterization of an NBS Spectrometric LowAlloy Steel Standard. NBS Misc. Publ. 260-3 (October 1964). COM74-I 1060** Hague, J. L. Mears, T. W., and Michaelis, R. E., Standard Reference Materials: Sources of

Information, NBS Misc. Publ. 260-4 (February

1965). COM74-11059

Alvarez, R., and Elitsch R., Standard Reference

Materials: Accuracy of Solution X-Ray Spectrometric Analysis of Copper-Base Alloys. NBS

Misc. Publ. 260-5 (March 1965). PB168068**

Shultz, J. I., Standard Reference Materials: Methods for the Chemical Analysis of White

Cast Iron Standards, NBS Misc. Publ. 260-6

(July 1975). COM74-1 1068**

Bell, R. K., Standard Reference Materials: Methods for the Chemical Analysis of NBS

Copper-Base Spectrochemical Standards. NBS

Misc. Publ. 260-7 (October 1965). COM74-

1 1067** Richmond, M.S., Standard Reference Materials: Analysis of Uranium Concentrates at the

National Bureau of Standards. NBS Misc. Publ. 260-8 (December 1965). COM74-I 1066** Anspach, S. C, Cavallo, L. M. Garfinkel, S. B. Hutchinson, J. M. R., and Smith. C. N., Standard Reference Materials: Half Lives of Materials Used in the Preparation of Standard Reference Materials of Nineteen Radioactive Nuclides

Issued by the National Bureau of Standards NBS Misc. Publ. 260-9 (November 1965). COM74-I 1065**

Yakowitz, H„ Vieth, D. L., Heinrich, K. F. J., and

Michaelis, R. E., Standard Reference Materials: Homogeneity Characterization on NBS Spectrometric Standards II: Cartridge Brass and LowAlloy Steel, NBS Misc. Publ. 260-10 (December

1965). COM 74- 1 1064** Napolitano, A., and Hawkins, E. G., Standard Reference Materials: Viscosity of Standard

Lead-Silica Glass, NBS Misc. Publ. 260-11 (November 1966). NBS Misc. Publ. 260-11** Yakowitz, H., Vieth, D. L., and Michaelis, R. E., Standard Reference Materials: Homogeneity

Characterization of NBS Spectrometric Standards III: White Cast Iron and Stainless Steel Powder Compact, NBS Misc. Publ. 260-12

(September 1966). NBS Misc. Publ. 260-12** Spijkerman, J. L., Snediker, D. K., Ruegg, F. C,

and DeVoe, J. R., Standard Reference Materials: Mossbauer Spectroscopy Standard for the Chemical Shift of Iron Compounds, NBS Misc. Publ. 260-13 (July 1967). NBS Misc. Publ.

260-13**

Menis, O., and Sterling, J. T., Standard Reference

Materials: Determination of Oxygen in Ferrous

Materials - SRM 1090, 1091, and 1092, NBS

Misc. Publ. 260-14 (September 1966). NBS Misc.

Publ. 260-14**

Passaglia, E., and Shouse, P. J. Standard Refer- ence Materials: Recommended Method of Use

of Standard Light-Sensitive Paper for Calibrating Carbon Arcs Used in Testing Textiles for Colorfastness to Light, NBS Misc. Publ. 260-15 (June 1967). (Replaced by NBS Spec. Publ.

260-41.) Yakowitz, H., Michaelis, R. E., and Vieth, D. L., Standard Reference Materials: Homogeneity

Characterization of NBS Spectrometric Standards IV: Preparation and Microprobe Characterization of W-20% MO Alloy Fabricated by Powder Metallurgical Methods, NBS Spec.

Publ. 260-16 (January 1969). COM74-1 1062** Catanzaro, E. J., Champion, C. E., Garner, E. L., Marinenko, G., Sappenfield, K. M., and Shields, W. R. Standard Reference Materials: Boric

Acid; Isotopic and Assay Standard Reference

Materials, NBS Spec. Publ. 260-17 (February

1970). Out of Print

v i
Geller, S. B., Mantek, P. A., and Cleveland, N.

G., Standard Reference Materials: Calibration of NBS Secondary Standard Magnetic Tape (Computer Amplitude Reference) Using the Reference Tape Amplitude Measurement

"Process A, "NBS Spec. Publ. 260-18 (November

1969). (See NBS Spec. Publ. 260-29.) Paule, R. C, and Mandel, J., Standard Reference

Materials: Analysis of Interlaboratory Measurements on the Vapor Pressure of Gold (Certifica- tion of Standard Reference Material 745). NBS

Spec. Publ. 260-19 (January 1970). PB190071**

Paule, R. C, and Mandel, J., Standard Reference

Materials: Analysis of Interlaboratory Measurements on the Vapor Pressures of Cadmium and

Silver, NBS Spec. Publ. 260-21 (January 1971). COM74-1 1359** Yakowitz, H., Fiori, C. E., and Michaelis, R. E., Standard Reference Materials: Homogeneity

Characterization of Fe-3 Si Alloy, NBS Spec.

Publ. 260-22 (February 1971). COM74-I 1357** Napolitano, A., and Hawkins, E. G., Standard

Reference Materials: Viscosity of a Standard

Borosilicate Glass, NBS Spec. Publ. 260-23 (December 1970). COM7 1-00 1 57**

Sappenfield, K. M., Marineko, G., and Hague, J. L., Standard Reference Materials: Comparison

of Redox Standards, NBS Spec. Publ. 260-24 (January 1972). COM72-50058**

Hicho, G. E., Yakowitz, H., Rasberry, S. D., and

Michaelis, R. E., Standard Reference Materials: A Standard Reference Material Containing

Nominally Four Percent Austenite, NBS Spec.

Publ. 260-25 (February 1971). COM74-1 1356** Martin, J. F., Standard Reference Materials:

National Bureau of Standards-US Steel Corportion Joint Program for Determining Oxygen and Nitrogen in Steel, NBS Spec. Publ. 260-26

(February 1971). 85 cents* PB 81 176620

Garner, E. L., Machlan, L. A., and Shields, W. R., Standard Reference Materials: Uranium

Isotopic Standard Reference Materials, NBS

Spec. Publ. 260-27 (April 1971). COM74-

I 1358**

Heinrich, K. F. J., Myklebust, R. L., Rasberry, S. D., and Michaelis, R. E., Standard Reference Materials: Preparation and Evaluation of SRM's 481 and 482 Gold-Silver and GoldCopper Alloys for Microanalysis, NBS Spec.

Publ. 260-28 (August 1971). COM7 1-50365**

Geller, S. B., Standard Reference Materials: Cali- bration of NBS Secondary Standard Magnetic Tape (Computer Amplitude Reference) Using

the Reference Tape Amplitude Measurement

"Process A-Model 2," NBS Spec. Publ. 260-29 (June 1971). COM71-50282

Gorozhanina, R. S., Freedman, A. Y., and

Shaievitch, A. B. (translated by M. C. Selby), Standard Reference Materials: Standard

Samples Issued in the USSR (A Translation from the Russian). NBS Spec. Publ. 260-30 (June

1971). COM71-50283**

Hust, J. G., and Sparks, L. L., Standard Reference

Materials: Thermal Conductivity of Electrolytic Iron SRM 734 from 4 to 300 K, NBS Spec. Publ. 260-31 (November 1971). COM71-50563**

Mavrodineanu, R., and Lazar, J. W., Standard

Reference Materials: Standard Quartz Cuvettes,

for High Accuracy Spectrophotometry, NBS

Spec. Publ. 260-32 (December 1973). 55 cents* SN003-003-OI213-1 Wagner, H. L., Standard Reference Materials: Comparison of Original and Supplemental

SRM 705, Narrow Molecular Weight Distri- bution Polystyrene, NBS Spec. Publ. 260-33 (May 1972). COM72-50526**

Sparks, L. L., and Hust, J. G., Standard Reference

Materials: Thermoelectric Voltage, NBS Spec.

Publ. 260-34, (April 1972). COM72-5037 1 ** Sparks, L. L., and Hust, J. G., Standard Refer- ence Materials: Thermal Conductivity of

Austenitic Stainless Steel, SRM 735 from 5 to 280 K, NBS Spec. Publ. 260-35 (April 1972.) 35 cents* COM72-50368**

Cali, J. P., Mandel, J., Moore, L. J., and Young, D.

S., Standard Reference Materials: A Referee Method for the Determination of Calcium in Serum, NBS SRM 915, NBS Spec. Publ. 260-36 (May 1972). CO M 72-50527**

Shultz, J. I. Bell., R. K. Rains, T. C, and Menis,

O., Standard Reference Materials: Methods of Analysis of NBS Clay Standards, NBS Spec.

Publ. 260-37 (June 1972). COM72-50692**

Richmond, J. C, and Hsia, J. J., Standard Refer- ence Materials: Preparation and Calibration of Standards of Spectral Specular Reflectance, NBS Spec. Publ. 260-38 (May 1972). COM72-

50528**

Clark, A. F., Denson, V.A., Hust, J. G., and

Powell, R. L., Standard Reference Materials The Eddy Current Decay Method for Resistivity Characterization of High-Purity Metals, NBS

Spec. Publ. 260-39 (May 1972). COM72-50529**

vi i
McAdie, H. C, Gain, P.D., and Menis, O., Standard Reference Materials: Selection of Thermal

Analysis Temperature Standards Through a Cooperative Study (SRM 758, 759, 760), NBS

Spec. Publ. 260-40 (August 1972.) COM72-

50776** Wood, L. A., and Shouse, P. J., Standard

Reference Materials: Use of Standard LightSensitive Paper for Calibrating Carbon Arcs Used in Testing Textiles for Colorfastness to Light, NBS Spec. Publ. 260-41 (August 1972) COM72-50775**

Wagner, H. L. and Verdier, P. H., eds.. Standard

Reference Materials: The Characterization of Linear Polyethylene, SRM 1475, NBS Spec. Publ. 260-42 (September 1972). COM72-

50944**

Yakowitz, H., Ruff, A. W., and Michaelis, R. E., Standard Reference Materials: Preparation and Homogeneity Characterization of an Austenitic Iron-Chromium-Nickel Alloy, NBS Spec. Publ. 260-43 (November 1972). COM73-50760**

Schooley, J. F., Soulen, R. J., Jr., and Evans, G. A.,

Jr., Standard Reference Materials: Preparation and Use of Superconductive Fixed Point

Devices, SRM 767, NBS Spec. Publ. 260-44 (December 1972). COM73-50037**

Greifer, B., Maienthal, E. J. Rains, T. C, and

Rasberry, S. D., Standard Reference Materials: Powdered Lead-Based Paint, SRM 1579, NBS

Spec. Publ. 260-45 (March 1973). COM73-

50226**

Hust, J. G., and Giarratano, P. J., Standard Refer- ence Materials: Thermal Conductivity and

Electrical Resistivity Standard Reference Materials: Austenitic Stainless Steel, SRM's 735 and

798, from 4 to 1200 k, NBS Spec. Publ. 260-46 (March 1975). SN003-003-0 1 278-5

Hust, J. G., Standard Reference Materials: Electrical Resistivity of Electrolytic Iron, SRM 797, and Austenitic Stainless Steel, SRM 798, from 5 to 280 K, NBS Spec. Publ. 260-47 (February

1974). COM74-50I76** Mangum, B. W., and Wise, J. A., Standard Refer- ence Materials: Description and Use of Precision Thermometers for the Clinical Laboratory,

SRM 933 and SRM 934, NBS Spec. Publ. 260-48 (May 1974). 60 cents* SN003-003-0 1 278-5 Carpenter, B. S., and Reimer, G. M., Standard Reference Materials Calibrated Glass Standards lor Fission Track Use, NBS Spec. Publ. 260-49 (November 1974). COM74-5U85

Hust, J. G., and Giarratano, P. J., Standard Refer- ence Materials: Thermal Conductivity and Electrical Resistivity Standard Reference Materials:

Electrolytic Iron, SRM's 734 and 797 from 4 to 1000 K, NBS Spec. Publ. 260-50 (June 1975). $1.00* SN003-003-0I425-7 Mavrodineanu, R., and Baldwin, J. R., Standard

Reference Materials: Glass Filters As a Standard Reference Material for Spectrophotometry;

Selection; Preparation; Certification; Use-SRM

930, NBS Spec. Publ. 260-51 (November 1975). $1.90* SN003-003-0148I-8

Hust, J. G., and Giarratano, P. J., Standard Refer- ence Materials: Thermal Conductivity and Electrical Resistivity Standard Reference Materials 730 and 799, from 4 to 3000 K, NBS Spec. Publ. 260-52 (September 1975). $1.05* SN003-003-

01464-8 Durst, R. A., Standard Reference Materials: Standardization of pH Measurements, NBS

Spec. Publ. 260-53 (December 1975, Revised). $1.05 SN003-003-01551-2

Burke, R. W., and Mavrodineanu, R. Standard

Reference Materials: Certification and Use of Acidic Potassium Dichromate Solutions as an

Ultraviolet Absorbance Standard, NBS Spec.

Publ. 260-54 (August 1977). $3.00* SN003-003-

01828-7

Ditmars, D. A., Cezairliyan, A., Ishihara, S., and

Douglas, T. B., Standard Reference Materials: Enthalpy and Heat Capacity; Molybdenum

SRM 781, from 273 to 2800 K, NBS Spec. Publ. 260-55 (September 1977). $2.20* SN003-003-

01836-8

Powell, R. L., Sparks, L. L., and Hust, J. G., Standard Reference Materials: Standard

Thermocouple Materials, Pt.67: SRM 1967, NBS

Spec. Publ. 260-56 (February 1978). $2.20* SN003-003-0 18864

Cali, J. P. and Plebanski, T., Guide to United

States Reference Materials, NBS Spec. Publ. 260-57 (February 1978). $2.20* PB 277173

Barnes, J. D., and Martin, G. M., Standard Refer- ence Materials: Polyester Film for Oxygen Gas

Transmission Measurements SRM 1470, NBS

Spec. Publ. 260-58 (June 1979) $2.00* SN003-

003-02077 Chang, T., and Kahn, A. H. Standard Reference

Materials: Electron Paramagnetic Resonance

Intensity Standard, SRM 2601, NBS Spec.

Publ. 260-59 (August 1978) $2.30* SN003-

003-01975-5

v i i i
Velapoldi, R. A., Paule, R. C, Schaffer, R., Mandel, J., and Moody, J. R., Standard Refer- ence Materials: A Reference Method for the Determination of Sodium in Serum, NBS Spec.

Publ. 260-60 (August 1978). S3.00* SN003-003

01978-0

Verdier, P. H., and Wagner. H. L., Standard Refer- ence Materials: The Characterization of Linear Polyethylene (SRM 1482, 1483, 1484), NBS

Spec. Publ. 260-61 (December 1978). S 1 .70* SN003-003-02006-I

Soulen, R. J., and Dove, R. B., Standard Reference

Materials: Temperature Reference Standard

for Use Below 0.5 K (SRM 768). NBS Spec.

Publ. 260-62 (April 1979). $2.30* SN003-003-

02047-8 Velapoldi, R. A., Paule, R. C, Schaffer, R. Mandel, J., Machlan, L. A., and Gramlich, J. W., Standard Reference Materials: A Reference Method for the Determination of Potassium in Serum. NBS Spec. Publ. 260-63 (May 1979). $3.75* SN003-003-02068

Velapoldi, R. A., and Mielenz, K. D., Standard

Reference Materials: A Fluorescence Standard

Reference Material Quinine Sulfate Dihydrate (SRM 936), NBS Spec. Publ. 260-64 (January

1980). $4.25* SN003-003-02 148-2 Marinenko, R. B., Heinrich, K. F. J., and Ruegg,

F. C, Standard Reference Materials: Micro- Homogeneity Studies of NBS Standard Reference

Materials, NBS Research Materials, and Other Related Samples. NBS Spec. Publ. 260-65

(September 1979). $3.50* SN003-003-02 1 14-1 Venable, W. H., Jr., and Eckerle, K. L., Standard

Reference Materials: Didymium Glass Filters for Calibrating the Wavelength Scale of Spectro- photometers (SRM 2009, 2010, 2013). NBS Spec.

Publ. 260-66 (October 1979). $3.50* SN003-003-

02127-0

Velapoldi, R. A., Paule, R. C, Schaffer, R., Mandel,

J., Murphy, T. J., and Gramlich, J. W., Standard

Reference Materials: A Reference Method for the Determination of Chloride in Serum, NBS Spec.

Publ. 260-67 (November 1979). $3.75* SN003-

003-02136-9 Mavrodineanu, R. and Baldwin, J. R., Standard

Reference Materials: Metal-On-Quartz Filters as a Standard Reference Material for Spectrophoto- metry-SRM 2031, NBS Spec. Publ. 260-68

(April 1980). $4.25* SN0O3-003-02 1 67-9

Velapoldi, R. A., Paule, R. C, Schaffer, R., Mandel, J., Machlan, L. A., Garner, E. L., and

Rains, T. C, Standard Reference Materials: A

Reference Method for the Determination of Lithium in Serum, NBS Spec. Publ. 260-69 (July)

1980). $4.25* SN003-003-02214-4 Marinenko, R. B., Biancaniello, F., Boyer, P. A., Ruff, A. W., DeRobertis, L., Standard Reference

Materials: Preparation and Characterization of an Iron-Chromium-Nickel Alloy for Microanalysis, NBS Spec. Publ. 260-70 (May 1981). $2.50* SN003-003-02328-1 Seward, R. W., and Mavrodineanu, R., Standard

Reference Materials: Summary of the Clinical Laboratory Standards Issued by the National Bureau of Standards, NBS Spec. Publ. 260-71 (November 1981). $6.50* SN003-003-0238 1-7 Reeder, D.J., Coxon, B., Enagonio, D., Christensen,

R. G., Schaffer, R., Howell, B. F., Paule, R. C,

Mandel, J., Standard Reference Materials: SRM

900, Antiepilepsy Drug Level Assay Standard, NBS Spec. Publ. 260-72 (June 1981). $4.25* SN003-003-02329-9

Interrante, C. G., and Hicho, G. E., Standard Refer- ence Materials: A Standard Reference Material Containing Nominally Fifteen Percent Austenite (SRM 486), NBS Spec. Publ. 260-73 (January

1982). $2.75* SN003-003-02386-8 Marinenko, R. B., Standard Reference Materials: Preparation and Characterization of K.-411 and

K-414 Mineral Glasses for Microanalysis: SRM

470. NBS Spec. Publ. 260-74 (April 1982). $3.50 SN003-003-023-95-7

Weidner, V. R., Hsia, J. J., Standard Reference

Materials: Preparation and Calibration of First Surface Aluminum Mirror Specular Reflectance Standards (SRM 2003a), NBS Spec. Publ. 260-75 (May 1982). $3.75 SN003-003-023-99-0

Hicho, G. E. and Eaton, E. E., Standard Reference Materials: A Standard Reference Material

Containing Nominally Five Percent Austenite (SRM 485a), NBS Spec. Publ. 260-76 (August

1982). $3.50 SN003-003-024-33-3 Furukawa, G. T., Riddle, J. L., Bigge, W. G., and

Pfieffer, E. R., Standard Reference Materials: Application of Some Metal SRM's as Jhermometric Fixed Points. NBS Spec. Publ. 260-77 (August 1982). $6.00 SN003-003-024-34-

1

i x
Hicho, Ci. E. and Eaton, E. E., Standard Reference

Materials: Standard Reference Material Containing Nominally Thirty Percent Austenite (SRM

4X7), NBS Spec. Publ. 260-78 (September 19X2). $3.75 SN003-003-024-35-0 Richmond, J. C, Hsia, J. J., Weidner, V. R., and

Wilmering, D. B., Standard Reference Materials: Second Surface Mirror Standards of Specular

Spectral Reflectance (SRM's 2023, 2024, 2025), NBS Spec. Publ. 260-79 (October 1982). $4.50 SN003-0O3-024-47-3

Schaffer, R., Mandel, J., Sun, T., Cohen, A., and

Hertz, H. S., Standard Reference Materials: Evaluation by an ID/ MS Method of the AACC

Reference Method for Serum Glucose, NBS

Spec. Publ. 260-80 (October 1982). $4.75 SN003-

003-024-43-1 Burke, R. W., Mavrodineanu, R. (NBS retired). Standard Reference Materials: Accuracy in Analytical Spectrophotometry, NBS Spec. Publ. 260-81 (In Press). Weidner, V. R., Standard Reference Materials: White Opal Glass Diffuse Spectral Reflectance Standards for the Visible Spectrum (SRM's 2015 and 2016). NBS Spec. Publ. 260-82 (In Press). Bowers,, G. N., Jr., Alvarez, R., Cali, J. P. (NBS

retired), Eberhardt, K. R., Reeder, D. J., Schaffer, R., Uriano, G. A., Standard Reference

Materials: The Measurement of the Catalytic

(Activity) Concentration of Seven Enzymes in NBS Human Serum SRM 909, NBS Spec.

Publ. 260-83 (In Press).

* Send order with remittance to Superintendent

ol Documents, US Government Printing Office Washington, DC 20402. Remittance from

foreign countries should include an additional one-fourth of the purchase price for postage.

** May be ordered from: National Technical Information Services (NT1S). Springfield

Virginia 22151.

X
TABLE OF CONTENTS

Page

PREFACE iii ABSTRACT fttffc&ad . 1 DISCLAIMER.

.

1 I. INTRODUCTION 2 A. Enzyme Project Backgound 4 B. General Criteria for Participants

andOperations 7 II. GENERAL COMMENTS ON ENZYME ACTIVITY

MEASUREMENTS 8 III. PROTOCOLS FOR ENZYME MEASUREMENTS 12

A. General Comments and Observations

on Enzyme Reference Methods Chosen .... 12

B. Reconstitution Protocol 14

IV. SPECIFIC ENZYME PROTOCOLS 16

A. Prostatic Acid Phosphate 16

B. Alkaline Phosphatase 24

C. Alanine Aminotransferase 34

D. Aspartate Aminotransferase 41

E. CreatineKinase 46

F. Lactate Dehydrogenase 54

G. Gamma -Gl ut amy 1 transfer ase 60

V. STATISTICAL ANALYSIS OF CATALYTIC CONCENTRATION

DATA 69 VI. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 78 VII. REFERENCES 81 APPENDIX - National Bureau of Standards

Certificate - SRM 909 87

xi
LIST OF FIGURES

Figure No . Page

1. ALP activity changes after reconstitution

at temperatures in the range 4-37 °C 33

2. Effect of time on CK activity after incubation

and reconstitution in the range 2-10 °C 52

3. Effect of time on CK activity after incubation

and reconstitution in the range 20-30 °C . . . . 52

4. Summary of ACP results for low activity (SRM 909)

material and for enriched material 71

5. Summary of AlaAT results, with and without

blank correcti on 72

6. Summary of results for ALP and AspAT . 73

7. Summary of results for CK and LDH 74

8. Summaryof results for y-GT 75

9. Summaryof all results 75

LIST OF TABLES

Table No . Page

1. Number of enzyme activity measurements, 1980-1981

Hartford Hospital 9 2. Catalytic (activity) concentrations in SRM 909 . . 11

3. Protocol for determination of y-Gl activity by

continuous monitoring 68

4. Summary of precision data, Round-Robin III . . . . 70

xi i
ABSTRACT

We determined the catalytic (activity) concentrations of seven enzymes (ACP, ALP, A 1 a AT , AspAT, CK, LDH and y-GT) in the

NBS lyophilized human serum Standard Reference Material (SRM 909).

SRM 909 enzyme activity values should provide the basis for

compatibility among methods for determining enzyme values in serum. Separate teams of clinical chemistry experts selected and carried out the analyses for the individual enzymes. The methods

used are primarily those recommended by the Standards Committees

of the AACC and/or the IFCC, as candidate reference methods;

however, a reaction temperature set-point of 29.77 °C (the

gallium melting point) was used. This monograph describes the serum reconst i t ut i on protocol, the specific enzyme methods, and the results of the third (and last) round of the interlaboratory test program, which we used to derive mean values for enzyme

catalytic (activity) concentrations in SRM 909 . Inter 1 aboratory

standard deviations of less than 10% were achieved. Measurements

made one year after the conclusion of the i nter 1 aboratory study

showed unchanged enzyme activities, within the uncertainties of the original assigned values.

Key words: enzyme; enzyme (catalytic) activity; human serum;

inter! aboratory reproducibility; reference method; Standard

Reference Mater i a 1

DISCLAIMER

In order to describe materials and experimental

procedures adequately, it was occasionally necessary to identify commercial products by the manufacturer's name

or label. In no instances does such identification imply endorsement by the National Bureau of Standards nor does it imply that the particular

products or equipment are necessarily the best

available for that purpose.

1
I. INTRODUCTION

Measurement processes have been studied extensively from

both an experimental and a statistical point of view.

Statistical quality control of industrial goods originated with

the work of Shewhart [1] who introduced the concept of control

charts over 50 years ago. In 1946, Wernimont [2] applied the use

of control charts to chemical analysis. The tremendous

post-World War II expansion of industrial and space-related

technology led to an unprecedented demand for better measurement

technology. In the period 1945 to 1965 many staff members of the

NBS devoted considerable effort to the systematic evaluation of the critical statistical parameters that describe measurement

processes, including precision, systematic error, and accuracy.

Also developed were a number of important statistical concepts

used in experimental design, interlaboratory testing, the

description of functional relationships, and the evaluation of experimental data. NBS Special Publication 300 [3] contains a collection of over 30 papers covering these topics along with an excellent set of general references on measurement science and

statistics. Particularly relevant to the general field of chemical analysis are the following papers contained in that

reference manu al :

1. C. Eisenhart's two papers on precision and accuracy of calibration systems, and the expression of the uncertainties of final results (see in particular [4])

2 . A series of papers by W. J. Youden on uncertainties in calibration, instrumental drift, experimental design, and

interlaboratory testing

3. M. 6. Natrella's paper on planning of experiments

4. The paper by J. Mandel and T. W. Lashof on interlaboratory evaluation of test methods

5. The paper on accuracy in chemical analysis using linear

calibration curves by J. Mandel and F. J. Linnig.

2
Our purpose here is not to present a detailed discussion

involving the statistical analysis of experimental data, but

rather to point out the key measurement concepts necessary to establish the validity of analytical data and, in particular, to establish measurement compatibility on a national scale. An important goal of the work cited above is to develop measurement

systems that will allow meaningful communication among different

measurement stations over long periods of time and large

geographical distances. This leads us to the concept of measurement compatibility. If measurements are to be useful in fostering meaningful economic or scientific communication between

two or more parties, the different parties must agree on the

results of the measurement and the meaning of the numbers

associated with the measurement [5-9]. This agreement should

take into consideration any imprecision and/or inaccuracies in the measurement process under consideration. Huntoon [10] in an extension of his earlier work [11] defining the National

Measurement System concept, has also cited the importance of measurement compatibility and described the general role of reference materials in achieving compatibility.

If a measurement process is to be meaningful, then the data

obtained should ideally be both precise and free of systematic

error within agreed upon end-use requirements. An accurate

measurement is both precise and free of systematic error [7].

Accurate measurement systems are necessarily compatible.

Additional requirements for measurement processes, such as wide

dynamic range, high sensitivity, or rapid operation, might also

be desirable. However, such requirements are not necessary in achieving accuracy but rather represent practical considerations.

If a measurement system is accurate, the numerical value obtained

for the properties being measured are "true values" within the

uncertainties of the measurement processes being used.

The true value determined by measurement processes is referred to as the "quaes i turn" by Dorsey and Eisenhart and extensively discussed in their paper on absolute measurements

[12]. A pragmatic operational definition of the "true value" of

3
a property is the value determined by precise measurement methods

that are free of systematic error, i.e., an accurate method.

Such values should ultimately be experimentally traceable to the

base units of measurement such as mass, length, and time.

Unfortunately, the state-of-the-art in enzyme activity

measurements is such that concepts such as accuracy and "true value" are not well-defined. Thus, an alternative must be sought

to bring about measurement compatibility. One approach is to set

up a measurement system based solely on interlaboratory precision

whereby all laboratories achieve compatibility through the use of defined, reproducible measurement methods. We used that approach

with mutually acceptable, well tested, stable methods of measurement, to provide "method -dependent " enzyme activity values

for a homogeneous, stable reference material.

The argument for demonstrating measurement compatibility is straightforward. If two laboratories obtain the same analytical

results for SRM 909, as stated herein (also given in the SRM 909

Informational Insert) for a specific enzyme when using the

reference method recommended, then with a fairly high degree of assurance, both can apply the recommended method to the same unknown with confidence that they will obtain the same result.

When this occurs, then measurement compatibility is achieved.

The need to demonstrate interlaboratory compatibility with enzyme

reference method measurement systems has been the rationale for

this project.

A . Enzyme Project Background

When NBS decided to undertake to certify the analytes in a human serum SRM, several clinical chemists suggested that the

utility of this SRM would be greatly enhanced if some of the most widely measured enzymes were also characterized with respect to activity. Primarily, with the advice and leadership of one of the authors (G. N. Bowers, Jr.), and the encouragement of many

other leaders in the U.S. clinical chemistry community, a network

of cooperating individuals from clinical laboratories was recruited to perform the enzyme activity measurements, according

to a well-defined, interlaboratory testing protocol.
The team leaders for each enzyme and the scientists who

contributed and their affiliations are given below:

Team #1 - Acid Phosphatase (ACP)

L. M. Ewen, Royal Columbian Hospital, New Westminster,

B. C., Canada, (Team Leader);

R. J. L. Bondar, Worthington Diagnostics, Freehold, NJ;

W . Miller, Du Pont - aca Division, Wilmington, DE;

G. N. Bowers, Jr. and M. Onoroski,

Hartford Hospital, Hartford, CT; and

L. M . Shaw, Hospital of the Univ. of Pennsylvania,

Philadelphia, PA

Team #2 - Alkaline Phosphatase (ALP)

G. N. Bowers, Jr., Hartford Hospital, Hartford, CT, (Team

Leader )

;

R. Miller, Technicon Instrument Company, Tarrytown, NY;

W. D. Fellows, Health Safety & Human Factors Laboratory, Kodak

Park, Rochester, NY;

J. P. Bretaudiere, New York Department of Health,

Albany, NY;

D. C. Hohnadel, Christ Hospital, Cincinnati, OH; and

W. Sowers, Hyland Division of Travenol, Round Lake, IL

5
Team #3 - A 1 a n i n e Aminotransferase (AlaAT)

R. Rej , New York State Department of Health, Albany, NY, (Team

Leader )

;

R. Miller, Technicon Instrument Company, Tarrytown, NY;

W. T. Ryan, Beckman Instruments, Inc., Brea, CA;

L. M. Shaw, Hospital of the Univ. of Pennsylvania,

Philadelphia, PA; and

G. M. Sims, Hycel Inc., Houston, TX

Team #4 - Aspartate Aminotransferase (AspAT)

R. Rej, New York State Department of Health,

Albany, NY, (Team Leader);

L. M. Shaw, Hospital of the Univ. of Pennsylvania,

Philadelphia, PA;

W. D. Fellows, Health Safety & Human Factors Lab., Kodak Park,

Rochester, NY;

N . W. Tietz, Univ. of Kentucky Medical Center, Lexington, KY; and

A. D. E. Bacharach, Strong Memorial Hospital, Rochester, NY Team #5 - Creatine Kinase (CK)

R. Elser, York Hospital, York, PA (Team Leader);

R. Davis, Du Pont - aca Division, Wilmington, DE;

W. T. Ryan, Beckman Instruments, Inc., Brea, CA;

J. H. Stromme, Ulleval Hospital, Oslo, Norway; and

J. F. O'Brien, Mayo Clinic, Rochester, MN

Team #6 - Lactate Dehydrogenase (LDH)

R. B. McComb, Hartford Hospital, Hartford, CT (Team Leader);

K. Y. Jackson, New York State Department of Health, Albany, NY;

B. Howell, National Bureau of Standards, Washington, DC;

S. Buhl, Technicon Instrument Company, Tarrytown, NY; and

D. S. Young, and J. F. O'Brien, Mayo Clinic, Rochester, NY

6
Team #7 - y-G 1 utamyl transferase (y-GT)

L. M. Shaw, Hospital of the Univ. of Pennsylvania,

Philadelphia, PA (Team Leader);

R. Miller, Technicon Instrument Company, Tarrytown, NY;

S. Osaki, Hycel Inc., Houston, TX;

J. H. Stromme, Ulleval Hospital, Oslo, Norway; and

A. W. Wahlefeld, Boehringer Mannheim, Tutzing, West Germany

B . General Criteria for Participants and Operations

The general requirements for individual team members and operation of the i nter 1 abor atory test program were:

(1) the team member must have experience in enzyme

activity measurements;

(2) the laboratory technical staff under the team members'

direction would follow the agreed-on testing protocol

in all regards;

(3) sufficient priority would be given the work to allow

reasonable scheduling;

(4) data would be collected and reported on an agreed-on

format on data sheets supplied by NBS;

(5) work would be done voluntarily and thus would not add

to the cost of SRM 909 to the ultimate user; and

(6) all results would be made available to the entire

clinical chemistry community through publication.

7
II. GENERAL COMMENTS ON ENZYME ACTIVITY MEASUREMENTS

Measurements of enzyme activity in body fluids have been

used in clinical medicine since the early 1900's when Wohlgemuth

[13] first introduced amylase measurements of urine as a means of diagnosing acute pancreatitis. Today a general clinical

laboratory may make measurements of about a dozen different

enzymes found in serum. The proportion of enzyme measurements

many laboratories report may be 15% of total test volume, as illustrated in Table 1 [14]. While analytical precision of day-to-day analyses within a laboratory may range between 5 and

15% (cv)*, the bet ween - 1 abor atory agreement is less satisfactory.

The data in reference [15] shows that in large national surveys,

i nter 1 abor atory cv' s for commonly measured enzymes, e.g. AspAT

can exceed 50%.

The Committee on Enzymes of the Scandinavian Society for

Clinical Chemistry and Clinical Physiology has shown that the use

of a single, strictly defined, "standardized" enzyme method in a network of 100-200 Scandinavian hospital laboratories can lead to much improved interlaboratory precision. In Scandanavia, it should be noted, most laboratories utilize similar instrumentation, have well-trained personnel, and purchase enzyme reagents

from a limited number of suppliers. In 1969, before the use of "standardized" methods had been initiated, the overall

interlaboratory cv for values for five enzymes (AspAT, AlaAT,

LDH, ALP and y- GT) ranged from 30 to 49%. In 1978, after the

widespread adoption of the "standardized" methods, cv's were in the range of 7 to 17% [16].

* cv denotes the coefficient of variation

8
TABLE 1

Number of Enzyme Activity Measurements in the Clinical

Chemistry Laboratory at Hartford Hospital - October 1980 through

September 1981.

Enzyme

Alanine aminotransferase (Ala AT) 39 612

Aspartate aminotransferase (AspAT) 38 986

Lactate dehydrogenase* (LDH) 37 253

Alkaline phosphatase* (ALP) 29 972

Creatine kinase* (CK) 19 767

Amy 1 ase 15 647

Lipase 3 576

Acid phosphatase* (ACP) 1 573

Gamma- g 1 utamyl transferase (y-GT) 1 465

Choi i nesterase 65 Ceru lop 1 asmi n 30

Enzyme Total 189 750

*Includes isoenzymes by electrophoresis or i mmu no i n h i b i t i on **Total clinical chemistry tests were 1,162,184 (excluding RIA,

qualitative urine and coagulation tests).

9
The achievement of enzyme standardization in the United States

would present a problem of greater magnitude from that in Scandinavia, since compatibility of results in 10 000 or more hospital and clinical testing laboratories would be necessary.

Furthermore, a wide variety of methods are used in the U.S.,

ranging from simple kits to complex multichannel instrument

systems, laboratory personnel vary markedly in training, and supplies of reagents for enzyme analyses are obtained from

literally hundreds of sources.

It has been suggested by the U.S. clinical chemistry

community that the "best" approach to achieve compatible results

in such an enormous number of laboratories is to develop and

promulgate for use, an interrelated hierarchy of reference

methods and reference materials [17]. The National Reference

System for Clinical Chemistry (NRSCC), which functions under the

administrative guidance of the National Committee for Clinical

Laboratory Standards (NCCLS), has been created to carry out this

task. For clinical enzymology, reference-type enzyme methods

would be needed. The methods would be used for assigning enzyme

activity values to the enzyme(s) in appropriate reference

materials, and these reference materials would be made readily

available to all.

The development of reference -type enzyme methods has reached

an advanced state within the Expert Panel on Enzymes of IFCC and standards groups in the U.S. As a consequence, it is now possible to write protocols reasonably like those that will

ultimately be recommended by these panels of experts.

The availability of the NBS Human Serum, SRM 909, which

contains several stable enzymes, provided a unique opportunity to examine the problems associated with interlaboratory use of these

reference type enzyme methods [18].

At the time when the selection of an appropriate human serum preparation for use as the SRM was begun, several manufacturers

were visited to assess their capabilities for the production of a suitable freeze-dried material that would meet target specif ica10
tions. The final choice was based on the low i n homogene i ty of pilot runs of the materials that were screened. The SRM would be

a freeze-dried serum in the form of multiple, small pellets, and would be dispensed into vials by weight.

Enzymes levels in the final manufactured lot of serum were specified in accord with the broad guidelines suggested by the

manufacturer, and in consultation with several clinical chemists.

The choice of tissue sources for the enzymes to be added was left

to the manufacturer.

The enzymes, their associated activities, and source of added enzymes are shown in Table 2. TABLE 2

Catalytic (Activity) Concentrations (U/L)

Total Base Amount

Enzyme Planned Level* Added*

Found

Here**

Tissue

Source of Supp 1 ement

ACP 2.2 ± 0.7 ND ND 0.23 None

ALP 100 ± 25 49 86 75 Calf Intestine

A 1 a AT 28 ± 8 5 31 24 Pig Heart

AspAT 28 ± 8 15

CK 150 ± 50 112

LDH 150 ± 50 97

33

179

144

31

123

229

Pig Heart

Pig Heart

Piq Heart

Y"GT 30 ± 15 ND ND 16

* at 37 °C, Dupont a_ca_ (automatic clinical analyzer)

None

at 29.77 °C

11
III. PROTOCOLS FOR ENZYME MEASUREMENTS

This chapter gives general information concerning the

specific enzyme methods chosen. It also presents details of the

protocol for reconstituting the lyophilized SRM 909 because this

part of the procedure was critical to the overall success of the

study. Chapter IV gives specific information on the protocols

used for each enzyme.

A . General Comments and Observations on the Enzyme Reference

Methods Chosen 1

The Expert Panel on Enzymes of the Committee on Standards of the IFCC has now published Parts 2-7 of recommendations [19-23]

on reference methods for the measurement of the catalytic

concentrations (activity per unit of volume) of the following

enzymes: aspartate aminotransferase, alanine aminotransferase,

y-glutamyltransferase, alkaline phosphatase and creatine kinase.

It has also published recommendations with regard to general

considerations on the determinations of enzymes [24] and methods

for preparation of enzyme reference materials [25]. In general,

the specific protocols used in this study closely follow these

IFCC recommendations if they were available in draft form in mid-1979. The major exception is our use of the melting point

temperature of gallium (29.77 °C), as recommended by Mangum [26]

and Bowers and Inman [27] as the reaction temperature set-point

instead of 30.00 °C.

Each team leader was responsible for the choice of reference

method after conferring with Dr. Bowers. The team leader wrote

the detailed protocol and circulated it among his team for

comment and concurrence. Thereafter, team members would follow

the final protocol without modification. Enzyme team members

were recruited mainly from among individuals who were previous

participants in other enzyme method development activities or transfer projects conducted, for example, by AACC, EPE/IFCC, CDC

or CAP. Team members were from governmental, academic, hospital,

and industrial laboratories.

1 2
The protocols for AlaAT and AspAT were prepared by Dr. Rej , for CK by Dr. Elser, and for y-GT by Dr. Shaw. These four

protocols were almost identical to EPE/IFCC methods already

published or in early draft stages in 1979. The modifications

introduced are discussed under the specific protocols in Chapter

IV.

The state of current prostatic acid phosphatase measurement

technology, which includes recently introduced radioimmunoassay methods, does not permit the selection solely of an activity concentration method as the reference method at this

time. Nevertheless, the method of Ewen and Spitzer [28] which

employs thymo 1 phthal ei n monophosphate as substrate and a 1-hour

incubation at 29.77 °C was used in our study for measuring the

activity of this enzyme. Dr. Shaw arranged for SRM 909 samples

to be analyzed concurrently by an immunological (RIA) prostatic

acid phosphoresis method. Unfortunately and inadvertently,

seminal fluid acid phosphatase was not added to the serum base

pool from which SRM 909 was made, and as a consequence, it contains only very low levels (background) of acid phosphatase.

In order to enhance our study of acid phosphatase, a special

small batch of lyophilized material similar in composition to SRM 909, but which contained added human seminal fluid acid

phosphatase, was analyzed in parallel with SRM 909. In addition

to the development of our ACP protocol, the purity of

thymolphthalei n monophosphate preparations and specifications

for use of this material as a high quality substrate were studied [29,30].

Alkaline phosphatase was based on the manual reference

method of Bowers and McComb [31]. However, the method was updated by the inclusion of specifications for p_-nitrophenol [32]

and £-ni trop heny 1 phosph ate [33]. Although originally planned,

no study was made to compare the 2-ami no -2-methy 1 -1 -propanol

preparations used as buffer in various laboratories,

with respect to potential inhibitor content [34]. The EPE/IFCC

draft recommendation for alkaline phosphatase [22] that has now been published, was unavailable at the start of this study.

13
The LDH reference method was developed further by Dr. McComb.

It utilizes the highly purified sodium pyruvate supplied by NBS

as SRM 939. The NADH supplied to each laboratory was from a single lot that was tested by Dr. McComb and found free of LDH - i nh i b i tor [35]. Special algorithms were employed to calculate the "initial" activity from the non-linear progress

curves

.

Amylase, although present in SRM 909, was not included in our study. The development of a consensus on a reference method

for amylase is not likely in the near future. There is neither

agreement on the methodologic principle for an amylase reference

method, nor especially an optimum substrate.

B . Reconst i tut i on Protocol

When determining the definitive values for the non -en zymat i

c

constituents of SRM 909, NBS reconstituted the material at room temperature. However, reactivation of the enzymes in a reproducible manner at lower reconstitution temperatures was needed .

Our study of activity versus the procedure for

reconstituting ( rehydr at i ng ) SRM 909 lyophilized serum led to adoption of a uniform reconstitution protocol. Some of the SRM's

catalytic activities were found to be dependent on reconstitution

variables and to change during storage at different temperatures.

Because we needed to reconstitute the lyophilized serum at below

room temperature to obtain reproducible enzymatic activity,

ice-water baths were used during the reconstitution process.

That provided an easily achievable uniform temperature for the

1-hour allowed for reconstitution. The separate enzyme protocols

provide individual details where needed for handling the

reconstituted material after 1 hour. The reconstitution

procedure is as follows:

1. Remove vials of SRM 909 lyophilized serum and diluent

from refrigerator. Keep the vial of diluent in an ice

14
water bath for 5 min prior to use. Ice and water (not

air and ice) should contact the vial.

2. Tap vial bottom of lyophilized serum on a solid surface

to dislodge freeze-dried particles adhering to the

stopper. Remove the metal cap and stopper; be careful

not to lose any of the dried particles.

3. Use a Class A volumetric pipette at room temperature to add exactly 10.0 mL of the ice-cold diluent to the vial

of lyophilized serum as follows: Dispense the diluent

slowly against the inside neck of the vial while

continually turning it, to wet all the serum particles.

4. Replace the stopper; swirl gently and invert the vial

10 times, then reimmerse it in the ice-bath. Repeat the

gentle swirling and inverting operations at 10 min

intervals for 1 hour, always returning the vial to the

i ce-bath

.

5. Before use, inspect to ensure the absence of undissolved particles.

6. Proceed to the directions given with individual enzyme

protocols.

1 5
IV. SPECIFIC ENZYME PROTOCOLS

This chapter contains the detailed descriptions of the

specific measurement protocols for the enzymes. Each section

is for one enzyme, and contains descriptions of (1) the

methodological principle, (2) instrumentation and equipment, (3)

reagents, (4) the analytical procedure, and (5) calculations. A summary of the data obtained in the interlaboratory testing for

each enzyme is given in Chapter V.

A . Prostatic acid phosphatase

(Orthophosphoric monoester phosphohydrolase, acid optimum,

EC 3.1.3.2)

Principle : This measurement of the catalytic (activity)

concentration of prostatic acid phosphatase (ACP) in human serum utilizes the method of Roy e_t aj_. [36] as modified by Ewen and Spitzer [28]. The reaction sequence is as follows:

ACP

Thymo 1 phthal ei n + H2O »- t hymo 1 p h t h a 1 e i n + H3PO4.

monophosphate

The spectrophotometry absorbance of thymolphthalein, the

product liberated from the substrate thymolphthalein

monophosphate (TMP) in proportion to the activity of the acid

phosphatase during its reaction, is measured after conversion of the thymolphthalein to its colored form at an alkaline pH.

16
Final Reaction Conditions:

Reaction temperature

PHAcetate buffer

29.77

5.4

Thymo 1 phthal ei n monophosphate (TMP)

Bri j - 35 Volume fraction (sample/total)

0.15 mol/L

1.0 mmol/L

1.5 g/L

1:12 (0.083)

Instrumentation and Equipment

Glassware : Volumetric glassware should meet the National Bureau

of Standards (NBS) Class A specification or be individually

calibrated prior to use. For pipetting serum specimens,

" to-conta i n

, " self-adjusting mi cropi pettes , accurate to within

±0.2% of volume are preferred. Alternatively, repetitive

dispensing devices with disposable tips may be used;

however, their precision and accuracy should be checked and

be within 0.5% (95% confidence limit). Tubes used in the

assay must be chemically clean and preferably made of borosilicate glass.

I nstrumentat i on : Specifications for the instrumentation, pH

meter, waterbath, and spectrophotometer are given in Notes 1, 2 and 3, respecti vely.

Reagents : Specifications for TMP and thymo 1 phth al e i n are given

in Notes 4 & 5, respectively [29]. All other chemical reagents

including water should meet American Chemical Society (ACS)

reagent grade specifications.

1. Acetic acid (5 mol/L). Use a buret to transfer 28.87 mL of glacial acetic acid into a 100 mL volumetric flask. Dilute

to volume with water. Mix well.

2. Sodium acetate (5 mol/L). Dissolve 68.0 g of sodium acetate

trihydrate ( C^COONa • 3^0 ) in water in a 100 mL volumetric

flask. Dilute to volume. Mix well.

17
3. Acetate buffer (5 mol/L, pH 5.4 at 25 °C). Add sufficient

5 mol/L sodium acetate (reagent 2) to 5 mol/L acetic acid

(reagent 1) to adjust to pH 5.4 at 25 °C.

4. Acetate buffer (0.25 mol/L, pH 5.4 at 25 °C). Pipette 5 mL

of acetate buffer (reagent 3) into a 100 mL volumetric flask.

Dilute to volume with water. Mix well.

5. Buffered substrate. Dilute a 300 g/L Brij-35 solution to a concentration of 3.24 g/L. Use 50 mL of this diluted

solution to dissolve the amount of Na2TMP* XH2O required to give a TMP concentration of 1.1 mmol/L when the volume of this reagent is brought to 100 mL; (eg., if using

Na2TMP» IIH2O, molecular weight 752.65, the appropriate

quantity is 82.8 mg). Add 1.92 g of sodium acetate

trihydrate to the solution, mix to dissolve, adjust the pH

of the solution to 5.4 at 25 °C with 0.1 mol/L HCL, and

finally bring volume to 100 mL with reagent grade water. Store in refrigerator.

6. Alkaline reagent (0.1 mol/L Na2C03, in 0.1 mol/L NaOH).

Dissolve 10.6 g of anhydrous Na2C03 and 4.0 g of NaOH in distilled water. Dilute to 1000 mL. Mix well.

7. Thymo 1 pht h a 1 e i n stock standard (3 mmol/L). Dissolve 129.2 mg

of t hymo 1 p h t h a 1 e i n in 70:30 (V/V) ii-prop ano 1 -water in a 100-mL volumetric flask. Fill to the mark. Mix well.

Analytical Procedure :

1. Preparation of standard curve (standardization to a catalytic concentration of 60 U/L): Into 10 mL volumetric flasks

pipette 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mL of thymo 1 phthal ei

n

stock standard (reagent 7). Fill each flask to the mark with

70:30 (V/V) ji-propanol -water mixture. Mix well. Into a set of seven tubes, dispense 550 y L of buffered substrate (reagent 5)

and 1.0 mL of alkaline reagent (reagent 6). Pipette 50 yL of the

70:30 ji-propanol-water mixture into the first tube of the set and

18
50 y L of one of each of the diluted standards into the remaining

tubes. Mix well. Read the absorbance of these solutions at 595 nm, using the first tube as blank. Plot absorbance versus

U / L on linear graph paper. These absorbances are equivalent to absorbance changes obtained by the action of 10, 20, 30, 40, 50,

and 60 U/L of acid phosphatase when a 30-minute assay incubation

time is used, or to one-half of these activities if a 60-minute

assay is used.

2. Measurement of the catalytic concentration of acid

phosphatase: Use six tubes for each specimen to be assayed, five

"test" tubes, labeled "15", "30", "45", "60" (for minutes) and "total", and also one "blank" tube. Into tubes labeled 15, 30,

45, 60 and blank, pipette 1.0 mL of the alkaline reagent

(reagent 6). Into the remaining empty tubes labeled total,

pipette 5.5 mL of the buffered substrate (reagent 5). Bring only

the tubes labeled total to the 29.77 °C assay temperature (see Note 2) in a water bath, and to each at convenient intervals

(e.g., 30 s) transfer 500 yL of a serum specimen, mix gently and allow to incubate at the assay temperature. At exactly 15, 30,

45 and 60 minutes after addition of specimen, withdraw aliquots

from each incubating total tube and transfer exactly 0.6 mL to the tubes correspondingly labeled 15, 30, 45, and 60.

While the total tubes are incubating, or following

i ncub at i on -- wh i c h ever is more con v en i ent -- add 550 yL of buffered

substrate (reagent 5) to each blank tube followed by 50 yL of each serum specimen, and mix.

Read the absorbances of test and blank solutions at 595 nm against reagent grade water; subtract blank from test absorbances; check that the reaction is linear with time for each

specimen; and obtain the catalytic (activity) concentration of acid phosphatase either by reference to the standard curve or calculation as described below.

Calculations:

The catalytic (activity) concentration of acid phosphatase

19
in SRM 909 measured by the above protocol at 29.77 °C, is calculated in terms of International Units per liter, (1.0 U/L

1.0 ymol "mi n~l • L"l) as follows:

Catalytic concentration (in U/L) = ( d

A

/ d

t

) V • e

1

•

b

l.h-I-u-1

where d_A is the change of absorbance at 595 nm over the specified

rate measurement interval, d_t is the measurement interval in minutes, is the volume of the final reaction mixture in liters,

e is the micro molar absorptivity of t hymo 1 pht h a 1 e i

n

(39 200 x 10~ 6 L • ymo

1 " * • cm" 1 ) under the final reaction

conditions, b is the cuvette pathlength in cm, and _y_ is the

sample volume in liters.

By substituting the numerical values for \/, e, b_, and y_: Catalytic concen- = (dA/dt) . (1.6 x 1Q- 3

) tration (in U/L)

(39 2 00 x 10-6) (i) (0.05 x 10"3)

= 816. 3 • dA per mi n

.

Catalytic (activity) concentration in katals per unit volume

(kat/L) can be calculated from the relationship 1.0 U/L = 16.67

nkat/L [37].

Notes

1. The pH measuring device should be calibrated at 25 °C against reference buffers from an authoritative source (e.g., NBS

or IUPAC).

2. Temperature of assay. The melting point of gallium

(29.77 °C) is the reaction temperature set-point for this

reference method. A water bath with a continuous agitation

capability is used to maintain ±0.05 °C control throughout the water mass.

3. The method is wavelength settingrelatively insensitive to variation in or spectral bandwidth if measurements are made

20
on spectrophotometers in which stray light is low. A wavelength

setting error of ±2 nm with a spectral bandwidth of 8 nm results

in an absorbance error of less than 1%. Cuvettes should have

parallel faces of optical glass that transmit light without

significantly reducing the light intensity due to absorption,

reflections or other scattering effects. The internal pathlength

should be 10.00+0.01 mm.

4. Specifications for TMP as substrate. The Na2TMP*xH20 from

different manufacturers may vary in content of thymolphthalein,

in content of water of crystallization and in color. To be acceptable as substrate, the supplier's analysis for the

Na2TMP , xH20 should show that the material meets the following

specifications [29]:

a. Formula: C 28 H 29° 7 PN a

* xH 2°

b. Molecular weight, anhydrous: 554.5.

c. Water content (by Karl Fischer method):

range 0.15-0.26 as mass fraction or 5.5:1

to 11:1 as mole fraction.

d. Appearance: white to pale yellow crystals;

clear, colorless to pale yellow, 25 g/L aqueous

solution.

e. pH of aqueous solution: range 8.0-8.5.

f. Apparent molar absorptivity: range 2150 - 2500

at 445 nm.

To check molar absorptivity, dissolve 100.0 mg of Na2TMP-x H2O in 0.1 mol/L Na2C03 in a 100 mL volumetric

flask. Dilute to volume with 0.1 mol/L Na2C03.

Prepare a 1 in 10 dilution with additional 0.1 mol/L

NaC03. Measure absorbance (A) at 445 nm with 0.1 mol/L

Na2C03 as reference. Calculate the apparent molar

absorptivity using label value for mole fraction of H2O

in Na2TMP'xH20 as follows:

21
Apparent molar _ (A) ( 554 . 5 ) absorptivity (1-mass fraction H2O) (0.100)

g. Thymo 1 phthal ei n content: less than 5 mmol/mol

To check mole fraction of t hymo 1 p h t h a 1 e i n in TMP,

add 1.0 mL of the alkaline reagent (reagent 6) to 0.5

mL of buffered substrate (reagent 5). The molar

absorptivity at 595 nm (25 0 C ) should be less than

0 .075 re 1 at i ve to the b lank .

h. Storage: 4 °C.

5. High purity t h ymo 1 p h t h a 1 e i n in a solution of 0.06 mol/L

Na2C03 solution (as under assay conditions) has been found to have a molar absorptivity of 40 500±200 L • mo 1

1

• cm" 1 at 600 nm

(at 25 °C and 10 mm pathlength). Test for this by transferring

25.00 mL of the t hymo 1 p ht h a 1 e i n stock standard (reagent 7) to a 1-

L volumetric flask. Dilute to volume at 25±1 °C with 70:30 (V/V)

in-propanol-water. A mixture of 3 ml of this solution and 5 mL of the alkaline color reagent (reagent 6) should have an absorbance

at 600 nm of 1 . 134±0 . 005 at 25 °C after blank correction.

6. Prepare tubes needed for serum sample preservation by

pipetting 25 y L of acetate buffer (reagent 3) into chemically

clean borosilicate glass tubes. Place tubes in an oven at 60 °C,

and allow buffer solution to evaporate but do not over-dry.

Stopper and store tubes at room temperature until required.

These tubes contain sufficient buffer for acidifying 0.5 mL of patient sample. Check tubes on initial preparation and

periodically throughout use by adding 0.5 mL of serum and measuring the pH after dissolving the buffer.

22
7 . Collect blood with minimal venous stasis. Plasma

obtained by collection into sodium heparin at a concentration of 0.2 g/L is preferred. Centrifuge whole blood for 10 minutes at 3000xG at 25 °C. Immediately thereafter transfer 0.5 ml_ of the

plasma to a tube containing dried acetate buffer. Agitate gently

to dissolve the buffer. Store specimens at -20 °C if they are

not to be assayed on the same day.

8. Alternative reconstitution of SRM 90 9: (for use if only

acid phosphatase is to be assayed). Follow reconstitution

protocol given above, except that 0.25 mol/L acetate buffer

(reagent 4) is used in place of the diluent supplied with SRM 909.

Store aliquots at -20 °C if required for use on subsequent days.

Comments by Team Leader (L. M. Ewen)

Final results were calculated from data submitted by R. J. L. Bondar, M. Onoroski/G. N. Bowers, Jr. and L. M. Ewen. The

prostatic acid phosphatase activity found in SRM 909 was extremely low.

We would have preferred to have an NBS material with a prostatic

acid phosphatase level at or above the upper limit of the reference

range for the method, e.g., to a level of 2 U/L.

Because of the low activity of this enzyme in SRM 909, no data were gathered on its reactivation. SRM 909 was not supplemented with prostatic acid phosphatase and the activity

found by our method was extremely low (the AA per 60 min was 0.02). This was confirmed by radioimmunoassay (RIA) performed by

L. M. Shaw which detected no prostatic acid phosphatase in the SRM.

As a check on the RIA methodology, samples similar to SRM 909,

but supplemented with prostatic acid phosphatase to a level of 5 U/L, were assayed simultaneously with SRM 909 by RIA, and the

level of prostatic acid phosphatase in the enriched material was found to be 11 yg/L.

2 3
The method used in this work should not be viewed by

standardizing agencies as an official recommendation of the

procedure. Several subcommittees such as the Enzyme Study Group

of the Standards Committee of the American Association for

Clinical Chemistry, are actively engaged in projects with the

objective of the development of a specific recommended method for

prostatic acid phosphatase. Their recommendations, while needed,

are not yet avai 1 ab le .

B . Alkaline Phosphatase

(Orthophosphoric monester phosphohydrolase, alkaline

optimum, EC 3.1.3.1)

Principle : This protocol for measuring the total catalytic

(activity) concentration of alkaline phosphatase in SRM 909 is a slightly modified version of the manual reference method

described by Bowers and McComb [31]. The method differs from

earlier published methods [38] only slightly in final reagent

conditions. The buffer pH is raised to 10.5, the substrate

concentration is increased to 16 mmol/L and the magnesium

concentration is increased to 1 mmol/L. However, the

procedural steps have been markedly altered. In order to avoid

prolonged exposure of the enzyme to 2A2M1P [39], the serum or other sample is preincubated with 3 mmol/L magnesium for at least

5 min before the activity is measured. Furthermore, the reaction

rate is followed only within the first two minutes to avoid

subsequent slower rates thought to be due to the removal of zinc

from the enzyme by chelation to 2A2M1P and/or to inhibitory

compounds in the buffer [34].

The appearance of the yellow color of p_- n i t rop hen o 1 (NP) is monitored continuously in a spectrophotometer at 402 nm as the

alkaline phosphat ase ( s ) catalyzes the hydrolysis of the

colorless substrate, p_-nitrophenyl phosphate (NPP). This

enzymatic reaction takes place in a tr ansphosphoryl at i ng buffer,

2-ami no-2-methyl -1-propanol (2A2M1P), with all reaction parameters

careful 1 y control 1 ed

.

24
ALP

NPP + 2A2M1P ^ NP + 2A2M1P-P.

Final reaction conditions:

Reaction temperature

PH2-Amino-2-methyl -1-propanol

(2A2M1P)

£-Nitrophenyl phosphate (NPP)

Magnesium acetate

Volume fraction ( s amp 1 e/ tot a 1 )

29.77 °C 10.5

1.0 mol/L

16.0 mmol/L

1.0 mmol/L

0.0164 (1:61)

25
Reagents : Specifications for the 2A2M1P [34], NP [33] and the

p_-nitrophenyl phosphate [32] are given in the Notes and should be met. All other chemical reagents and water should meet American

Chemical Society (ACS) r e agen t

- g r ad e specifications. Glassware

should meet the National Bureau of Standards (NBS) Class A specification or be individually calibrated prior to use.

1. Magnesium solution (3 mmol/L): Dissolve 650 mg of magnesium

acetate*4H20 in water in a 1-L volumetric flask. Dilute to volume. Mix well. This solution is stable indefinitely at

4 °C.

2. 2A2M1P buffer (1.5 mo 1 / L , pH 10.50 at 29 . 77 °C): Warm the

2A2M1P (see Note 1) at 30-35 °C until completely liquefied.

Weigh 135 g of this liquid directly in a 1-L volumetric

flask. Add about 500 ml_ of reagent-grade water, mix, and then add 190 mL of 1.000 mol/L HC1. Allow solution to cool

to 2 5 ± 1 °C, and adjust to volume with reagent-grade water.

Mix well. At 29.77 °C, this buffer has a pH of 10.5 0+0.05 when measured after calibration, using special

Tris-type electrodes (see Note 2) and NBS buffers, and sloping (i.e., adjusting the slope controls) of the pH meter.

When stored at room temperature with protection from

atmospheric CO 2, this buffer is stable for 1 month.

3. p- N i t r op heny 1 phosphate buffered substrate (24.5 mmol/L in 2A2M1P buffer): For each 1 mL of reagent 3 required,

dissolve 9.1 mg of Na2NPP«6H

2 0 (see Note 3) in 2A2M1P buffer

(reagent 2). Make fresh daily.

4. Stock standard NP (1 mmol/L): Dissolve 139.1 mg of high-purity NP (see Note 4) in water. Dilute to volume in 1-L volumetric flask at 25±1 °C with water. Mix well. This

solution is stable for many months if protected from light.

26
5. Working standard NP (25 ymol/L in 2A2M1P buffer): Place

900 mL of 2A2M1P buffer (reagent 2) in a 1-L volumetric

flask. Add exactly 25.00 mL of stock standard NP (reagent

4). With contents at 25±1 °C, fill flask to the mark with water and mix. The absorbance of this reagent at 402 nm (at

29.77 °C in a 10.00 mm cuvette) is 0.476 when a blank made

from 9 parts of reagent 2 and 1 part of water is subtracted.

This is equivalent to a molar absorptivity of 19 050

L«mol~l«cm-l. If the value obtained differs by more than

±1% from this value, the observed value should be used to calculate the activity. The user should be able to explain

the reasons for the difference. Reagent 5 has been shown to

be stable for at least 8 weeks.

Analytical Procedure:

Into a test tube containing 1.0 mL of magnesium solution

(reagent 1), add 50 y L of sample. A calibrated mechanical

diluter/pipette or a "to-contain" pipette may be used to deliver

sample; however, adequate wash-out of the pipette must be ensured.

Mix tho-roughly and place tube in a water bath at 29.77 °C for

exactly 5 min so that magnesium activation and temperature

equilibration occur before the rate measurement is made.

Immediately on completion of the 5-minute preincubation with

magnesium, add 2.0 mL of buffered substrate (reagent 3) which has

been prewarmed to 29.77 °C. Mix well. Immediately transfer the

reaction mixture into a 1.000-cm cuvette in a spectrophotometer

set to 40 2 ±1 nm (see Note 6) and equipped to allow the cuvette

and contents to be rapidly adjusted to a reaction temperature of 29.77±0.05 °C (see Note 5). Immediately record absorbance

at 402 nm vs. time continuously or at regular intervals (e.g., 10

s) for a total of 120 s against an air blank. Obtain the rate of absorbance change per minute from the spectrophotometry data by

graphic or mathematical means.

27
Calculations:

The catalytic (activity) concentration of alkaline

phosphatase in SRM 909 measured by this protocol at 29.77 °C is calculated in terms of International Units per liter (1.0 U/L = 1.0 ymol 'mi n~l • L~l ) as follows:

Catalytic concen- = dA/dt . v -l. £ -l. b -l. v -l tration (in U/L)

where d_A is the change of absorbance at 402 nm over the specified

rate measurement interval, d_t is the measurement interval in minutes, is the total volume of the final reaction mixture in liters, y_ is the sample volume in liters, is the cuvette

pathlength in cm, and e_ is the micro molar absorption coefficient

of NP, which is 19 050 x 10" 6 [..mol^-cm -1 under the final

reaction conditions. By substituting numerical values,

Catalytic concen- _ (dA/dt) (3 .o 5 x 10 -3)

tration (in U/L) " (lg Q50 x 10 _ 6) (1) (0 . 05 x 10 -3)

= 3203'dA per min.

Catalytic (activity) concentration in katals per unit volume

(kat/L) can be calculated from the relationship 1.0 U/L = 16.67

nkat/L [37].

Notes :

1. 2A2M1P has been shown by many workers to contain

inhibitors. The 2A2M1P used should be inhibitor-free by

both enzymatic and special testing, as described by Rej

e_t aj_. [34]. They suggest that 5 - ami n o - 3 - azo - 2

, 2 , 5 - tr i me t hy 1 - hexanol is one of these inhibitors.

2. Measurements of pH at 10 and above in amino propanol

solutions require special "Tris-type" electrodes, which can be obtained either from Sigma Chemical Co., St. Louis, M0 63178, as "Trisma" electrode No. E4878, or from the Beckman Instrument Co.,

Fullerton, CA 92634, as described in Applications Research

Technical Report No. 542.

28
3. The quality of Na2NPP«6H20 may vary from lot to lot and manufacturer to manufacturer [33]. Variations in water content

may cause differences of ±5%. The specifications for this

substrate are as follows:

a. Molar absorptivity: At 311 nm and 2 5 °C in 10 mmol/L

NaOH, the molar absorptivity has been found to be 9866 L

o mol-l'cm - l. If all other specifications are met, Na2NPP«xH20, when x is in the range of four to six, may be acceptable. The molar concentration of NPP in solution is established from its

absorbance in NaOH at 311 nm.

b. Test for NP: The mole fraction of NP in NPP should be less than 0.1%. The absorbance of a 100 mmol/L

solution of NPP in a 1.000-cm cuvette at 432 nm should

be less than 0.300 [32].

c. Inorganic phosphate in Na2NPP«xH

2 0. Amount

allowable is less than 1%. Test for inorganic

phosphate in a 100 mmol/L Na2NPP solution by methods

used normally in a clinical chemistry laboratory to determine phosphorous in human serum.

4. High-purity NP after being recrystallized from water,

dichloro me thane, and again water [32] has been found to have a molar absorptivity of 18 380±90 L • mo 1

1

• cm" 1

, at 401 nm and 25 °C

in 10 mmol/L NaOH (1.000-cm pathlength). Check by transferring

25.00 mL reagent 4 into a 1-L flask and diluting to volume at 25 ±1 °C with 10 mmol/L NaOH. The absorbance of this 25 ymol/L

solution after blank correction should be 0.460±0.002.

5. Temperature of assay: The melting point of gallium,

29.77 °C, is the reaction-temperature set point for this

reference method. A water bath with a continuous agitation

capability is used to maintain ±0.05 °C-control in the reaction

mixture [27].

29
6. Temperature factors for this reference method for alkaline

phosphatase on pooled human serum at 2 5, 30, and 37 °C relative

to 29.77 °C are 0.79, 1.01 and 1.33, respectively. Correction is not made for thermal expansion or temperature effects on absorbance. Similar temperature-conversion factors have been

found with a crude preparation of alkaline phosphatase prepared

from human liver and added to human serum pool.

7. The method is relatively insensitive to variation in wavelength setting or spectral bandwidth when measurements are made on spectrometers in which stray light is low. A wavelength

setting error of ±3 nm with a spectral bandwidth of 5 nm results

in an absorbance error of less than 1% [40],

8. The reference intervals for healthy individuals for this

method at 29.77 °C (see Note 5) are as follows:

3 0
Popu 1 at i on Catalytic concentration, (U/L)

Newborns, 1 week Up to 250

Girls

1-12 years

>15 years

Up to 350

See adu Its

Boys

1-12 years

12-15 years

>20 years

Up to 350

Up to 500

See adults

Adults 25-100

Comments by Team Leader (G. N. Bowers, Jr.)

Final results (RRII) were calculated from data supplied by

all members of the team (see list in the Introduction). The

human alkaline phosphatase activity level in the base pool after

the initial processing, but before supplementation, was 49 U/L at

37 °C as measured by the manufacturer on a Du Pont aca . Partly

purified, ca 1 f - i n test i na 1 alkaline phosphatase was added to the

base pool by the manufacturer prior to lyophilization, to give a total activity of about 100 U/L (by aca_ at 37 °C). Since the

activity factor for altering the reaction temperature from

29.77 °C to 37 °C with most alkaline phosphatase methods and materials is approximately 1.33, we expected the activity for SRM

909 to be about 75 U/L. Many other factors, such as pH and buffer concentration, preincubation with magnesium, and length of exposure to buffer before and during assay would also affect the

activity [36]. Fortuitously, the activity found by use of this

protocol (at 29.77 °C) was near 75 U/L.

One laboratory's initial difficulty with the protocol is worth noting. Its results for the two prelimininary test rounds

31
were markedly different from all of the others. The laboratory

discovered two causes for its discrepant results: (1) the

temperature at the cuvette was too low because of insufficient

water flow, and ( 2 ) instead of air or water, a substrate/

magnesi um/2A2MlP had been used as the blank. Mg(0H)2 had

precipitated, which caused high blank readings. With these

problems avoided, its results for RR III, then corresponded with

the other laboratories.

RRI and RRII were carried out in the spring and summer of 1980. The i nter 1 abor atory mean values were 73.2±4.3 U/L (S.D.

for individual laboratories) and 7 4 . 4± 3 . 5 U/L respectively. For

RR III data, which were obtained during the winter of 1980-81 , the mean value was 75.4±2.0 U/L. Using the above protocol, a European government R&D clinical laboratory found a value of

76 . 5 ±1 . 0 U/L in the spring of 1981.

The participants and instruments used in RRI 1 1 are given

below.

Participant

G . N . Bowers , Jr

.

W. N. Fellows

D. C. Hohnadel

J. P. Bretaudiere

R . Miller

W. Sowers

Instrument Used

Van' an Cary 219

Beckman ASTAR

Gilford 300N

Varian Cary 219

Varian Cary 219

Varian Cary 219

The greatest variable one faces in measuring alkaline

phosphatase activity in lyophilized material is the increase in activity seen with time and incubation temperature after

reconstitution. Figure 1 shows the SRM 909 alkaline phosphatase

activity increases observed as a function of time and

temperature, the samples being held at various temperatures after

undergoing a uniform 1-h reconstitution in an ice-water bath.

Although higher activities can be reached more rapidly by

incubating the reconstituted material at higher temperatures, the

maximum activities thus obtained are not stable. In fact a
</)

(1/n) OoZZ"63 »B Rw\o\f d"IV

33
completely reproducible procedure was not found. By warming

samples for 8 h at 37 °C, a peak activity of 95 U/L was reached;

but this activity level subsequently decreased slightly.

Observations were made over an 18 month period on 19 reconstituted samples that were held at 4 °C. Initially, these

activities were 75±2 U/L, but after 8 months, the range was from 26 to 104 U/L. In the absence of a satisfactory means for

stabilizing activity, we decided to reconstitute and hold samples

at 0 °C in an ice-water bath. This would reduce variation in the

temperature and consequently, the rate of reactivation among

laboratories. The decision to use 0 °C for reconstituting was applied to all enzymes, and was in effect for Round Robin III. For the non - en zymat i c constituents of SRM 909, see Certificate of Analysis for SRM 909, Appendix 1; NBS independently released its

final recommendations for reconst i tut i on at room temperature

(23 ±2 °C). A systematic comparison of the differences in enzyme reactivation resulting from these two procedures has not been made, but one can expect to add 2 U/L of phosphatase

activity from reconstituting at room temperature for 1-h.

Steady increases in activity with time would occur thereafter, as shown in Figure 1.

C . Alanine Aminotransferase

( L-Al an i ne : 2-oxog 1 utar ate aminotransferase, EC 2.6.1.2)

Principle : Alanine aminotransferase (AlaAT), formerly called

glutamate pyruvate transaminase (GPT), catalyses the following

reaction:

L-alanine + 2-oxog 1 utar ate AllAI^ pyruvate + L-glutamate

The activity of AlaAT is measured by converting the pyruvate thus

34
formed into lactate with reduced nicotinamide adenine

dinucleotide (NADH) and lactate dehydrogenase (LDH) as indicator

enzyme [20, 41]:

pyruvate + NADH + H + UL^ L-lactate + NAD +

The quantity of NADH oxidized, which is equivalent to the amount

of pyruvate formed, is measured by the decrease in absorbance at 339 nm.

Final Reaction Conditions :

Reaction temperature

L - A 1 a n i n e PH30 °C Tris buffer

2-0xog 1 utar ate Pyridoxal 5'-phosphate

NADH (assuming NADH- Na£- 2H 2 0

)

LDH Volume fraction (sample/total)

Reagents :

1. L-Alanine (615 mmo 1 / L ) in Tris (110 mmol/L, pH 7.30): To about 800 mL of de ionized water in a 1-L or larger beaker,

add 13.32 g of Tris and 54.80 g of L-alanine. Stir contents

with beaker immersed in a water bath to achieve a temperature of 30±2 °C. While monitoring the temperature,

adjust pH to 7.30±0.02 with 1 mo 1 / L HC1. Transfer

quantitatively to a 1.00-L class A volumetric flask.

Equilibrate at 20±2 °C and bring volume to mark. Mix well.

Refrigerate working solution; freeze the remainder.

2. 2-0xogl utarate (180 mmol/L) in Tris (110 mmol/L, pH 7.30):

To about 600 mL deionized water in a 1-L or larger beaker,

add 26. 30 g of 2 -oxog 1 ut ar i c acid and 13.32 g of Tris. Stir

contents with flask immersed in a water bath to achieve a temperature of 30±2 °C. While monitoring the temperature,

29.77 °C 500 mmol/L

7.30

100 mmol/L

15 mmol/L

0.10 mmol/L

0.165 mmol/L

1200 U/L (25 °C)

0.083 (1:12)

35
adjust pH to 7.30±0.02 with 10 mol/L and 1 mol/L NaOH. Transfer quantitatively to a 1.00-L class A volumetric flask.

Equilibrate at 20±2 °C and bring volume to mark. Mix well.

Freeze in aliquots of about 10 ml_.

3. Tris (110 mmol/L, pH 7.30): To about 800 mL of deionized

water in a 1-L or larger beaker, add 13.32 g of Tris. Stir

contents with flask immersed in a water bath to achieve a temperature of 30 ±2 °C. While monitoring temperature,

adjust pH to 7.30±0.02 with 1 mol/L add HC1 . Transfer

quantitatively to a 1.00-L class A volumetric flask.

Equilibrate at 20±2 °C and bring volume to mark. Mix well.

Store at 4 °C.

4. Pyridoxal phosphate (5.0 mmol/L) in Tris (110 mmol/L, pH

7.30): For each 100 mL of reagent 4 required, dissolve

133 mg of pyridoxal 5'-phosphate in 100.0 mL of reagent 3. Portion out aliquots of about 10 mL. Store at £-20 °C. This reagent can be used for 5 days when kept at 4 °C.

5. Lactate dehydrogenase (3.06 kU/mL): Purchase hog muscle LDH containing 550 U/mg at 25 °C as a 10 mg/mL solution in glycerol (See Note 3). Add 0.8 mL of a 50:50 (V/V)

glycerol: water mixture to each 1.0 ml of enzyme solution.

LDH activity should be verified by the method given in reference [41].

6. Combined assay mixture: To prepare sufficient mixture for

the number of assays to be performed, combine the following

in the quantities shown:

36
No. of assays: 10 20 30 50 100

Reagent 1, (in mL) 20 40 60 100 200

(Alanine)

Reagent 5, (in mL) 0.01 0.02 0.03 0.05 0.1

(LDH)

Reagent 4, (in mL) 0.5 1.0 1.3 2.5 5.0

(Pyridoxal -P)

Na 2 NADH- 2H 2 0, ( in mg) 3.0 6.0 9.0 15.0 30.0

(dry powder)

Analytical Procedure:

Add 0.2 mL of serum to a cuvette. Pipette 2.00 mL of the

combined assay mixture (reagent 6) into the same cuvette and incubate at 29.77 °C for 15 min. When the absorbance of the

mixture at 339 nm is stable, add 0.2 mL of 2-oxogl utarate

(reagent 2), mix, and record absorbance changes. The reaction

must be followed continuously. The reaction rate is obtained

over the linear portion of dA/dt, usually after 2 or 3 minutes.

One blank reaction must be assessed as follows: Combine 2.0 mL

of reagent 6 as above and 0.2 mL of distilled water as a sample.

Assay in triplicate each day. The value for this blank

measurement should be less than 5 U/L.

37
Calculations:

The catalytic (activity) concentration of alanine aminotransferase in SRM 909 measured by this protocol at 29.77 °C is calculated in International Units per liter, 1.0 U/L = 1.0 (ymol »mi

n~ 1

• L~ 1

) as follows:

Catalytic concen- . ( dA/d

t

)

.

v . 1

• b" 1• v' 1 tration (in U/L)

where d_A is the change of absorbance at 339 nm over the specified

rate measuring interval, d_t is the measuring interval in minutes,

is volume of the final reaction mixture in liters, y_ is the

sample volume in liters, b is the cuvette pathlength in cm, and

£ is the micromolar absorptivity of NADH which is 6 300 x 10~6

L'ymol"l'cm - l under the final reaction conditions. By

substituting the known values:

(dA/dt) • (2.4 x 10" 3

) (6.30 x 10" 3

) (1) (0.20 x 10" 3

) 1905*dA per min.

Calibration of reagent blank activity is identical; the final result

is calculated by deducting the value of the reagent blank (U/L) from

that found wi th SRM 909 (U/L) .

Catalytic (activity) concentration in katals per unit volume

(kat/L) can be calculated from the relationship 1.0 U/L = 16.67

nkat/L [37].

Catalytic concentration (in U/L)

38
Notes

:

1. Reagents 1 and 4 should be filtered through microporous

filter (0. 22 ym) .

2. Assay volume may be reduced to 1.00 mL of reagent 6 and

0.100 mL each for sample and reagent 2.

3. Boehr i nger-Mannhei m hog muscle LDH (catalog 127.868 or 127.221) is suitable for preparation of reagent 5.

Comments by Team Leader (R. Rej)

The method selected for use in these round robins is based

on that recommended by the AlaAT Study Group of the Standards

Committee of the American Association for Clinical Chemistry [41]

and the Expert Panel on Enzymes of the International Federation

of Clinical Chemistry [20], This procedure was developed with

considerable cooperation and collaboration between these two groups and therefore represents both a national and international

reference method for measurement of AlaAT activity. There are slight mechanical and methodological differences between these

two methods. However, in terms of substrate concentrations and measurement pH, they are identical. The major difference lies in the measurement of four different catalytic rates for each

specimen in the IFCC procedure. The AACC method -- and that used

in these round robins -- recommends the measurement of a single

blank measurement: a reagent blank using distilled water as a specimen. The major sources of catalytic activity in this blank

measurement are contaminant AlaAT activity within the LDH (reagent 5) and the reduction of 2-oxog 1 utarate by LDH itself:

LDH 2-oxog 1 utarate + NADH + H + 2-hydroxyg 1 ut ar ate + NAD +

The muscle type isoenzyme of LDH (LDH-5, M4) catalyzes this

reaction at a rate several-fold slower than that catalyzed by the

heart type isoenzyme (LDH-1, H4); therefore this isoenzyme was recommended for these measurements. The K m of this isoenzyme for

39
pyruvate, however, is larger than that of LDH-1 and the lag phase

(the time required after addition of 2-oxogl ut ar ate before a linear change in absorbance with time is observed) is measurable.

Reaction rates for NBS SRM 909 were determined 3 min after

addition of 2-oxog 1 utar ate . The magnitude of the reagent blank

was always measurable and the mean of triplicate measurements

within each laboratory was deducted from the catalytic rate measured with SRM 909 (see Figures 5 and 5b). The rates given in Table 4 are those determined with such blank correction.

The additional two blanks proposed by the IFCC [20] may compensate for interferences in some patient sera. No such interferences were observed with NBS SRM 909 after the required 15 min preincubation.

The preincubation before addition of 2 -oxog 1 ut ar at e may be performed at 30±2 °C rather than the rigorously controlled 29.77

needed for measurement of catalytic activity -- this is particularly important if a t hermost at t ed flow-cell is used for

measurements. All laboratories used the NBS Gallium Cell

(SRM 1968) for calibrating laboratory thermistors. NBS SRM 922

(Tris base) was used for preparation of all buffers and

substrates. The temperature of reconstituting liquid did not appear to affect results. Individual spheres of the dry serum

were found to be attracted to the stopper and neck of vials. A piezo-electric anti-static pistol (Zerostat by Discwasher) was used to reduce static charges.

The participants and instruments used in the all round

robins ar e

:

R. Rej

L. Shaw

Varian Cary 219

Gilford Stasar III

G. Sims & S. Osaki Beckman 24 and 34

W. Ryan

R. Miller

Beckman DU-8

Varian Cary 219

40
D. Aspartate Aminotransferase

( L-Aspartate : 2-oxog 1 utarate aminotransferase, EC 2.6.1.1)

Principle : Aspartate aminotransferase (Asp AT), formerly

glutamate oxalacetate transami nase-GOT, catalyzes the following

react i on

:

The activity of AspAT is measured by converting the oxalacetate

into L-malate with reduced nicotinamide adenine di nucleotide

(NADH) and malate dehydrogenase (MDH) as indicator enzyme:

The quantity of NADH oxidized, which is equivalent to the amount

of oxalacetate formed, is measured by the decrease in absorbance

at 339 nm.

The assay employs the final conditions recommended by the AACC Standards Committee working group [42]; this assay

correlates well with the proposed IFCC methods [19], Three

modifications are incorporated: 1) the melting point of gallium,

at 29.77 °C, is the measurement temperature; 2) the volume

fraction of specimen was changed from 0.0667 (1:15) to 0.083

(1:12) to allow the use of convenient volumetric glassware for

all additions, i.e., 2.00 and 0.200 mL or 1.00 and 0.100 mL pipettes can be used for dispensing reagents and specimen; and

3) LDH is not used. The catalytic rate of the reaction mixture

prior to the addition of 2-oxoglutarate is monitored in the spectrophotometer prior to adding the 2-oxoglutarate.

Final reaction conditions:

AspAT

L-aspartate + 2-oxoglutarate oxalacetate + L-glutarate.

MDH oxalacetate + NADH + H + L-malate + NAD +

.

Reaction Temperature

pH

Tr i s buffer

29.77

7.80

89 mmo 1 /

L

L- Asp art ate 175 mmo 1 /

L

41
2-0xog 1 ut ar ate Pyridoxal 5' -phosphate

NADH (assuming NA 2 NADH- 2H 2O

)

MDH (EC 1. 1 . 1 . 37 ) Volume fraction ( samp 1 e/ tot a 1 )

15 mmo 1 /

L

0.11 mmo 1 / 0. 165 mmo 1 /

L

0.95 U/ml_(25

(1:12)

°C)

0.083

Reagents :

1. L-aspartate (216 mmol/L) in Tris (100 mmol/L, pH 7.8 at

30 °C): Add 28.8 g of L-aspartic acid and 12.1 g of Tris

to 700 mL of deionized water in a 1-L beaker. Stir

continuously and monitor the pH. While temperature is controlled to 30+2 °C, add NaOH pellets slowly (no more than

5 g). Then adjust pH to 7.8±0.02 at 30 °C with 10 mo 1 /

L

NaOH. Cool solution to ambient temperature and

quantitatively transfer to a 1.0-L class-A volumetric flask.

Bring to the mark. Mix well. Stable for at least 2 weeks.

2. Malate dehydrogenase (235 U/mL): To 10 mL of 50:50 (V/V)

glycerol -water , add 0.2 mL of porcine MDH 10 mg/mL in glycerol (about 1200 U/mg at 25 °C). Mix gently by

inversion. Refrigerate.

3. Pyridoxal phosphate (5.44 mmol/L) in water: Dissolve 288 mg

of pyridoxal phosphate monohydrate in 200 mL of distilled

water. Portion out in about 10 mL aliquots and freeze at £ 20 °C. Can be used for 4 days when refrigerated (at

^4 °C).

4. 2-0xog 1 utarate (180 mmol/liter) in Tris (100 mmol/liter,

pH 7.8): Add 2.63 g of 2 - oxo g 1 ut ar i c acid and 1.21 g of Tris to about 70 mL of deionized water. While controlling

the temperature at 30±2 °C, adjust pH to 7.80±0.02 with

1.0 mol/L and 10 mmol/L NaOH. Quantitatively transfer into

a 100.0 mL class A-volumetric flask and bring to mark at ambient temperature. Stable at 4 °C for at least 2 days.

Freeze in portions of about 10 mL (£-20 °C).

42
5. Combined assay mixture: To prepare sufficient assay

for the tests to be performed, combine the following

quantities shown below:

mi xt u r

i n

Reagent 1 (Aspartate) 60 mL Reagent 2 (MDH) 0.3

Reagent 3 (Pyridoxal phosphate) 1.5

Na2NADH-2H 2 0 (dry powder) 9.0

mL

mL

mg

Analytical Procedure:

Add 0.2 mL of serum to a cuvette. Pipette 2.00 mL of the

combined assay mixture (reagent 5) into the same cuvette;

incubate at 29.77 °C at least 15 min. When absorbance at 339 nm

is stable, add 0.2 mL of 2 - o xo g 1 u t ar ate (reagent 4), mix, measure

rate at 339 nm. One blank reaction must be assessed as follows.

Combine 2.0 mL of reagent 5 and 0.2 mL of distilled water as sample. Assay in triplicate each day. The value for the blank

should be less than 0.8 U/L.

Calculations :

The catalytic (activity) concentration of aspartate aminotransferase in SRM 909 measured by this protocol at 29.77 °C is calculated in International Units per liter

(1.0 U/L =1.0(ymol •min" 1 -L- 1

) as follows:

Catalytic concentration (in U/L) = dA/d t • V • e" 1

• b" 1

• v" 1

where d_A is the change of absorbance at 339 nm over the specified

rate measurement interval, d_t is the measuring interval in minutes, is the total volume of the final reaction mixture in liters, v_ is the sample volume in liters, b> is the cuvette

pathlength in cm, and £ is the micromolar absorption coefficient

of NADH which is 6300 x 10" 6 L • u mo 1

1

• cm" 1 under the final

reaction conditions.

43
By substituting values,

Catalytic concend A/dt (2.4 x 1Q-3)

tration (in U/L) " (6>3 x 1Q . 3) (;) x 1Q _ 3)

= 1905 • dA per mi n

.

Reagent blank is calculated in an identical manner. The reagent

blank is typically 0.4+0.4 U/L and need not be deducted from the

overall rate measurement.

Catalytic (activity) concentration in katals per unit volume

(kat/L) can be calculated from the relationship 1.0 U/L = 16.67

nkat/L [37].

Notes :

1. Reagents 1 and 4 should be filtered through microporous

f i Iter (0. 22 urn)

.

2. Boehringer-Mannheim porcine heart MDH (product 127892 and

1 2 7906 ) meets these specifications. The g 1 yc ero 1 - b as ed material must also show low Asp AT contamination.

3. Assay volume may be reduced to 1.00 mL of assay mixture

(reagent 5) and 0.100 mL each of serum and 2 - o xo g 1 ut ar at

e

( reagent 4 ) .

Comments by Team Leader (R. Rej)

The method selected for use in these round robins is based

on those recommended by the AspAT Study Group of the Standards

Committee of the American Association for Clinical Chemistry [42]

and the Expert Panel on Enzymes of the International Federation

of Clinical Chemistry [19]. There are several methodological

differences between these two methods. These include differing

substrate concentrations and different procedures for measurement

44
of blank activity. As with the A 1 a AT procedure described

previously, four separate measurements are determined for each

specimen in the IFCC method. Because of the interference due to addition of LDH (also see AlaAT procedure) LDH was not added to the procedure used for the NBS SRM 909 study. LDH is often added

to AspAT assays to more quickly remove endogenous pyruvate. The

pyruvate concentrations of SRM 909 do not appear excessive so that there was no requirement to correct for this potential

interference with SRM 909. This is not necessarily the case with

patient specimens. Reagent blanks (measurement of catalytic)

rate with distilled water as a specimen) were performed by all participants but the magnitude of these catalytic rates was less

than the overall imprecision for these determinations. In the

majority of laboratories this blank rate was the equivalent of <0.8 U/L. It was therefore decided to use the measurement of the

catalytic rate found with SRM 909 without deduction of this

reagent blank (Table 4). The reagent blank was, however,

determined in each round robin to insure that its magnitude did

not exceed 0.8 U/L. The omission of LDH from the procedure used

likely contributed to the insignificance of the reagent blank.

Two additional blanks proposed by the IFCC [19] may

compensate for interferences in some patient sera. No such

interferences were observed with NBS SRM 909 after the the

required 15 min preincubation.

The preincubation before addition of 2-oxoglutarate may be performed at 30±2 °C rather than the rigorously controlled 29.77 c needed for measurement of catalytic activity -- this is particularly important if a thermost atted flow-cell is used for

measurements. All laboratories used the NBS Gallium Cell

(SRM 1968) for calibrating laboratory thermistors. NBS SRM 922

(Trrs base) was used for preparation of all buffers and substrates. The temperature of reconstituting liquid did not appear to affect results. Individual spheres of the dry serum

were found to be attracted to the stopper and neck of vials. A piezo-electric ant i -static pistol (Zerostat by Discwasher) was used to reduce static charges.

45
The participants and instruments used in all three round

robins are:

R. Rej Varian Cary 219

Beckman ACTA C 1 1

1

N . Tietz

A. Bacharach Varian Cary 118

W. Fellows Beckman ACTA C I 1

1

L. Shaw Gilford Stasar III

E . Creatine Kinase

(ATP :Creatine N-phosphotransf erase , EC 2.7.3.2)

Principle : This protocol was developed for measuring the total

creatine kinase (CK) catalytic (activity) concentration in SRM

909 lyophilized human serum. It was adapted with two modifications from the Scandinavian Committee on Enzymes (SCE)

method [43]. The modifications include: 1) changing the

reaction temperature from 37 °C to the melting-point of gallium,

29.77 °C, and 2) omitting adenylate kinase (AK) inhibitors by

using instead a discrete measurement of a blank to correct for

the presence of AK in the reference material.

The appearance of NADH is continuously monitored in a spectrophotometer at 340 nm as the enzyme creatine kinase

catalyzes the transfer of a phosphate group from creatine

phosphate to adenosine diphosphate (ADP) to form adenosine

triphosphate (ATP), which is coupled to the indicator reaction,

according to the following scheme.

CK Creatine phosphate + ADP Creatine + ATP

H K ATP + D-'glucose ADP + D-gl ucose-6-phosphate

G6PD

D-glucose-6- + NAD + phosphateD-gl uconate-6- + NADH + H + phosphate

46
F i nal reaction conditions:

Reaction temperature

pH (at 29.77 °C)

Imidazole acetate

Creatine phosphate

Ad enosine-5' -diphosphate (ADP)

D-G 1 ucose Nicotinamide adenine di nucleotide

(NAD +

) Hexokinase (HK) (EC 2.7.1.1)

D-Glucose-6-phosphate

dehydrogenase (G6PD) (EC 1.1.1.49)

(L. mesenteroides)

Magnesium acetate

N-Acetyl cystei ne ( N AC

)

Ethylenediaminetetracetic acid,

sodium salt, Na2 ( EDTA)

Volume fraction (sample total)

29.77

6.60

100

30

2 20

2

2500

1500

10

20

2

0.043

mmo 1 /

L

mmo 1 /

L

mmo 1 / mmo 1 /

L

mmo 1 /

U/L

U/L

mmo 1 /

L

mmo 1 / mmo 1 / L'

(1:23)

1.5

t T h i s concentration is not critical and may range

3 . 0 mmo 1 / 1 i ter . from

This method differs from other CK methods by eliminating the

use of adenylate kinase inhibitors [43, 44]. A blank measurement

must be made in the absence of creatine phosphate to account for

adenylate kinase activity [45]. EDTA has been included [44].

Sample and incomplete reagent (the latter lacks creatine

phosphate) are pre incubated for 5 minutes before activity

measurements are begun.

Reagents :

All reagents should meet American Chemical Society

reagent-grade (A.R.) specifications. Water should be at Type II grade (2.0 x 10 6 ohm cm at 25 °C). east

47
1. Imidazole acetate stock solution [128 mmol/L in pH 8.0

buffer (25 °C) with magnesium acetate (12.8 mmol/L) and EDTA

(2.55 mmol/L)]: Dissolve imidazole (8.72 g), disodium

ethyl enedi ami ne t et r ac e t at e • 2H 2O (0.95 g) and magnesium

acetate-4H 2 0 (2.75 g) in about 950 mL of Type II H 2 0. Add

acetic acid (1 mol/L) to adjust to pH 8.0 (at 25 °C). Then

add more H 2 0 to a total volume of 1000 mL. Reagent is stable for 2 months at +4 °C and for a minimum of six months

at -20 °C.

2. Working reagent A. Imidazole acetate (115 mmol/L) pH 8.0

(25 °C), magnesium acetate (11.5 mmol/L), EDTA (2.3 mmol/L).

N-acetyl cysteine (23.0 mmol/L), ADP (2.3 mmol/L), D-glucose

(23.0 mmol/L), N AD + (2.3 mmol/L), hexokinase ( 2900 U/L),

D-g 1 ucose -6-phosphate dehydrogenase (1750 U/L): To prepare

100 mL, combine 90 mL of reagent 1 with N-acetylcysteine

( 375 mg), adenos i ne -5 1 -d i p hos p hor i c acid (98 mg), D-glucose

(414 mg), and nicotinamide adenine dinucleotide (153 mg).

Mix well. Adjust temperature to 29.77 °C before adjusting

pH to 6.60 with 1 mol/L acetic acid. Then add between

260-290 U (25 °C) of hexokinase and approximately 175 U (20 °C) of gl ucose-6-phosphate dehydrogenase (L.

mesenteroi des ) . Type II water is then added to the 100 mL volume (see notes 1 and 2). Store at +4 °C or -20 °C. The reagent is stable for 6-h at room temperature; for 24 -

h

at +4 °C; and for a minimum of 1 week at -20 °C.

3. Working reagent B. Creatine phosphate (345 mmol/L):

Dissolve 1.125 g of creatine phosphate tetrahydrate disodium

salt in 7 mL of type II water. Dilute to 10.0 mL. Mix well.

See note 3. Store at +4 °C or -20 °C. Stable for 3 months

at +4 °C, and for a minimum of 1 year at -20 °C.

Analytical Procedure : Measurement of total CK activity plus adenylate kinase

(AK) activity.

48
Pipette 100 y L of serum into a test tube containing 2.000 mL

of reagent 2 that has been prewarmed to approximately 30 °C. Mix

and incubate for 5 mi n at approximately 30 °C. Add 200 u L of reagent 3 that has been prewarmed to approximately 30 °C. Mix

and pour into a 1.000-cm cuvette. Place the cuvette in a controlled cell compartment maintained at 29.77 °C of a spectrometer set to read at 340 nm. The rate of the steady state reaction which follows the lag phase of up to 120 s, is determined by continuous recording of the 340 nm absorbance.

Record the absorbance vs. time for a total of 300 s against a blank made from 2.000-mL of reagent 2, 100 yL of Type II water

and 200 yL of reagent 3.

Procedure for blank (AK activity only): Into a test tube

containing 2.000-mL of reagent 2 that has been prewarmed to approximately 30 °C, pipette 100 y L of serum. Mix and incubate

for 5 min at approximately 30 °C. Add 200 u L of Type II water

that has been prewarmed to approximately 30 °C. Mix and pour

into a 1.000-cm cuvette. Place the cuvette into the

spectrophotometer's controlled cell compartment. The rate of the

steady. state reaction which follows the lag phase of up to 120 s,

is determined by continuous recording of absorbance at 340 nm. Record the absorbance vs. time for a total of 300 s against a blank made of 2.000 mL of reagent 2, and 300 yL of Type II water. Calculations :

The catalytic (activity) concentration of creatine kinase in SRM 909 measured by this protocol at 29.77 °C, is calculated in International Units per liter, (1.0 U/L = 1.0 y mo 1 • mi n " 1 • L"

1 ) as follows [46].

49
Catalytic concen- q

-, = dA/dt- V- e-l« b- 1 tration (in U/L)

where dA is the change of absorbance at 340 nm over the specified

rate measuring interval, d;t is the measuring interval in minutes, is the total volume of the final reaction mixture in liters, £ is the micro molar absorption coefficient of NADH, which

is 6317 x 10"6 L*ymol"l'cm~l under the final reaction conditions

[47], b is the cuvette pathlength in centimeters, and v_ is the

sample volume in liters. Therefore:

Catalytic concen-_ (dA/dt) (2 . 3 x 1Q-3)

tration (in U/L) " (6317 x 10 _ 6) (1) {QA x 10 - 3)

= 3641*dA per min.

Catalytic (activity) concentration in katals per unit volume

(kat/L) can be calculated from the relationship

1.0 U/L = 16.67 nkat/L [37].

Notes :

1. ATP concentration in reagent 2 should be less than

10 y. mo 1/1 iter; as a consequence the increase in absorbance

on addition of the auxiliary enzymes should be less than

0.060.

2. The absorbance of reagent 2 at 340 nm should be less

than 0.200. Certain lots of imidazole have been found to contain an unknown substance which contributes significantly

to absorbance at 340 nm. These lots should be identified

and not used in the preparation of stock imidazole buffer.

Although at present, it is not clear whether the recovery of activity is affected, the range of linearity of measurement

is compromised by starting with a high initial absorbance.

3. The absorbance of reagent 3 at 340 nm should be less than

0.150.

50
COMMENTS BY TEAM LEADER ( R . C. Elser)

The amount of residual CK activity present in the base pool

after processing but before supplementation with exogenous enzyme

was estimated to be 112 U/L. Partly purified porcine heart CK was added to the base pool prior to 1 yoph i 1 i zat i on to increase

the final activity to the 150-200 U/L range when assayed on a DuPont a_ca at 37 °C.

The participants and instruments used in the final round

robin (III) are given below for the participants who submitted

results for Round Robin 3. Participant Instrument Used

R. C. Elser Gilford Stasar III

J. H. Stromme GEMSAEC

R. H. Davis Gilford 250

J. F. O'Brien GEMSAEC

The reconsti tution protocol followed for CK was identical to that recommended for reconstitution of this material in the assay

of other enzyme constituents of SRM 909. A systematic comparison

of the differences in reactivation from reconstituting SRM 909 at

0 °C and at room temperature has not been made, but it is to be expected that approximately 20% less activity might be recovered

with reconstitution at room temperature.

Davis, et a 1 . [44] have shown that the temperature at which

lyophilized sera are reconstituted has an effect on the recovery

of CK activity. SRM 909 was reconstituted and incubated at 2, 6, 10, 20, 25 and 30 °C and assayed at 30, 60, and 90 minutes

following reconstitution. The assay method, which was performed

at 30 °C, used the DuPont aca optimized CK formulation. Figures

2 and 3 show the combined effects of time and temperature on recovered values. It is clear that both time and temperature,

especially in the 2-10 °C range, affect recovery. It appears

that no particular time and temperature combination is superior.

51

However, the reproducibility of the temperature of an ice bath meets the needs of temperature constancy from laboratory to laboratory and from time to time. Time of rehydration at this

temperature may vary from 30 to 90 minutes with less than 2% variation in recovery of activity.

The stability of CK-3 (CK-MM) has been examined by Whitner,

et a 1 . [47a]. The presence of ADP and N-acetylcysteine increased

the half-life of CK activity by approximately 30-fold when

lyophilized material was stored at -20 °C.

53
F . Lactate Dehydrogenase

( L-Lactate : NAD + oxi doreductase , EC 1.1.1.27)

Principle : Lactate dehydrogenase (LDH) catalyzes the following

reaction:

LDH pyruvate + NADH + H + ^L-lactate + NAD +

.

Lactate dehydrogenase activity is determined by measuring

the decrease in absorbance at 339 nm due to the oxidation of NADH

as pyruvate is reduced to L-lactate [48-51].

Final reaction conditions :

Reaction temperature

pH (at 29.77 °C)

Tris buffer

Sodium pyruvate

NADH Volume fraction (sample/total)

Reagents :

1. Tris buffer (0.15 mol/L): In a 1-L volumetric flask,

dissolve 18.22 g of tr i s ( hydroxymethy 1 ) ami nometh ane (NBS SRM

922) in 800 mL of deionized water. To this solution add

exactly 124.5 mL of 1.000 mol/L HC1 and mix thoroughly.

Dilute to mark with deionized water. Verify that the pH at 29.77 °C is 7.35±0.02. This reagent should be stable for 2 weeks at room temperature.

2. NADH (0.229 mmol/L): Weigh the appropriate amount of dry

Na2NADH'xH20 (Note 1) into a 200 mL volumetric flask and dissolve in Tris buffer (reagent 1). Adjust volume to the

mark and mix. The absorbance of this solution at 339 nm should be 1.447±0.05 and the pH should be 7.35±0.02 at 29.77 °C . The solution is stable at room temperature for 4 hours and at 4 °C for 24 hours.

29.77 °C

7. 35 0.0968 mol/L

1.2 mmol/L

0.15 mmol/L

0.016 (1:61)

54
3. Sodium pyruvate (3.66 mmol/L): In a 100 mL volumetric

flask, dissolve 40.2 mg of sodium pyruvate (NBS SRM 910) in 50 mL of reagent grade water. Dilute to volume with

additional water and mix. Stable for 8 hours at room temperature or for 24 hours at 4 °C.

4. Potassium dichromate (0.205 mmol/L): Dissolve 60.1 mg of K2O2O7 (NBS SRM 136) in a 1-L volumetric flask in approximately 500 mL of deionized water. Add 0.10 mL of concentrated HCIO4 and dilute to volume with water.

Analytical Procedure:

To a test tube containing 2.0 mL of reagent 2, add 50 yL of sample (Note 2). Incubate this mixture for 10 min at 29.77 °C. Initiate the LDH reaction by adding 1.00 mL of reagent 3 that has been preincubated at 29.77 °C for at least 5 min. Mix and quickly transfer a portion of this

mixture to a 1.00 cm cuvette (Note 3) that is at 29.77±0.1 °C (Note 4). Record the 339-nm absorbance of the

reaction mixture at 30 s intervals for a total of 180 s starting 30 s after addition of the pyruvate.

Calculations :

The time course of the LDH reaction must be corrected for

non-linearity [52]. The initial rate is calculated from the

corrected curve. The value obtained is used to calculate the

activity as follows:

1. The natural logarithm (In) corresponding to each 30 s data point (InA) is plotted vs. time (in min). From the resulting straight line, calculate the slope

and y intercept (In A 0 ). A 0 is obtained from the

antilog of lnA

Q (see Note 5).

2. Calculate from these constants, the initial rate of absorbance change.

55
(dA/dt

)

0

= slope x A 0

= initial rate

From this initial rate of absorbance change, the

catalytic (activity) concentration of lactate

dehydrogenase in SRM 909 measured by the above protocol

at 29.77 °C is calculated in International Units per liter (1.0 U/L = 1.0 ymo 1 -mi n" 1

• L" 1

) as follows:

Catalytic concen- = (dA/dt) . v

. £

- 1 . b

~ 1

.

v

- 1

( tration (in U/L) 0

where d_A is the change of absorbance at 339 nm over the

specified rate measuring interval, d_t is the measuring

interval in minutes, is the total volume of the final

reaction mixtures in liters, v_ is the sample volume in liters, b is the cuvette pathlength in cm, and £ is the

micro molar absorption coefficient (molar absorptivity)

of NADH which is 6300 x 10~ 6 L • umo

1

1

• cm" 1 under the

final reaction conditions. Therefore:

Catalytic concen-

= (dA/dt )o tration (in U/L) (6300 x 10 -6) (1 ) (0 .05 x 10" 3

) = 9 68 2 »dA„ per min.

o

Catalytic (activity) concentration in katals per unit

volume (kat/L) can be calculated from the relationship

1.0 U/L = 16.67 nkat/L [37] . Notes :

1. Some suppliers of NADH (and catalogue numbers) are Boehr i nger-Mannheim (107727), Sigma (7179), and P-L Biochemicals (6500). The quality and formula

weight of commercially available NADH vary

considerably from lot to lot. The material to use should appear as dry white crystals and, according

56
to the labeling, should contain a minimum of 98%

NADH as d i sodium salt hydrate. Water content is different even among the best lots of NADH so that

amounts used to prepare reagent 2 will vary.

The amounts of NADH given below are needed to prepare 100 mL of reagent 2, based on formula

weights (F.W.) stated on the labeling provided

with every lot of NADH. The amounts needed

compensate for water of hydration and ethanol

content

F.W. Amount Needed (mg)

750 17.3

755 17.4

760 17.5

765 17.6

770 17.7

775 17.8

780 17.9

785 18.0

790 18. 2 795 18. 3 800 18.4

The delivery of each sample from the TC pipette

should be followed by 2 washes with the diluted

sample mixture. A mechanical p i petor -d i 1 utor may

be used as an alternative, if the accuracy and precision of this apparatus can be shown to be within 0.5% (95% confidence) for both sample and diluent.

The pathlength of this cuvette should be 1.00 ±0.01 cm. An indirect check of this dimension

can be made by measuring the absorbance of acid

dichromate at 350 nm [53]. The absorbance of a 0.205 mmol/L solution at 25 °C against distilled

water should be 0.645±0.006 (±2SD).

57
4. The temperature of the reaction mixture should be checked just before the 30 s reading and just

after the 300 s reading by use of a small-mass

temperature probe that is connected to a measuring

system which is calibrated against a gallium cell.

If the set temperature is different from 29.77 °C (e.g., 30.0 °C), activities should be corrected to 29.77 °C. Reaction temperatures that differ by more than 0.3 °C from the 29.77 °C reference point

should not be used

.

5. As an alternative, the time course data can be curve fitted to the exponential equation y = a bx . In this case a = A 0 and b = slope; thus

axb = slope x A 0

= initial rate or (dA/dt) 0

. COMMENTS BY TEAM LEADER (R. B. McComb)

The average LDH catalytic concentration increased

substantially between RRI, RRII, and RRIII. This probably

reflects changes in the protocol that were introduced to provide

more accurate estimates of the initial reaction rates. We decreased the measurement intervals from five 1-min readings to five 30 s readings. In RRIII, all participants used the same

high quality NADH preparation. The NADH was supplied by Dr. J. Siegal (P. L. Bi ochemi cal s , Cat. No. 6500). Based on high

performance liquid chromatography, 95% of the 260 nm absorbing

material in this preparation was present in the NADH peak. (NBS

has developed specifications for SRM NADH, but material meeting

these specifications is not available as an SRM.)

The lab-to-lab variation in results was least in RRIII. The

coefficient of variations were 4.9%, 8.2% and 6.4% in RRIII,

RRII, and RRI, respectively. Results obtained by performing the

assay at temperatures other than 29.77±0.3 °C are corrected to this reference temperature by using the correction factor 7.2%

per degree.

58
The effects of r ec on s t i t ut i on and storage temperature on LDH activity were tested during RRIII. One vial of SRM 909 was reconstituted according to the SRM 909 protocol and stored at

0 ° C . A second vial was reconstituted in an identical fashion

but both reconsti tution and storage was at 22 °C. LDH activity

was measured in triplicate on samples taken from each vial at 1, 6, and 24 hours. Average activity increased 4% over the 24 hour

period in both vials. Activity in the vial that was reconstituted at 0 °C was consistently less by an average of 1.6% than

that in the vial reconstituted at 22°C. These are small

differences but suggest the need for strict adherence to the

reconst i tut i on protocol when using SRM 909.

A report from France [60] suggests that the inclusion of 200

mmol/L sodium chloride in the reaction mixture eliminates

non-linearity in the time-course of the P to L reaction. If this

effect can be confirmed, this modification would constitute a major improvement in methodology for the measurement of LDH activity.

59
G . y- Gl uta my 1 transferase

[ ( y-G 1 u t amy 1

) -pep t i de : amino acid y-g 1 utamyl transfer ase

,

EC 2.3.2.2]

Principle : The biochemical and clinical chemistry literature

shows that a number of donor substrates have been used for the

assay of y-GT activity. We use L -y -g 1 u t amy 1 - 3 -c ar bo xy

4- n i t ro an i 1 i d e (GluNA-CO?) and g 1 ycy 1 g 1 yc i ne (glygly), and this

conforms with the recommendation of the Expert Panel on Enzymes

of IFCC [21]. GluNA-CO? is the preferred substrate since it has

the practical advantage of being readily soluble in aqueous

solution. The rate of absorbance change at 410 nm found with

human sera is 21% higher with GluNA-C02 than with an equivalent

concentration of L-y- gl u tamy 1 - 3- su 1 f on i c - 4 - n i troa n i 1 i de , another

possibly utilizable water-soluble substrate. The absorbance

contribution of 4-nitroanilide substrates is significant at 405

nm, but decreases substantially as wavelength is increased.

G 1 ycy 1 g 1 yc i ne is the preferred acceptor substrate since a significantly higher catalytic rate (and, therefore, more readily

observable change in absorbance) is obtained using it as compared

with other acceptor substrates.

y-GT in human serum catalyzes two reactions simultaneously,

namely, tr anspept i dat i on and autotr ansf er . In the former

reaction glygly is the y -glutamyl acceptor substrate; in the

latter reaction G 1 u NA-CO2 is the acceptor substrate:

( 1 ) TRANSPEPTIDATION

y-GT

G 1 u NA-CO2 + glygly Y -gl uglygly + NA-CO2

(2) AUTOTRANSFER

y-GT

GIUNA-CO2 + GIUNA-CO2 GlugluNA-C02 + NA-CO2

In the case of human serum y-GT, the reaction rate obtained at

a concentration of 10 mmol/L of GIUNA-CO2 in the absence of glygly is 13% of the rate obtained at the same GIUNA-CO2

60
concentration but with 150 mmol/L of glygly present. The

relative rate of transpeptidation to autotransfer at substrate

concentrations used in our method, 6 mmol/L GluNA-C02 and 150 mmol/L glygly, is less than 1%. The substrate g 1 ycy 1 g 1 yc i ne also serves as buffer.

Final Reaction Conditions:

Reaction temperature

pH (29.77 °C)

L-y-glutamyl -3-carboxy-4-

n i t r 0 a n i 1 i d e Glycyl glyci ne Volume fraction (sample/total)

29.77 °C 7.90

5 mmo 1 /

150 mmol/L

0.091 (1:11)

The assay is initiated by the addition of serum to the

combined substrate/buffer mixture, equilibrated to 29.77 °C. The

assay is followed at 410 nm for at least 300 s. Venous blood is collected under conditions that minimize stasis. Serum is the

preferred specimen. Plasma obtained from whole blood ant i coagulated with any anticoagulant tends to give lower values than the

corresponding serum specimen. Investigations with this method

have shown that y- g 1 u t amy 1 1 r an sf er ase in serum remains stable for

a minimum of 5 days at +4 °C and for a minimum of 7 months at -80 °C. Instrumentation and Equipment :

Glassware: Volumetric glassware should meet the National

Bureau of Standards (NBS) Class A specification or be individually calibrated prior to use. For pipetting control

material or serum specimens, mechanical pipettes with disposable

tips may be used, but the accuracy and precision of the device

used should be checked and be within 0.5% (95% confidence

interval )

61
Instrumental on

:

1) pH meter. Calibrate meter at 25 °C against

reference buffers from a recognized authoritative source

(NBS or IUPAC), but use at 29.77±0.3 °C.

2) Temperature of the assay. The melting point of gallium,

29.77 °C, is the set point temperature for this

reference method. A water bath is required, having

continuous agitation capability to maintain uniform

conditions throughout the water mass. Temperature

should be controlled at the set-point ±0.05 °C.

3) Spectrophotometer. A wavelength setting error maximum of ±2 nm with a spectral bandwidth of 8 nm is required. Cuvettes should have parallel faces of optical glass which transmits light without

significantly reducing the light intensity due to absorption, reflections or other scattering effects.

The internal pathlength should be 1.00±0.01 cm.

62
Reagents:

To prevent the growth of micro-organisms in the solutions,

sterilized containers should be used. All solutions should be prepared in calibrated flasks with freshly distilled and

deionized water.

1. N-Glycylglycine (183.3 mmol/L, pH 7.90 at 29.77 °C):

Dissolve 24.222 g of g 1 ycy 1 g 1 yc i ne (free base, MW 132.12) in approximately 800 mL water in a 1-L volumetric flask, adjust

pH to 7.90 at 29 . 77 °C with 2 mo 1 / L NaOH against

standardized reference buffers (NBS or IUPAC). Adjust

volume to the mark with water. Mix well. (See Note 1.)

2. L-Y-G 1 ut amy 1 -3-car boxy- 4-n i t roan i 1 i de (66 . 00 mmol/L):

Dissolve 2.286 g of L-y-gl utamyl -3-carboxy-4-ni troani 1 ide

monoammonium salt monohydrate ( MW 364.30) in water in a 100 mL volumetric flask. Fill to the mark. Mix well. (See Note 2.)

3. Rea'gent mixture L-Y-gl utamyl -3-carboxy-4-ni troani 1 i de (6.6 mmol/L) and g 1 ycy 1 g 1 yc i ne (165 mmol/L, pH 7.90 at 29.77 °C): Combine 90.00 mL of reagent 1 and 10.0 mL of reagent 2 in a 100 mL volumetric flask. Mix well. The

absorbance at 410 nm of this solution measured against water should not exceed 0.500. (See Note 3.)

4. Reagent solution g 1 ycy 1 g 1 yc i ne (165 mmol/L, pH 7.90 at 29.77 °C: Combine 90.0 mL of reagent 1 and 10.0 mL of water

in a 100 mL volumetric flask. Mix well.

5. Sodium chloride solution (154 mmol/L): Dissolve 9.00 g sodium chloride in 1000 mL water. Analytical Procedure : Subprocedures that constitute one measurement:

63
Measurements of the rates of two reaction mixtures are required for determining the Y-GT activity of the serum sample:

A, the rate of the overall reaction; and

B, the rate due to the reagents (as a reagent blank).

The two measurements are made on mixtures having a 2.20 ml_ final

volume; the cuvette pathlength is 1.00±0.01 cm; the cuvette

compartment of the spectrophotometer is thermostated at 29.77±0.05 °C; and absorbances are measured at 4 1 0± 1 nm.

The two mixtures are prepared as follows:

for rate A, add 0.200 mL of serum to 2.000 mL of reagent 3; for rate B, add 0.200 mL of water to 2.000 mL of reagent 3.

The temperatures of the serum, water, and reagents 3 and 4 are at 29.77 °C prior to being combined. After pipetting the

separate components into the cuvette and mixing them, the

absorbances are recorded for 300s.

For sera with catalytic concentrations of y-GT up to about

300 U/L, the values of dA/dt_ are constant over an interval of at least 300 s. If the values of dA/dt^ are found to be greater than

0.215 per minute, dilute the serum 5- or 10-fold with reagent 5 and repeat the measurements.

Corrections for blank reactions

The overall y-GT reaction rate (A) is corrected for the

reagent blank rates (B) as follows:

(dA/dt) corrected = (dA/dt)

A

- (dA/dt)

B The corrected value of (dA/dt_) is used in the following

calculations. It corresponds to the initial rate of the reaction

catalyzed by y-glutamyltransferase. The serum blank is usually

zero. The reagent blank (due to autohydro 1 ys i s of L-y - g 1 ut amy 1 -3 -car bo xy-4-n i t roan i 1 i de ) is very small, but 64
measurable. It should be within the range of 0.0002 - 0.0005A

per min under the conditions of the assay.

Calculations :

The catalytic (activity) concentration of Y-gl utamyl transferase in SRM 909 measured by the above protocol

at 29.77 °C, is calculated in International Units per liter (1.0

U/L = 1.0 ymo 1 *mi

n

-1

• L" 1 ) as follows:

Catalytic concen- = (dA/dt ) . v

.. e-1 VW"l

tration (in U/L)

where dA is the change of absorbance at 410 nm over the specified

rate measuring interval, dt_ is the measuring interval in minutes,

is the total volume of the final reaction mixture in liter, y_ is the sample volume in liters, b is the cuvette pathlength in cm, and e is the micro molar absorption coefficient of 5-amino-2-nitrobenzoate which is 790 x 10 "6 L • mo 1 ~ ^ • cm" 1 under

the final reaction conditions (see Note 4). Therefore,

Catalytic concen- _ (dA/dt) (2 . 2 x 10 -3)

tration (in U/L) " (7<g x 10 _ 4) (1) (0 . 2 x 10-3)

= 1391 • dA per min

.

Catalytic (activity) concentration in katals per unit volume

(kat/L) can be calculated from the relationship 1.0 U/L = 16.67

nkat/L [37]. Notes :

1. Glycine can be a contaminant of g 1 ycy 1 g 1 yc i ne preparations.

Glycine is an inhibitor of y-GT (competitive with the donor

substrate) and its level must be kept to the minimum

achievable (less than 0.1% on a molar basis). We have found

that currently available lots of this substrate from Sigma

or BMC satisfy this requirement.

65
The pure L-steroi somer is required since the D-form is only

35% as reactive. Other minor contaminants found in preparations of this substrate, L-a-gl utamyl -3-carboxy-

,

4-ni troani 1 ide and 5 -ami no-2-n i troben zoate , inhibit they-GT

reaction minimally or not at all. However, their

proportions in GluNA-C02 preparations must, nevertheless, be

as low as practically achievable (i.e. less than 0.5% and

0.1% by weight, respectively. L-substrate that meets the

above criteria has been available from BMC. Other vendors

of the highly pure substrate may be used. When ordering,

specify pure L substrate.

An analysis report must be obtained from the manufacturer of this substrate. A pale yellow color is obtained when

dissolved in aqueous solution. The absorbance at 410 nm of

a 1 mmol/L solution of this substrate in 150 mmol/L

glycyl glyc i ne (pH 7.9) has been found to be 0.045-0.050.

The reagent solutions should be stoppered and stored either

in a refrigerator at 4 °C (short term) or in a freezer at -20 or -80 °C (long term). No measurable decrease in catalytic activity of y-gl utamyl transferase in serum pools

was obtained after 1 month of storage of reagents 1 and 2 at

4 °C or after 7 months at either -20 or -80 °C. At room temperature, bacterial contamination is a limiting factor.

Moreover, at room temperature spontaneous hydrolysis of L-Y-gl utamyl -3-carboxy-4-n i troan i 1 i de produces a gradual

increase in absorbance at 410 nm. (After 5 days at room temperature an absorbance increase of about 0.3 is produced.)

The rate of spontaneous hydrolysis is even greater in reagent 3 at room temperature.

66
4 . High purity 5-amino-2-nitrobenzoic acid is available from

BMC. This material, which has ^0.2% water by weight,

was found to be chromatographically pure (TLC, GLC), but is greater than 98% pure on the basis of nitrogen analysis.

The value for the molar absorption coefficient of 2-amino-2-nitrobenzoate under the final reaction conditions

has been reported to be 790 !_• mo 1 " ^ • cm" 1 [59].

Comments by Team Leader (L. Shaw)

The stability of Y-GT catalytic activity in SRM 909 was tested in each of the three round robins by Drs. Rauscher and Wahlefeld. After r ec on s t i t u t i on according to the SRM 909

protocol, Y-GT activity was determined 5 times. SRM 909 was then

stored in the refrigerator at 4 °C and 5 replicate assays were performed at the following times; 30-min, 60-min, 2-h and 7-h.

The results of round robin 3 are included in Table 3. The

results show good short-time stability of Y-GT in SRM 909. This

result is, of course, consistent with the well-known stability

(in human serum) of Y-GT.

67
Table 3. Protocol for determination of Y-glutamyltransferase

activity by continuous monitoring - From RR III

DATE REPLICATE Y-GT-ACTI VITY (U/L) FOUND AFTER

NUMBER RECONSTITUTION TIME

30-mi n 6 0 - m i n 2 h 7 h week 1 1 15.1 16.8 18.8 15.5

Haw 1 u ay i 9C1 K P. ID . o1 £ D 1 D . O1 7 A 1 K P. 1 D . O

3 16.4 17.4 17.4 16.8

A 9 P. 9 1 K A 17 A 1 p 0 lo. J 17 K 1 / . D

5 16.0 16.8 16.4 16.0

X 16.1 17.0 17.6 16.5 16.8

C V % 4.0 1.9 4.5 4.9 3.6

week 1 1 15.5 16.0 15.1 15.1 Haw 9 a ay c

9

c

it; 10.0k 1 D . 1 10.1 110.1 K 1 3 15. 5 16.8 16.8 16.0

D 9 K

DC. 0

1 K /I 1 A Q ID . o 1 D . D1 K c 10.0

5 15.5 16.0 15.5 16.4

15.7 16.1 15.6 15.6 15.8

cv % 2.6 4.4 4.5 3.7 3.8

week 2 1 15.1 14.7 16.0 16.0

H T> W 1 day l 9C

i c n 1 0 . J 1 o . u

i k n ID . U

3 15.1 15.5 16.0 16.8

A 9 Q A £ O id . d

1 (T C 17 9 1 K Q

1 0 . O

5 15.5 15.5 16.4 17.2

x~ 1.4 15.3 16.3 16.6 15.9

C V % 1.2 1.2 1.6 1.6 3.7

week 2 1 16.0 16.0 15.1 15.5

day 2 2 15.5 16.0 14.7 16.0

3 15.5 16.0 14.7 16.0

A2 54 4 16.8 16.0 15.1 15.5

5 16.8 17.6 16.0 16. 4

x~ 16.1 16. 3 15.1 15.9 15.9

C V % 2.0 2.2 1.8 1.2 3.9

68
V. STATISTICAL ANALYSIS OF CATALYTIC CONCENTRATION DATA

ON SRM (909) ROUND ROBIN III

The data summarized in this chapter are only from RRIII of the i nter 1 aboratory enzyme study. The raw data were taken

directly from the data reporting forms, each form showing five

replicate values the lab obtained for each of four vials of reconstituted SRM 909. One lab in the ACP team had not participated in RRII, but submitted results for ACP ( LO and HI)

in RRIII. Since these results were highly discordant with the RRIII results from the other labs in the team, they are not included in this evaluation.

The raw data were reduced by taking the arithmetic mean of the replicate values for the catalytic concentration of individual vials. This produced a reduced data set in which,

within a given enzyme -team, each lab is represented by four

values-one for each vial.

Figures 4-8 provide graphical summaries of the reduced data

set for each of the seven enzyme-teams. The data for ACP were summarized separately for the enriched (non-SRM) and unenriched

(SRM 909 ) material (Figures 4a and 4b). The data for the A 1 a AT team were summarized both with and without use of the blank

correction (Figures 5a and 5b). The data corresponding to the 19 individual participating laboratories are coded by letters A through U (I and 0 are not used). In Figs. 4-8, the four values

from each lab are plotted about a horizontal solid line segment

which indicates the mean concentration (i.e. mean of four mean values) for that lab. Distances along these lines have no meaning except to show, from left to right, that the four values

were derived from four individual vials of serum analyzed by that

lab on (week 1, day 1), (week 1, day 2), (week 2, day 1), (week

2, day 2), respectively. A dashed line is plotted at the overall

mean value for the team, and dotted lines are plotted above and below this mean at a distance of 1 "reproducibility" standard

deviation (see Table 4). Table 4 gives a numerical summary of the results in a form which follows ANSI/ASTM E 691-79, "Standard

69
CO LU

>—< O o w ^ . Q Q O I—

CC C/O

00

I—t—I i_ _Q I— CD TJ >- -Q '

—I E

<C 4- C O

Q_ f0

C_5 r- <

•«- S_ \ S- Si- Si.

«-n O O

00 I O 00 I (_) oo JO -Q

Q_ A3 |— J*: (T3 |— _^ (O _l .— < C — <Cc>—

^£> i— i— un i— i— lo

<C -Q JO

O. <T3 ^ T3

00 i— C_> i

CO Q > CO

CM LO LO LO LO <x> CNJ ZD ^t- o i—l (£> CO CNJ CNJ oo CNJ a; LU >

LU CO LU o fr)3<Q J <JD .—1 r-.

LU. ^ 1— —1 OO CNJ co cjo OO oo LO 1—1

LU ro CT> LO

>- CO Q O CNJ i—i

LO i—l CNJ 1—1

oo

cc 1—

<cs:

s:

OO O O CNJ O OO 00 o

=DCO

>- > o I— o CXI co LO LO CNJ CNJ CTi CNJ LO

LU

_i oo —1 > ^ CNJ CNJ OO CNJ CNJ 1—1

CO i—i LU OO

C_) '—* CO O CO

h- LU CNJ <c <

a: w h- • —1 i- o LO oo

o_ <: Q OO oo CT> <«o oo LO

LU 1- 2 i—i O oo co

o_ OO i—i o cn co LO oo *£> o CNJ ^ LU \

ai zs o o o O O cr, LO o

70
0.30- ^ 0.28

0.26-

0.24

25 0.22

0.20-

0. 18- Lab A Lab B Lab C

Figure 4a.

^ 5.6-

5.4-

5.2-

5.0-

4.8-

4.6- Lab A Lab B Lab C

Figure 4b, Figures 4a and 4b: Summary of ACP results for low activity (SRM 909) material

(4a), and for enriched material (4b), showing the mean value per vial (closed

circle, •) , the mean values per laboratory (horizontal solid lines, ), the mean value for all laboratories (dashed line, ), and the 1-sigma

"reproducibility" standard deviation limits (dotted lines, ).

71
: ^

L

1 1 1 1 1 1 1 1 11 ill i Lab F Lab G Lab J Lab K Lab L

Figure 5a.

29

27

23-

21 Lab G Lab J Lab K Lab L

Figure 5b,

Figures 5a and 5b: Summary of results for AlaAT, with blank correction (5a) and without blank correction (5b), showing the mean value per vial (closed

circle, •) , the mean values per laboratory (horizontal solid lines, ), the mean value for all laboratories (dashed line, ), and the 1-sigma

"reproducibility" standard deviation limits (dotted lines, ).

72
f

" -

, -J,

u IT

ii i i i i i i i i i i i i i i i i iiii

Lab C Lab D Lab E Lab F Lab G Lab H

Figure 6a.

<Ca

Q 28—1 I'll iiii iiii iiii iiii

Lab D Lab G Lab J Lab M Lab N

Figure 6b. Figures 6a and 6b: Summary of results for ALP (6a), and for AspAT (6b), showing

the mean value per vial (closed circle, •) , the mean values per laboratory (horizontal

solid lines, ), the mean value for all laboratories (dashed line, ), and the 1-sigma "reproducibility" standard deviation limits (dotted lines, ).

73
140

130-

120-

110

90

XJL

Y

±

Lab P Lab Q Lab R

Figure 7a. Lab S

~ 260- JN

D^ 250 H

240

230-

220-

210 rV

1 1 1 1 ii i i i i iiii —

Lab C Lab G Lab Q

Figure 7b. Lab T

Figures 7a and 7b: Summary of results for CK (7a), and for LDH (7b), showing the mean value per vial (closed circle, •) , the mean values per laboratory (horizontal

solid lines, ), the mean value for all laboratories (dashed line, ), and the 1-sigma "reproducibility" standard deviation limits (dotted lines, ).

74
0h

0

I |5_J 1 i i i i i i i

i i i i i i i i i i i i Lab F Lab J Lab L Lab P Lab U

Figure 8: Summary of results for v-GT , showing the mean value per vial

(closed circle, •) , the mean values oer laboratory (horizontal solid lines,

), the mean value for .all laboratories (dashed line, ), and the 1-sigma "reproducibility" standard deviation limits (dotted lines, ).

10-

Figure 9: Summary of all results in terms of percent deviations from enzyme team means. Each laboratory is represented by its mean value

(closed circle , •)

.

75
Practice for Conducting an Interlaboratory Test Program to Determine the Precision of Test Methods."

Table 4, column (1), contains the overall mean value of concentration in U/L ( ymo 1 Mni n

" ^ • L" ^ ) . The "repeatability"

standard deviation (S r

) and coefficient of variation (cv) are given in column (2). The S r value is obtained by calculating the

standard deviation of the four values representing each lab and

pooling the results across labs for each enzyme-team.

The standard deviation S[_ and corresponding cv are given in column (3). S[_ estimates the bet we en-lab component of variation

from a random-effects model, one-way analysis of variance. This

quantity represents the variability due strictly to between-lab

differences, after the wi thin-lab variability is taken out. The

formula is:

S L = / S(Xj-X)2 - (S r ) 2 \ P-l 4

where

p = number of labs in a given enzyme-team

X-j = mean value for lab i (i = l,...,p)

X = mean value among all labs in an enzyme-team.

The "reproducibility" standard deviation in column (4)

combines the within-lab and between-lab standard deviations. It represents the typical variability between results obtained by two different laboratories analyzing one vial each under the

present protocol (which involves averaging five replicate values).

The corresponding coefficient of variation for reproducibility

(as a percent of the mean value) is given in parentheses.

Figure 9 presents a graphical summary of some of the information

in Table 4. The values plotted against the vertical axis are percent deviations from the enzyme-team means. All enzyme-teams

are represented on this graph. The values plotted represent

individual lab means, although the labs are not individually

identified. The formula for the "percent deviations" plotted is: 76
% deviation = (lab, mean) - (enzyme-team mean) x \ q

q

(enzyme-team mean)

These statistical results show that very good interlaboratory reproducibility of results may be achieved using the

analytical methods described here. Except for measurements of the unenriched and therefore exceptionally low acid phosphatase

level, the teams achieved reproducibility standard deviations of less than 9% for the enzyme catalytic (activity) concentrations.

We emphasize that the mean values (Table 4) for the

catalytic activity concentrations of the seven enzymes in SRM 909 are consensus values, obtained by the cooperating laboratories

through the use of the protocols described in this monograph.

The assigned values for the catalytic (activity) concentrations

in SRM 909 are "method dependent values" given for information

purposes only. They are not certified by NBS.

In order for SRM users to achieve agreement with the

assigned values, they must follow exactly 1) the serum

recons-titution protocol at 0 °C for 1 - h and 2) exactly adhere to every detail of the analytical methods protocols described in this document.

The long-term stability of the assigned enzyme catalytic

(activity) concentration values also is not characterized.

Several of the enzyme team leaders are monitoring the stability

of the activity of these enzymes. Results obtained by the team

leaders in the winter of 1982-1983 for all enzymes (except

ACP-low) confirmed the values found in column (1) of Table 4 within the reproducibility standard deviations shown. SRM 909 users will be kept informed of any stability problems that are identified.

77
VI. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

This study of the catalytic concentration of seven enzymes

in SRM 909 was made possible by the voluntary cooperation of 28 scientists and their staffs in 17 U.S. laboratories and 3 elsewhere (in Canada, Norway and West Germany).

Two round robins like the final, third round robin, which

was the one used to assemble data for assigning the catalytic

(activity) concentration values, gave the participants pragmatic

preliminary experience with the protocols and statistical design

for these analyses. The reference type method protocol was chosen by each Team Leader who was very alert to the current

status of IFCC and AACC recommendations for the enzyme reference

method for which he (or she) was responsible. Although not entirely identical to IFCC methods, our methods are generally

little different from them in final assay conditions. For the

phosphatase and lactate dehydrogenase, no IFCC recommendations

existed at the initiation of this study in 1979 to guide our selections; hence, these are exceptions.

The reaction temperature set-point was 29.77 °C, the gallium

melting-point. NBS/SRM 1968 gallium cells were sent to each Team

to assure standardization at 29.77 °C reaction temperature. The

fact that 29.77 °C was used as the reaction temperature set point

in this study should not be taken to signify that either NBS or

all of the cooperating team leaders and members endorse this

temperature as the optimum for determining enzyme catalytic

activities. We used this means for standardizing reaction

temperature to achieve interlaboratory reproducibility of enzyme

catalytic concentration values. The temperature set point was thus rigorously established and controlled for our study.

The mean values for the catalytic (activity) concentration

of the seven enzymes in SRM 909 represent method dependent values

obtained through the use of a cooperating laboratory network.

The assigned values are given for informational purposes only,

they are not certified by NBS.

78
Information on changing the reaction temperature from 29.77

to 30 or 37 °C was not obtained as a mu 1 t i 1 abor atory cooperative

effort. Such information was added subsequently to make the SRM 909 more useful. The information on "temperature factors"

for SRM 909 enzymes was gathered only in the laboratories of the team leaders so identified and have not been checked or endorsed

by the other scientists in the cooperating laboratories or by NBS personnel

.

The main conclusion of this study is that excellent

interlaboratory reproducibility of results (interlaboratory

standard deviations of less than 10%) may be achieved for

measurement of enzyme catalytic (activity) concentrations of stable, reference type materials, if participating laboratories

adhere exactly to a rigorously defined reconst i tut i on protocol

and reference method protocol including the assurance of high

quality and uniform reagent control.

Information obtained in this study should also be useful in the development of future NBS human serum SRM's. The study

participants made the following Recommendat i ons :

1. When securing serum material for certification, NBS procurement contracts should specify the enzyme additives in terms of species, tissue, purity, stability and activity per mL

or mg protein.

2. The supplier should give details on all enzyme

additives (as above).

3. Acceptable enzyme sources are as follows: ACP seminal fluid, ALP-beef kidney or liver (not placenta or intestine of any species), porcine heart for ASAT, ALAT,

CK and beef or porcine kidney for y-GT.

The authors also recommend that all reference enzyme

activity measurements should be made using the reference methods

described in this monograph until they may be replaced by

-human

LDH, and

79
reference methods adopted by the International Federation for Clinical

Chemistry and/or the National Reference System for Clinical Chemistry.

The authors further recommend that temperature factors relating enzyme

activity at 29.77 °C--to activities at 25, 30 and 37 °C be established

as part of any future study.

80
REFERENCES

CI]

[2]

[3]

[4]

[5]

[6]

[7]

C8]

Shewart, W . A. Economic control of quality of manufactured product. New York: Van Nostrand; 1931

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statistical concepts and procedures. Natl. Bur. Stand.

(U.S.) Spec. Publ. 300; 1969.

Eisenhart, C. Realistic evaluation

accuracy of instrument calibration

Bur. Stand. (U.S.) 67C(2): 161

of the precision and

systems . J . Res . Nat 1 187; 1963 Apri 1 -June. Cali, J. P. et . a 1 . The role of standard reference

materials in measurement systems. Natl. Bur. Stand. (U.S.)

Monogr. 148; 1975.

Uriano, G. A.; Cali, J. P. Role of reference materials

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ACS Symposium Series 63, American Chemical Society,

Washington, DC; 1977.

Cali, J. P chemi s tryA systematic

Med . I ns t r u m

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(8): 17; to accuracy

1974 . in clinical

Uriano, G. A.; Gravatt, C. C. The role of reference

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Crit. Rev. in Anal. Chem. (7): 362; 1977.

[9] Cali, J. P.; Stanley, C. L. Measurement compatibility and

standard reference materials. Ann. Rev. Mater. Sci. (5):

329; 1975.

[10] Huntoon, R. D. Standard reference materials and meaningful

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[11] Huntoon, R. D. Science. (158): 67; 1967.

[12] Dorsey, N. E.; Eisenhart, C. Sci. Mon. (77): 102; 1953.

Also paper 1.3 in NBS Spec. Publ. 300.

[13] Wohlgemuth, J. Uber eine neue methode zur quatitativen

bestimmung des diastatischen ferments. Biochem. Z. (9): 1; 1908.

[14] Bowers, G. N., Jr.; McComb, R. B.; Burnett, R. W.

Begansky, T. Annual report for clinical chemistry fiscal

year 1981. Department of Pathology, Hartford Hospital.

Submitted December, 1981 to the Chairman, Dr. W. Bronson.

81
[15] Elevitch, F. R., ed. Analytical goals in clinical

chemistry. Proceedings of the 1976 Aspen Conference;

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78-79.

[16] Horder, M. Personal communication to Bowers, G. N. Jr. [17] Boutwell, J. H., ed . A national understanding for the

development of reference materials and methods for

clinical chemistry. Am. Assoc. Clin. Chem.; Washington,

DC; 1978.

[18] Bowers, G. N., Jr.; Cali, J. P.; Elser, R.; Ewen, L. M.

;

McComb, R. B.; Rej , R.; Shaw, L. M. Activity measurements

for seven enzymes in lyophilized human serum SRM No. 909.

Clin. Chem. (26): 969; 1980.

[19] IFCC methods for the measurement of the catalytic

concentration of enzymes, Part 2, IFCC method for

aspartate aminotransferase ( L-Aspartate : 2-oxog 1 utar ate aminotransferase, EC 2.6.1.1); Revised conditions from

prior publication in Clin. Chim. Acta (70): F19 - F42

following page 470; 1976. Stage 2.2 Provisional

Recommendtion - 1979; Clin. Chim. Acta (80): F21 - F22

following page 39 4; 1977.

[20] IFCC methods for the measurement of the catalytic

concentration of enzymes, part 3; IFCC method for alanine

aminotransferase (L-Alanine: 2-oxog 1 utarate aminotransferase, EC 2.6.1.2); Stage 1.2 - Provisional

Recommendation - 1979; Clin. Chim. Acta (105): 147F-154F;

1980.

[21] IFCC methods for the measurement of the catalytic

concentration of enzymes, part 4; IFCC method for y-g 1 utamyl transferase (y-g 1 utamy 1 ) - peptide: amino acid

Y-gl utamyltransf erase , EC 2.3.2.2); Stage 1, Draft 3 Recommendation - 1981; obtained from EPE/IFCC M . Horder

(chairman), Dept. of Clinical Chemistry, Univ. of Odense,

Odense , Denmark . [22] IFCC methods for the measurement of the catalytic

concentration of enzymes, part 5; IFCC method of alkaline

phosphatase ( or t hop hos p hor i c monoester phosphohydro 1 ase (alkaline optimum) EC 3.1.3.1); Stage 1, Draft 3 Recommendations - 1981; ibid . [23] IFCC methods for the measurement of the catalytic

concentration of enzymes, part 7; IFCC method for creatine

kinase (ATP: creatine phosphotransferase, EC 2.7.3.2);

Stage 1, Draft 2 Recommendation - 1981; ibid .

82
[24] IFCC methods for the measurement of the catalytic

concentration of enzymes, part 1, General considerations

concerning the determination of the catalytic concentration of an enzyme in the blood serum or plasma of man; Stage 4 - Approved Recommendation - 1978; Clin. Chim. Acta (98): 163-174F; 1979.

[25] IFCC methods for the measurement of the catalytic

concentration of enzymes, part 6; IFCC methods for the

preparations of reference enzyme materials; Stage 1, Draft

2, Recommendations - 1981; ibid . [26] Mangum, B. W.; The gallium melting-point standard: its

role in our temperature measurement system. Clin. Chem.

(23): 711-718; 1977.

[27] Bowers, G. N . , Jr.; Inman, S. R. The gallium

melting-point standard: its application and evaluation

for temperature measurements in the clinical laboratory.

Clin. Chem. (23): 733-737; 1977.

[28] Ewen, L. M. ; Spitzer, R. W. Improved determination of prostatic acid phosphatase (sodium t hymo 1 p h t h a 1 e i

n

monophosphate substrate). Clin. Chem. (22): 627-632;

1 976 . [29] Bowers, G. N., Jr.; Onoroski, M . ; Schifreen, R. S.; Brown,

L. R., Klem, R. E., and Ewen, L. M. S pec t r op h o t o me t r i

c

and liquid-chromatographic studies of t hymo 1 ph t h a 1 e i

n

monophosphate. Specifications for high-quality substrate

for the measurement of prostatic acid phosphatase

• activity. Clin. Chem. (27): 1372-1377; 1981.

[30] Kessner, A.; Woodard, E. J.; Bowers, G. N . , Jr. Effects of substrate concentration on the results of determination of prostatic acid phosphatase with thymo 1 phthal ei

n

monophosphate. Clin. Chem. (29): 148-151; 1982.

[31] McComb, R. B.; Bowers, G. N . , Jr.; Posen, S. Alkaline

phosphatase. New York: Plenum Publishing Corp.; 1979.

903-907 . [32] Bowers, G. N . , Jr.; McComb, R. B.; Christensen, R. G.; Schaffer, R. High-purity 4-nitrophenol: purification,

characterization, and specifications for use as a spectrophotometry reference material. Clin. Chem. (26):

724-729; 1980.

83
[33] Bowers, G . N . , Jr.; McComb, R. B. and Upretti, A. 4-Ni trophenyl phosphate - Characterization of high-purity

materials for measuring alkaline phosphatase activity in human serum. Clin. Chem. (27): 135-143; 1981.

[34] Re j , R.; Bret aud i ere , J. P.; Jenny, R. W. ; Jackson, K.

Y. Measurement of alkaline phosphatase activity:

characterization and identification of an inactivator in 2-ami no-2-methyl -1-propanol . Clin. Chem. ( 27 ): 1401 -1409 ; 1981

.

[35] McComb, R. B.; Gay, R . J. A comparison of reduced-NAD

preparations from four commercial sources. Clin. Chem.

(14): 754-763; 1968.

[36] Roy, A. V.; Bhower, M. E.; Hayden, J. E. Sodium

thymo 1 pht h a 1 e i n monophosphate: a new acid phosphatase

substrate with greater specificity for the prostatic

enzyme in serum. Clin. Chem. (17): 1093-1102; 1971.

[37] Bechtler, G.; Haeckel, R.; Horder, N . ; Kuffer, H . ; Porth, A. J. Provisional Guidelines (1980) for the

calculation and evaluation of reaction rates in clinical

chemistry. IFCC draft document of August 2, 1980 from

Expert Panels on Instrumentation and Expert Panel on Enzymes

.

[38] Bowers, G. N . , Jr.; McComb, R. B., Measurement of total

alkaline phosphatase activity in human serum. Clin. Chem.

(21): 1988-1995; 1975, AACC Selected Method.

[39] Bowers, G. N . , Jr.; McComb, R. B.; Kelley, M. L. Effect of magnesium on alkaline phosphatase activity in human serum. Clin. Chem. (23): 1149-1150; 1977. Abstract.

[40] Schifreen, R. S.; Burnett, R. W. Effect of wavelength

error and spectral bandwidth on measurement of alkaline

phosphatase activity. Clin. Chem. (25): 429-431; 1979.

[41] Re j , R., et a 1 . Recommendations of the Alanine

Aminotransferase Study Group, Subcommittee on Enzymes,

Committee on Standards, American Association for Clinical

Chemistry. Clin. Chem. (26): 1023; 1980 and personal

communication from author.

[42] Rodgerson, D. 0.; Tetz, N. W. ; Burtis; C. A., Rej , R.; Rinker, A. A. A recommended method for the measurement of aspartate aminotransferase activity in human serum. Study

Group on ASAT, Subcommittee on Enzymes, American

Association for Clinical Chemistry. Clin. Chem. (26):

1023-1024; 1980.

84
[43] The Committee on Enzymes of the Scandinavian Society for

Clinical Chemistry and Clinical Physiology. Recommended

method for the determination of creatine kinase in blood.

Scand. J. Clin. Lab. Invest. (39): 1-5; 1979.

[44] Davis, R. A., Jr.; Ward, J. U.; Fellner, W. H . ; Effect of temperature and timing on the reconstitution of creatine

kinase in the NBS reference sera no. 909. Clin. Chem.

(27): 1028; 1981. Abstract.

[45] Szasz, G . ; Gruber, W . ; Bernt, E. Creatine kinase in serum: 1, determination of optimum reaction conditions,

Clin. Chem. (22): 650-656; 1976.

[46] Recommendation of the Study Group on Creatine Kinase,

Subcommittee on Enzymes, Committee on Standards, American

Association for Clinical Chemistry. Personal

communication with R. Elser; 1979.

[47] McComb, R. B.; Bond L. W.; Burnett, R. W.; Keech, R. C;

Bowers, G. N., Jr. Determination of the molar

absorptivity of NADH. Clin. Chem. (22): 141-150; 1976.

[47a] Whitner, V. S., Morin, L. G., McKneally, S. S., and Sampson, E. J. Stability of creatine kinase-3 in lyophilized materials. Clin. Chem. (28): 41; 1982.

[48] Buhl, S. N . ; Jackson, K . Y.; Graffunder, B. Optimal

reaction conditions for assaying human lactate

dehydrogenase pyr u v at e - to - 1 ac t at e at 25 , 30 , and 37 °C. Clin. Chem. (24) : 261-266; 1978.

[49] Howell, B. F . ; McCune, S., Schaffer, R. Lactate-topyruvate or pyr u v at e - to - 1 ac t at e assay for lactate

dehydrogenase: a re-examination, Clin. Chem. (25):

269-272; 1979.

[50] Gay, R., McComb; R. B.; Bowers, G. N .

, Jr. Optimum

reaction conditions for human lactate dehydrogenase

isoenzymes as they affect total lactate dehydrogenase

activity. Clin. Chem. (14): 740-753; 1968.

[51] Scandinavian Society for Clinical Chemistry and Clinical

Physiology. Recommended methods for the determination of four enzymes in blood. Scand. J. Clin. Lab. Invest. (33):

291-306; 1974.

[52] Statland, B. E.; Louderback, A. L., Nonlinear regression

analysis approach for determining "true" lactate

dehydrogenase activity in serum with the centrifugal

analyzer ( "Rotoc hem" ) . Clin. Chem. ( 18 ): 845-849; 19 72.

85
[53] Burke, R. W. ; Deardorff, E. R.; Menis, 0. Liquid

absorbance standards. J. Res. Natl. Bur. Stand. (U.S.).

76(A): 469-482; 1972.

[54] Orlowski, M. ; Meister, A. Isolation of y-glutamyltranspept i dase from hog kidney. J. Biol. Chem. (240):

338-347; 1965.

[55] Szasz, G. A Kinetic photometric method for serum

Y-gl utamyl transpepti dase . Clin. Chem. ( 15 ): 1 24-136 ; 1969 . [56] Shaw, L. M . ; London, J. W. ; Fetterolf, D.; Garfinkel,

D., y-Glutamyltransferase: kinetic properties and assay

conditions when y-glutamyl-4-nitroanilide and its

3-carboxy derivative are used as donor substrates. Clin.

Chem. (23): 79-85; 1977.

[57] Solbert, H. E.; Theodorsen, L.; Stromme, J. H. y-Glutamyltransferase in human serum: an analysis of kinetic models. Clin. Chem. (27): 303-307; 1981.

[58] London, J. L.; Shaw, L. M. ; Theodorsen, L . ; Stromme, J. H. The application of response surface methodology to the

Y-gl utamyltransf erase assay. Clin. Chem. (28): 1140-1143;

1982 . [59] Wu, A. H. B.; Bowers, G. N., Jr. The linear molar

absorptivity coefficient of 5-amino-2-nitrobenzoate under

the final reaction conditions of the IFCC method.

Personal communication between authors and Dr. Shaw.

[60] Vassault, A.; Maire, I.; Seb i lie, L.; Bozon, D. Recommendations for determining the catalytic concentration of lactate dehydrogenase in human serum at +30 °C. Ann. Biol . Clin. ( 40 ) : 160; 1982 .

86
U. S. Department of Commerce Malcglpfe&aldrige „ _ rt _ .. _ _ .. OT t appendix

National Bureau of Standards SEE ADDENDUM

Ernes. ^bfr.^tor (/\ REVISED SRM 909 CERTIFICATE WILL BE AVAILABLE SrVERAL MONTHS AFTER

THIS MONOGRAPH IS PRINTED. COPIES OF THE REVISED CERTIFICATE CAN BE OBTAINED FROM NBS.)

jNattmtal ^mtmx of ^tnndnvds

Certificate

Standard Reference Material 909

Human Serum

This Standard Reference Material (SRM) is intended for use in evaluating the accuracy of clinical procedures for the determination of specified constituents in serum, in calibrating instruments and equipment used in these procedures, and

in validating working or secondary reference materials. CERTIFIED VALUES OF ANALYTES: The concentrations of the analytes were determined by methods having the

highest accuracy, i.e., definitive methods. The certified values are given in two ways, depending on whether

reconstitution is done with or without weighing the freeze-dried serum contents of a vial. Concentration values having

smaller uncertainties may be obtained by weighing the freeze-dried serum and multiplying this mass by the certified concentration values of the analytes per unit mass of dried serum. These certified values appear in Table 1. When the contents of a vial are not weighed, the mean values for the certified concentrations and their uncertainties should be used. These values, which apply to all vials of reconstituted SRM 909, appear in Table 2. NOTICE AND WARNINGS TO USERS

USE: HANDLE AS IF CAPABLE OF TRANSMITTING HEPATITIS! Although this product was tested with

licensed third generation reagents and found nonreactive for the presence of hepatitis B surface antigen (HBsAG), no known test method can offer assurances that products derived from human blood will not transmit hepatitis. SRM 909 IS INTENDED FOR "IN VITRO" DIAGNOSTIC USE ONLY. STORAGE: The freeze-dried serum should be stored in a refrigerator at a temperature between 2 and 8 °C. It should not be frozen nor exposed to sunlight or ultraviolet radiation. Under the recommended storage conditions, this SRM is expected to be stable for at least one year; should evidence indicate a more rapid degradation of the certified properties, purchasers will be notified by NBS. The material is not certified for use after one year from date of purchase. The statistical analysis of the data was performed by K.R. Eberhardt of the Statistical Engineering Division. The overall direction and technical measurements leading to the certification were under the chairmenship of E. Garner, H.S. Hertz, T.J. Murphy, D.J. Reeder, R. Schaffer and E. White V. The technical and support aspects concerning the preparation, certification, and issuance of this Standard Reference

Material were coordinated through the Office of Standard Reference Materials by R. Alvarez. Washington, D.C. 20234 George A. Uriano, Chief September 15, 1980 Office of Standard Reference Materials January 13, 1981 (revision) (over) October 8, 1982 (revision) 87
Instructions for Use HANDLE AS IF CAPABLE OF TRANSMITTING HEPATITIS! SRM 909 is supplied as a set of six vials of

freeze-dried human serum and six vials of high-purity, diluent water for use in reconstituting the serum. Two procedures for reconstituting SRM 909 are described. Selection of a procedure depends on the uncertainties required for the concentrations of the analytes. If lower uncertainties than those shown in Table 2 are required, the

freeze-dried serum contents of a vial must be weighed as described in Procedure A. Procedure A. Reconstitution with weighing of the freeze-dried serum: Completely remove label and adhesive by

scraping the vial and then wiping it with a tissue moistened with a solvent, such as acetone or ethanol. Scratch an

identification on vial. Remove metal closure and lightly tap bottom of vial to dislodge any serum particles adhering to the

stopper. Dislodge stopper to equalize air pressure, then replace, wipe surface of vial, and weigh to the nearest 0. 1 mg.

(Use a clean empty vial of the same size as a tare.) Carefully remove stopper to avoid possible loss of serum particles. Use

a Type 1 Class A volumetric transfer pipet or other dispenser of known accuracy to slowly add 10.00 ± 0.02 mL of the

diluent water at 20 - 25 °C to the sides of the vial while continually turning the vial. Replace stopper, swirl vial two or

three times, and let stand for 10 minutes. Mix contents by gently swirling, let stand for approximately 30 minutes, swirl again, let stand 10 minutes, and finally invert the vial several times. Do NOTshake vigorously because this will cause

frothing. Total time for reconstitution is approximately 1 hour. After reconstitution, use contents as soon as possible. If not used immediately, store between 2 and 8 °C until ready for use, preferably within 8 hours. After the reconstituted serum has been used, clean and dry the vial and its stopper. Reweigh after replacing stopper. The tare is reweighed at the same time to compensate for changes in temperature and humidity. The mass of dry serum is given by the difference between the original and final weighings. The concentration of an analyte, after the contents of a vial is weighed and reconstituted with 10.00 mL of diluent water,

is calculated by multiplying the mass of freeze-dried serum, in grams, by the certified concentration of the analyte per gram of freeze-dried serum given in Table 1. For example, if the mass of freeze-dried serum in a vial is 0.8703 g, the concentration of uric acid in this vial would be: 0.5681 mmol-L'V1 * 0.8703 g = 0.4944 mmol/L.

The uncertainty is also calculated similarly and for this example would be: 0.0050 mmol-L~V x 0.8703 g = 0.0044 mmol/L.

Table 1. Certified Analyte Concentrations and Uncertainties per Gram of Freeze-Dried Serum after Reconstitution of SRM 909 according to Procedure A. Analyte

1 Concentration,2 per gram, Uncertainty,

3 per gram, mmol L

_1 g

_1 mmolL ' g

1 Calcium

3 3.560 ±0.013

Chloride

3 128.0 ± 1.5 Glucose

3 7.74 ±0.11

Lithium

3 1.945 ± 0.033 Potassium3 4.1546 ± 0.0098

Uric Acid

3 0.5681 ±0.0050

1. Analytical Methods

a. Isotope dilution mass spectrometry.

2. The certified concentrations apply to reconstituted serum at room temperature (20-25 °C).

3. The uncertainties represent 95%/ 99% statistical tolerance limits for the concentrations per gram of dry serum reconstituted with 1 0.00 mL of high-purity water. The tolerance limits are constructed so that, at a confidence level of 95%, they will contain

the concentration/ mass ratios for at least 99% of the vials.

Page 2 SRM 909

88
Procedure B. Reconstitution of SRM 909 without weighing freeze-dried serum: Remove metal closure and lightly tap bottom of vial to dislodge any serum particles on stopper. Carefully remove stopper to avoid possible loss of serum

particles. As described in Procedure A, reconstitute with 10.00 ± 0.02 mL of the diluent water, and use immediately or

store between 2 and 8 °C until ready for use, preferably within 8 hours. Table 2 gives the certified concentrations of the

constitutents and the tolerance limits for use with this procedure. Table 2. Certified Concentrations and Uncertainties for Analytes in Reconstituted SRM 909 for Use with Procedure B. Analyte

1

Calcium4

Chloride

3

Glucose

4

Lithium

3

Potassium

3

Uric Acid

3

1. Analytical Methods:

a. Isotope dilution mass spectrometry.

2. The certified concentrations apply to reconstituted serum at room temperature (20-25 °C).

3. The uncertainties represent 95%/ 95% statistical tolerance limits for the concentrations, and reflect the combined

effects of measurement imprecision and the variability of the mass of dry serum among vials. They are constructed so

that, at a cpnfidence level of 95%, they will include the concentrations of at least 95% of all vials of SRM 909, when

reconstituted according to Procedure B. The major source of uncertainty in the certified concentrations shown in Table 2 is caused by vial-to-vial variability in the mass of freeze-dried serum. Information on the relation between mass variability and concentration was obtained

directly during the course of experimentation by weighing the contents of each vial as an intermediate step in determining

the concentration of an analyte. Supplementary information on the variability in mass was obtained from a differential refractometry experiment in which over 170 vials were analyzed. The data obtained from these two sources indicate that the distribution of masses is non-Gaussian and skewed in the direction of high mass. The largest two masses encountered were 5.2% and 2.7% above the mean mass, 0.8469 g. All other masses were contained in an interval from 1.5% below to 1.8% above the mean.

Source of Material: The human serum for SRM 909 was processed, vialed, and packaged by Hyland Division, Travenol

Laboratories Inc., Round Lake, Illinois. Analyses were performed in the NBS Center for Analytical Chemistry by I.L. Barnes, K. A. Brletic, R.G. Christensen, A. Cohen, J.W. Gramlich, W.R. Kelly, L.R. Machlan, J.R. Moody, L.J. Powell, L.T. Sniegoski, and M.J. Welch (Research

Associate, College of American Pathologists). This Standard Reference Material has been measured and certified at the Laboratories of the National Bureau of Standards, Gaithersburg, Maryland. All inquiries should be addressed to: Office of Standards Reference Materials Room B311, Chemistry Building National Bureau of Standards Washington, D.C. 20234 The date of issuance and certification of this Standard Reference Material is September 15, 1980.

Page 3 SRM 909 89

Concentration , mmol-L

-1

3.02

108

6,55

1.65

3.52

0.481

Uncertainty

3

, mmol-L

-1 +0.17 -0.06 +7

-3

+0.46

-0.21 +0.12 -0.06

+0.19 -0.06

+0.031 -0.012
U. S. Department of Commerce

Addendum to

Rational ^mtm of jltamlartte

Certificate of ^nalgsts

Standard Reference Material 909

Human Serum

ADDITIONAL CERTIFICATION

The following certified values are to be added to Tables 1 and 2. Table 1. Certified Analyte Concentrations and Uncertainties per Gram of Freeze-Dried Serum after Reconstitution of SRM 909 according to Procedure A. Analyte

1 Concentration,

2per gram, Uncertainty,

3per gram, mmolL^g 1 mmol-L

-1 g_1

Cholesterol* 4.346 ±0.030 Magnesium8 1.425 ±0.072

Uncertainties for Analytes in Reconstituted SRM 909 for Use with Procedure B. Uncertainty

3

,

Table 2. Certified Concentrations and

Analyte

1

Cholesterol

8

Magnesium8

Washington, D.C. 20234 November 14, 1980

3.68

1.21

+0.22

-0.08 +0.14

-0.10

George A. Uriano, Chief

Office of Standard Reference Materials

90
INFORMATION ON ENZYMES

Enzymes - The catalytic concentrations in terms of U / L* of seven enzymes were determined in an interlaboratory study, and are given in Table 3. The results are in a form that follows ANSI/ASTM E691-79 "Standard Practice for Conducting an Interlaboratory Test Program to Determine the Precision of Test Methods".

Because of the state-of-the-art for the determination of enzymes, their concentrations cannot be stated on an accuracy

basis. The enzyme values shown below are material and method-dependent. Thus, any laboratory using this SRM and

the catalytic concentrations of these enzymes must follow the methods as described. The values given are consensus

values, not certified by NBS, and were obtained through the interlaboratory study. They are provided for information

only.

Catalytic concentration is expressed in terms of International enzyme units per liter (U/L) where U is given as micromoles of substrate converted per minute. (1.0 U equals 16.67 nkat.) Table 3 Catalytic Concentrations of Enzymes in Reconstituted SRM 909

Enzyme Number of Laboratories

Overall Mean, U/LRepeatability

Std. Dev.

Reproducibility'

Std. Dev. Acid Phosphatase

3 3 0.23 0.02 0.04 Alkaline Phosphatase 6 75.4 1.9 2.2 Alanine Aminotransferase 5 24.2 0.8 1.8 Aspartate Aminotransferase 5 30.7 0.4 0.9 Creatine Kinase 4 123.0 9.7 10.2 Lactate Dehydrogenase 4 229.2 5.0 9.6 Y-Glutamyltransferase 5 16.4 0.3 0.4

1. Repeatability (within-laboratory standard deviation) for each enzyme was obtained by calculating the standard

deviation of the four daily mean values (5 replicates) obtained in each laboratory and pooling the results across

laboratories!

2. Reproducibility standard deviation combines the within-laboratory and between-laboratory standard deviations. It represents the typical variability between results obtained by two different laboratories analyzing one vial each under the present protocol (which involves averaging five replicate values).

3. Native catalytic concentrations. Prostatic acid phosphatase was not added to the serum base.

Reconsitution Procedure for Enzyme Determinations : The enzyme concentrations listed inTable 3 were measured after the lyophilized serum had been reconstituted with ice-cold water for about one hour as described below. This procedure

is different from those given on pages 2 and 3 of the Certificate.

1. Remove the vials of SRM 909 lyophilized serum and diluent water from the refrigerator.

2. Immerse the vial of water in an ice-water slush for 5 minutes. Water-ice should contact the vial, not air-ice.

3. Tap the SRM 909 vial lightly on a solid surface to dislodge any serum particles adhering to the stopper. Remove

metal cap and stopper, being careful to avoid loss of serum particles.

4. Transfer 10.0 mL of the cold diluent water* to the vial of serum as follows: Using a class A, 10-mL pipette at room

temperature, dispense the water slowly to the side of the vial while continually turning the vial to wet all serum

particles and enhance dissolution.

5. Replace the stopper, swirl to wet beads of serum, gently invert vial 10 times, and immerse the vial in ice water. Repeat the gentle swirling and inverting operations at 10 minute intervals for one hour, always returning the vial to the ice-water bath.

6. Inspect the reconstituted SRM 909 visually. Undissolved serum particles should not be evident. Invert the reconstituted SRM gently several times before use for specific enzyme procedures.

* A slight excess over 10.0 mL is actually delivered in this procedure. For enzyme concentration measurements, the error

is not considered significant.

91
Enzyme Methods - The methods used in this study are outlined below. Details of the methods and the experience with

their use during the interlaboratory study are to be written for publication in the NBS 260 Series. (This series is used to disseminate information on the preparation, measurement, certification, and use of NBS SRM's.) All methods use a

reaction temperature of 29.77 Q C, which can be verified by using a Gallium Melting Point Cell, SRM 1968 (1).

1. Acid Phosphatase fOrthophosphoric monoester phosphohydrolase (acid optimum), EC 3. 1.3.2]. The catalytic concentration of acid phosphatase in U/L is determined using thymolphthalein monophosphate as

substrate. Enzymatic hydrolysis at pH 5.4 yields a colorless product, thymolphthalein, which becomes a self-indicating chromogen (A= 595 nm) when the enzymatic reaction is terminated by the addition of alkali. The method of Ewen and

Spitzer (2) is employed with the following modifications: a) the reaction temperature is 29.77 °C and b) multiple tests are performed at 15, 30, 45, and 60 min. The final reaction conditions are: Reaction temperature 29.77 °C pH 5.4 Acetate buffer 0.15 mol/L

Thymolphthalein monophosphate 1.0 mmol/L

Brij-35 1.5 g/L Volume fraction (sample/total) 0.083 (1:12)

2. Alkaline Phosphatase [Orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1]. The catalytic concentration of alkaline phosphatase in U/L is measured by following the liberation of 4-nitrophenol from the substrate 4-nitrophenyl phosphate in the transphosphorylating buffer, 2-amino-2-methyH-propanol. It utilizes the method of Bowers and McComb (3) with the single modification of changing the reaction temperature from

30 °C to 29.77 °C. The final reaction conditions are: Reaction temperature 29.77 °C pH 10.5 4-Nitrophenylphosphate 16.0 mmol/L

2-Amino-2-methyl-l-propanol 1.0 mol/L

Magnesium acetate' 1.0 mmol/L

Volume fraction (sample/ total) 0.0164 (1:61)

3. Alanine Aminotransferase (L-Alanine: 2 oxoglutarate aminotransferase, EC 2.6.1.2). The catalytic concentration of alanine aminotransferase (AlaAt) in U/L is measured by coupling pyruvate production

with reduced nicotinamide adenine dinucleotide (N ADH) and lactate dehydrogenase (LDH). It utilizes the IFCC/ EPE

and AACC/ AlaAt Study Group reference method conditions (4,5) at a reaction temperature of 29.77 °C instead of 30 °C. The volume fraction of serum allows convenient use of standard volumetric glassware. The starting reagent is 2-oxoglutarate, added after 15 minutes preincubation of specimen with an otherwise complete reaction mixture at 29.77 0 C and after a stable absorbance is reached at 339 nm. The coupled catalytic reaction is followed for 300 seconds. A reagent blank reaction rate (water as specimen) is deducted from the overall rate. The final reaction conditions are: Reaction temperature pH

^-Alanine

2-Oxoglutarate

Pyridoxal-5'-phosphate

Tris buffer NADH (assuming NADH Na2 2H2 0) LDH (EC 1.1.1.27) Volume fraction (sample/ total)

29.77

7.3 500

150.11 89

0.16

2.2

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

U/mL(25 C)

0.083 (1:12)

92
4. Aspartate Aminotransferase (L-Aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1). The catalytic concentration of aspartate aminotransferase in U / L is measured by coupling oxalacetate production with reduced nicotinamide adenine dinucleotide (NADH) and malate dehydrogenase (MDH). It utilizes the 1FCC/EPE

reference method conditions (6) at a reaction temperature of 29.77 °C instead of 30 °C and with minor modifications suggested by the AACC Study Group (7). In addition, lactate dehydrogenase is omitted from the reagent system. The

starting reagent is 2-oxoglutarate, added after 15 minutes preincubation at 29.77 °C and after a stable absorbance is reached at 339 nm. The coupled catalytic reaction is followed for 300 seconds. The final reaction conditions are: Reaction temperature 29.77 °C pH 7.8 L-Aspartate 175 mmol/ L

2-Oxoglutarate 15 mmol/L

Pyridoxal-5'-phosphate 0.11 mmol/ L

Tris buffer 89 mmol/L

NADH (assuming NADH-Na2 -2H 2 0) 0.16 mmol/ L MDH (EC 1.1.1.37) 0.95 U/mL(25°C)

Volume fraction (sample/ total) 0.083 (1:12)

5. Creatine Kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2). The catalytic concentration of creatine kinase (CK.) is measured by coupling adenosine triphosphate production with nicotinamide adenine dinucleotide (NAD ) through the use of intermediate reactions catalyzed by hexokinase and

glucose-6-phosphate dehydrogenase. It follows the method recommended by the IFCC/EPE (8) and the AACC/CK

Study Group (9) with the following modifications: a) adenylate kinase inhibitors are removed; b) a blank reaction is run;

c) disodium ethylenediamine tetraacetate (Na2 EDTA) is added to the system; and d) the reaction temperature is 29.77 °C. The final reaction conditions are: Reaction temperature 29.77 °C PH 6.6 Imidazole acetate 100 mmol/L

Creatine phosphate 30 mmol/L

Adenosine-5'-diphosphate 2 mmol/L

JD-Glucose 20 mmol/L

NAD+ 2 mmol/L

Hexokinase (EC 2.7.1.1) 2500 U/L

Xl-Glucose-6-phosphate dehydrogenase 1500 U/L

(EC 1.1.49) (from Leuconostoc

mesenteroides) Magnesium acetate 10 mmol/L

N-Acetyl cysteine 20 mmol/L

Na2 EDTA 2 mmol/L

Volume fraction (sample/total) 0.043 (1:23)

6. Lactate Dehydrogenase (L-Lactate: NAD oxidoreductase, EC 1.1.1.27). The catalytic concentration of lactate dehydrogenase in U/L is measured by following the oxidation of reduced

nicotinamide adenine dinucleotide (NADH) at 339 nm. It utilizes the method of Bowers (10) with minor modifications at a reaction temperature of 29.77 °C. The final reaction conditions are: Reaction temperature 29.77 °C pH 7.2 Sodium pyruvate 1.2 mmol/L

NADH 0.15 mmol/L

Tris buffer 96.8 mmol/L

Volume fraction (sample/ total) 0.016 (1:61)

93
7. y-Glutamyltransferase [ (7-Glutamyl)-peptidase: amino acid 7-glutamyltransferase, EC 2.3. 2.2.]. 1 he catalytic concentration of 7-glutamyltransferase in U/L is measured by following the liberation of the

4-nitroaniline at 410 nm. The acceptor substrate glycylglycine also serves as the buffer. This assay utilizes the

I FCC/ EPE proposed reference method (11) with a modification in reaction temperature from 30 °C to 29.77 °C. This

assay is initiated by the addition of serum to the combined substrate/ buffer mixture equilibrated to 29.77 °C. It is followed at 410 nm for at least 300 seconds. The final assay reaction conditions are: Reaction temperature 29.77 °C pH 7.90 JL-7-Glutamyl-3-carboxy-4-nitroanilide 6 mmol/L

Glycylglycine 150 mmol/L

Volume fraction (sample/total) 0.091 (1:11)

References:

(1) Mangum, B. W. and Thornton, D. D., Eds., Standard Reference Materials: "The Gallium Melting Point Standard", NBS Spec. Publ. 481 (June 1977); also Clin. Chem. 23, 71 1-724 (1977).

(2) Ewen, L. M. and Spitzer, R. W., Improved determination of prostatic acid phosphatase (sodium

thymolphthalein monophosphate substrate), Clin. Chem. 22, 627-632 (1976).

(3) McComb, R. B., Bowers, Jr., G. N., and Posen, S., Alkaline Phosphatase. Plenum Press, New York, N.Y. (1979), pages 903-907, or also Clin. Chem. 2], 1988-1995 (1975).

(4) 1FCC Methods for the measurement of catalytic concentrations of enzymes, Part 3. IFCC Method for alanine aminotransferase (L-alanine 2-oxoglutarate aminotransferase, EC 2.6. 1.2) (Stage 2, Draft 1, 1979-1 1), Clin. Chim.

Acta 105, 147F-172F (1980).

(5) Recommendations of the Alanine Aminotransferase Study Group, Subcommittee on Enzymes, Committee

on Standards, American Association for Clinical Chemistry - Rej, R., Clin. Chem. 26, 1023 (1980) and personal communication from the author.

(6) IFCC Methods for the measurement of catalytic concentrations of enzymes. Part 2. IFCC Method for aspartate aminotransferase (L-Aspartate 2-oxoglutarate aminotransferase, EC 2.6. 1.1) (Stage 2, Draft, 1977), Clin. Chim. Acta 80, F21, F22, following page 394 (1977) Redefinitions of conditions previously published as Part 2

in Clin. Chim. Acta 70, F19-F42, following page 470 (1976).

(7) Recommendations of the Aspartate Aminotransferase Study Group, Subcommittee on Enzymes, Committee

on Standards, American Association for Clinical Chemistry - Clin. Chem. 26,, 1023-1024 (1980).

(8) IFCC Methods for the measurement of catalytic concentrations of enzymes, Part 7. Method for creatine kinase (ATP : creatine N-phosphotransferase, EC 2.7.3.2) (Stage 1, Draft 1981) obtained from IFCC Expert

Panel on Enzymes, M. Horder (Chairman), Department of Clinical Chemistry, University of Odense, Odense, Denmark.

(9) Recommendation of the Study Group on Creatine Kinase, Subcommittee on Enzymes, Committee on

Standards, American Association for Clinical Chemistry, 1979 - personal communication with R. Elser. (10) Bowers, G. N., Jr., Lactic Dehydrogenase. In Standard Methods of Clinical Chemistry (Vol. 4J, Seligson, D.,

Ed., Academic press, New York 1963, p. 163. (11) IFCC Methods for the measurement of catalytic concentrations of enzymes, Part 4. IFCC Method for 7-glutamyltransferase [ (7-glutamyl)-peptide: amino acid 7-glutamyltransferase, EC 2.3.2.2] (Stage 1, Draft 1981-3) - personal communication with L. M. Shaw.

94
Team 1 - Acid Phosphatase (ACP)

L. M. Ewen, Royal Columbian Hospital, New Westminister, B. C, Canada (Team Leader)

R. Bondar, Worthington Diagnostics, Freehold, N.J. W. Miller, Dupont - ACA Division, Wilmington, Del. G. N. Bowers, Jr., and M. Onoroski, Clinical Chemistry Laboratory, Hartford Hospital, Hartford, Conn.

L. M. Shaw, HUP/ Pepper Laboratory, Philadelphia, Pa. Team 2 - Alkaline Phosphatase (ALP)

G. N. Bowers, Jr., Hartford Hospital, Hartford, Conn. (Team Leader)

R. Miller, Technicon Instrument Company, Tarrytown, N.Y. W. D. Fellows, Health Safety & Human Factors Laboratory, Kodak Park, Rochester, N.Y.

J. P. Bretaudiere, N.Y. State Department of Health, Albany, N.Y.

D. C. Hohnadel, Christ Hospital, Cincinnati, Ohio W. Sowers, Hyland Division of Travenol, Round Lake, 111. Team 3 - Alanine Aminotransferase (AlaAT)

R. Rej, N.Y. State Department of Health, Albany, N.Y. (Team Leader)

R. Miller, Technicon Instrument Company, Tarrytown, N.Y. W. Ryan, Beckman Instruments, Inc., Brea, Calif.

L. M. Shaw, HUP/ Pepper Laboratory, Philadelphia, Pa. G. Sims, Hycel Inc., Houston, Texas Team 4 - Aspartate Aminotransferase (AspAT)

R. Rej, N.Y. State Department of Health, Albany, N.Y. (Team Leader)

L. M. Shaw, HUP/ Pepper Laboratory, Philadelphia, Pa. W. D. Fellows, Health Safety & Human Factors Lab., Kodak Park, Rochester, N.Y.

N. W. Tietz, University of Kentucky Medical Center, Lexington, Ky.

A. Bacharach, Strong Memorial Hospital, Rochester, N.Y. Team 5 - Creatine Kinase (CK)

R. Elser, York Hospital, York, Pa. (Team Leader)

R. Davis, Dupont - ACA Division, Wilmington, Del. W. Ryan, Beckman Instruments, Inc., Brea, Calif.

J. H. Str^mme, Ulleval Hospital, Oslo, Norway

J. F. O'Brien, Mayo Clinic, Rochester, Minn. Team 6 - Lactate Dehydrogenase (LDH)

R. B. McComb, Hartford Hospital, Hartford, Conn. (Team Leader)

K. Y. Jackson, N. Y. State Department of Health, Albany, N.Y.

B. Howell, National Bureau of Standards, Washington, D.C.

S. Buhl, Technicon Instrument Company, Tarrytown, N.Y.

D. S. Young and J. F. O'Brien, Mayo Clinic, Rochester, Minn. Team 7 - 7-glutamyltransferase (7-GT)

L. M. Shaw, HUP/ Pepper Laboratory, Philadelphia, Pa. (Team Leader)

R. Miller, Technicon Instrument Company, Tarrytown, N.Y.

S. Osaki, Hycel Inc., Houston, Texas

J. H. Stro'mme, Ulleval Hospital, Oslo, Norway

A. Wahlefeld, Boehringer Mannheim GmbH, Tutzing, West Germany.

95 *U.S. GOVERNMENT PRINTING OFFICE : 1983 0-380-997/5095
NBS-114A (REV. 2-BC)

1. PUBLICATION OR 3. Publication Date BIBLIOGRAPHIC DATA

' REPORT NO. SHEET (See instructions) NBS SP 260 -83 June 1983

4. TITLE AND SUBTITLE Standard Reference Materials:

The Measurement of the Catalytic (Activity) Concentration

of Seven Enzymes in NBS Human Serum SRM 909

5. author(S) G.N. Bowers, Jr., R. Alvarez, J. P. Cali, K.R. Eberhardt, O.J. Reeder, R. Schaffer, G.A. Uriano, R. Elser, L.M. Ewen , R.B. McComb, R. Re j , L.M. Shaw

6. PERFORMING ORGANIZATION (if joint or other than NBS. see instructions) 7. Contract/Grant No. NATIONAL BUREAU OF STANDARDS DEPARTMENT OF COMMERCE 8. Type of Report & Period Covered WASHINGTON, D.C. 20234 Final

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City. State. ZIP/ Same as in item 6 above.

10. SUPPLEMENTARY NOTES Library of Congress Catalog Card Number: 83-600543

I Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant bibliography or literature survey, mention it here) We determined the catalytic (activity) concentrations of seven enzymes (ACP, ALP, A 1 a AT , AspAT, CK, LDH and y-GT) in the

NBS lyophilized human serum Standard Reference Material (SRM 909) SRM 909 enzyme activity values should provide the basis for

compatibility among methods for determining enzyme values fh serum. Separate teams of clinical chemistry experts selected and carried out the analyses for the individual enzymes. The methods

used are primarily those recommended by the Standards Committees

of the AACC and/or the IFCC, as candidate reference methods;

however, a reaction temperature set-point of 29.77 °C (the

gallium melting point) was used. This monograph desc ri be's 't he serum r ec o n s t i t u t i on protocol, the specific enzyme methods, and

the results of the third (and last) round of the interlaboratory test program, which we used to derive mean values for enzyme

catalytic (activity) concentrations in SRM 909. Inter 1 aboratory

standard deviations of less than 10% were achieved. Measurements

made one year after the conclusion of the i nter 1 aboratory study

showed unchanged enzyme activities, within the uncertainties of the original assigned values.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolon s) enzyme; enzyme (catalytic) activity; human serum; interl aboratory reproducibility;

reference method; Standard Reference Material.

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