乳酸脱氢酶的测定1

Clin Chem Lab Med 2002; 40(6):643–648 © 2002 by Walter de Gruyter · Berlin · New York International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)1,2) Scientific Division Committee on Reference Systems for Enzymes (C- RSE)3) Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase [L-Lactate: NAD+ Oxidoreductase (LDH), EC 1.1.1.27] Gerhard Schumann1, Roberto Bonora2, Ferruccio Ceriotti3, Pascale Clerc-Renaud4, Carlo A. Ferrero3, Georges Férard5, Paul F.H. Franck6, F.-Javier Gella7, Wieland Hoelzel8, Poul Jørgen Jørgensen9, Takashi Kanno10, Art Kessner11, Rainer Klauke1, Nina Kristiansen12, Jean-Marc Lessinger5, Thomas P.J. Linsinger12, Hideo Misaki13, Mauro Panteghini2, Jean Pauwels12, Heinz G. Schimmel12, Arlette Vialle4, Gerhard Weidemann14 and Lothar Siekmann15,4) 1 Klinische Chemie, Medizinische Hochschule Hannover, Hannover, Germany 2 Clinical Chemistry Laboratory 1, Azienda Ospedalliera “Spedali Civili”, Brescia, Italy 3 Laboratorio Standardizzazione, Istituto Scientifico H. S. Raffaele, Milano, Italy 4 Laboratoire de Biochimie Pédiatrique, Hôpital Debrousse, Lyon, France 5 Laboratoire de Biochimie appliquée, Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France 6 Department of Clinical Chemistry, Leyenburg Hospital, The Hague, The Netherlands 7 BioSystems, S.A., Barcelona, Spain 8 Roche Diagnostics GmbH, Tutzing, Germany 9 Department of Clinical Chemistry, Odense University Hospital, Odense, Denmark 10 Department of Laboratory Medicine, Hamamatsu University, School of Medicine, Japan 11 Beckman Coulter, Inc. Brea, CA, USA 12 European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium 13 Asahi Kasei Corporation, Fine Chemicals & Diagnostics Division, Tokyo, Japan 14 Institut für Klinische Chemie und Laboratoriumsmedizin, Klinikum der Stadt, Nürnberg, Germany 15 Institut für Klinische Biochemie, Universität Bonn, Bonn, Germany This paper is the third in a series dealing with refer- ence procedures for the measurement of catalytic ac- tivity concentrations of enzymes at 37°C and the certi- fication of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measure- ment of Catalytic Concentration of Creatine Kinase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotrans- ferase; Part 6. Reference Procedure for the Measure- ment of Catalytic Concentration of �-Glutamyltrans- ferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of �-Glu- 1) The exclusive © for all languages and countries is vested in the International Federation of Clinical Chemistry and Labora- tory Medicine. 2) IFCC Sections reprinted in J. Clin. Chem. Clin. Biochem. are listed in the Cumulative Index, which appeared in connection with the contents of this journal Volume 27, 1989./Since 1991 have been printed in (Eur.) J. Clin. Chem. Clin. Biochem./Since 1998 have been printed in Clin. Chem. Lab. Med. IFCC 1991/1 Vol. 29, 435–457 IFCC 1991/2 Vol. 29, 531–535 IFCC 1991/3 Vol. 29, 577–586 IFCC 1991/4 Vol. 29, 767–772 IFCC 1992/1 Vol. 30, 901–905 IFCC 1994/1 Vol. 32, 639–655 IFCC 1995/1 Vol. 33, 247–253 IFCC 1995/2 Vol. 33, 399–404 IFCC 1995/3 Vol. 33, 623–625 IFCC 1995/4 Vol. 33, 627–636 IFCC 1995/5 Vol. 33, 637–660 IFCC 1997/1 Vol. 35, 317–344 IFCC 1997/2 Vol. 35, 345–349 IFCC 1997/3 Vol. 35, 805–831 IFCC 1997/4 Vol. 35, 833–843 IFCC 1998/1 Vol. 36, 57–65 IFCC 1998/2 Vol. 36, 185–203 IFCC 1998/3 Vol. 36, 887–893 IFCC 2000/1 Vol. 38, 363–370 IFCC 2000/2 Vol. 38, 1301–1314 IFCC 2001/1 Vol. 39, 175–179 IFCC 2001/3 Vol. 39, 283–289 IFCC 2002/1 Vol. 40, 78–89 IFCC 2002/2 Vol. 40, 631–634 IFCC 2002/3 Vol. 40, 635–642 3) Members: L. Siekmann (DE), F. Ceriotti (IT), G. Férard (FR), T. Kanno (JP), G. Schumann (DE) 4) Reprint requests and inquiries should be addressed to: Prof. Dr. Lothar Siekmann, Institut für Klinische Biochemie, Univer- sität Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany, Phone: +49 228 2875911, Fax: +49 228 287 5033, E-mail: Lothar.Siekmann@ukb.uni-bonn.de Received for publication 2002-4-4 IFCC Primary Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes at 37°C IFCC 2002/4 lenovo 高亮 乳酸脱氢酶 lenovo 高亮 乳酸盐 lenovo 高亮 氧化还原酶 lenovo 高亮 实用校正工作 lenovo 高亮 参考制品 lenovo 高亮 肌酸激酶 lenovo 高亮 丙氨酸氨基转移酶 lenovo 高亮 天冬氨酸氨基转移酶 644 Schumann et al.: IFCC reference procedure for lactate dehydrogenase tamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37°C. A document describing the determination of prelim- inary upper reference limits is also in preparation. The procedure described here is deduced from the previ- ously described 30°C IFCC reference method (1). Dif- ferences are tabulated and commented on in Appen- dix 1. Clin Chem Lab Med 2002; 40(6):643–648 Key words: IFCC reference procedure; Lactate dehy- drogenase; Preliminary reference interval. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydroge- nase; NAD, β-nicotinamide adenine dinucleotide; NADH, β-nicotinamide adenine dinucleotide, reduced form. Reaction Principle L-(+)-Lactate + NAD+ –LDH→ Pyruvate + NADH + H+ Specimens Calibration materials, control specimens and human sera. Measurement Conditions Concentrations in the final reaction mixture and the measurement conditions are listed in Tables 1 and 2. Reagents 1. N-Methyl-D-glucamine, (C7H17NO5), Mr=195.22 2. L(+)-Lactic acid, monolithium salt (C3H5O3Li), Mr=96.01 3. β-Nicotinamide adenine dinucleotide (NAD), free acid (C21H27N7O14P2), Mr=663.4 4. NAD, lithium salt, dihydrate (C21H26N7O14P2Li . 2 H2O), Mr=705.4 5. Hydrochloric acid (HCl), Mr=36.46, 2 mol/l 6. Sodium chloride (NaCl), Mr=58.44 Note: NAD can contain inhibitors for LDH. The absence of inhibitors should be declared by the manufacturer. Reagents of the highest purity must be used. If a chemical is suspected of containing impurities affect- ing the catalytic activity of the analyte, further investi- gations must be performed, e.g. comparisons with products from different manufacturers and different lots. It is recommended to use reagents which have al- ready been tested and approved in comparisons. Charts for the Adjustment and the Control of the pH Values (Procedure for the Adjustment of pH Values at Temperatures Diverging from 37°C) Both the thermometer and the pH electrode are sus- pended in the mixed solution simultaneously. The stirred solution is then titrated to the pH value listed in the chart for the currently measured temperature. The speed of agitation should be the same during the cali- bration, the control and the adjustment of the pH value. The pH electrode should be positioned in the centre of the stirred solution. The fact that the temperature can change during the titration must be taken into account. For this reason, the temperature in the proximity of the target value should be controlled again and the target pH value corrected according to Table 3, if necessary. The same applies to the adjustment of the temperature compensation of the pH meter. Preparation of Solutions The given mass of the compounds for the preparation of solutions refers to 100% content. If the content of the reagent chemical employed is less (e.g. yz %), the amount equivalent to the given mass is calculated by the use of a factor: Fcontent=100 / yz Highly purified water with a quality comparable to bi-distilled water (conductivity < 2µS/cm, pH 6–7, sili- cate < 0.1 mg/l) shall be used for the preparation of the reagent solutions. The expanded (k=2) combined uncertainty (normally distributed) of each weighing procedure (including the uncertainty of the purity of the substance) shall be ≤ 1.5%. Table 1 Concentrations in the final complete reaction mix- ture for the measurement of LDH. N-Methyl-D-glucamine 325 mmol/l pH (37°C) 9.40 ± 0.05* L-(+)-Lactate 50 mmol/l β-NAD+ 10 mmol/l (free acid 3.15 mmol/) (lithium salt 6.85 mmol/l) Volume fraction of sample 0.0435 (1:23) * expanded (k=2) combined uncertainty Table 2 Measurement conditions for the measurement of LDH. Temperature 37.0°C ± 0.1°C* Wave length 339 nm ± 1 nm Band width ≤ 2 nm Light path 10.00 mm ± 0.01 mm* Incubation time 180 s Delay time 90 s Measurement interval 180 s Readings (measurement points) ≥ 6 * expanded (k=2) combined uncertainty lenovo 高亮 试剂 lenovo 高亮 温度计 lenovo 高亮 同时发生的 lenovo 高亮 滴定的 lenovo 高亮 搅拌 lenovo 高亮 刻度 lenovo 高亮 近似 Schumann et al.: IFCC reference procedure for lactate dehydrogenase 645 Reaction solution 7.30 g (373.8 mmol/l) N-Methyl-D-glucamine 0.552 g (57.50 mmol/l) Lactic acid, monolithium salt – Dissolve in about 80 ml water. – Adjust to pH (37°C) 9.4 with 2 mol/l hydrochloric acid. – Transfer to a 100 ml volumetric flask. – Equilibrate the volumetric flask and water to 20°C. – Fill the water (20°C) up to the calibration mark of the volumetric flask. Stability at 2°C–8°C: 1 month Start reagent solution 0.240 g (36.23 mmol/l) NAD, free acid 0.556 g (78.78 mmol/l) NAD, lithium salt, dihydrate Note: The use of NAD free acid without NAD lithium salt for the preparation of the start reagent solution (as stipulated for the IFCC 30°C reference method) reason- ably decreases the pH value of the final complete reac- tion mixture. A start reagent solution containing a mixture of NAD free acid and NAD lithium salt has two advantages: 1. The pH value of the final complete reaction mixture does not decrease. 2. The absorbance of NAD depends strongly on the pH value but the change of the absorbance due to a change of the pH value occurs not spontaneously. Therefore, the reagent blank rate reaction remains non- linear for about 4–6 minutes if NAD free acid as the start reagent solution is added to the alkaline reaction solution. This effect is considerably reduced if the start reagent solution contains a mixture of NAD free acid and NAD lithium salt. Note: The above recommended NAD free acid/NAD lithium salt mixture dissolves rather slowly. Raising the temperature (up to 40°C) speeds up the dissolving process. – Dissolve in about 6 ml water. – Transfer to a 10 ml volumetric flask. – Equilibrate the volumetric flask and water to 20°C. – Fill water (20°C) up to the calibration mark of the vol- umetric flask. Stability at 2°C–8°C: 1 week Table 3 Dependence of the pH value of the reaction solution upon temperature. Temperature (°C) pH Temperature (°C) pH Temperature (°C) pH 15.00 9.976 23.50 9.741 32.00 9.522 15.25 9.969 23.75 9.734 32.25 9.516 15.50 9.962 24.00 9.728 32.50 9.509 15.75 9.955 24.25 9.721 32.75 9.503 16.00 9.948 24.50 9.714 33.00 9.497 16.25 9.940 24.75 9.708 33.25 9.491 16.50 9.933 25.00 9.701 33.50 9.485 16.75 9.926 25.25 9.695 33.75 9.479 17.00 9.919 25.50 9.688 34.00 9.472 17.25 9.912 25.75 9.681 34.25 9.466 17.50 9.905 26.00 9.675 34.50 9.460 17.75 9.898 26.25 9.668 34.75 9.454 18.00 9.891 26.50 9.662 35.00 9.448 18.25 9.884 26.75 9.655 35.25 9.442 18.50 9.877 27.00 9.649 35.50 9.436 18.75 9.870 27.25 9.642 35.75 9.430 19.00 9.864 27.50 9.636 36.00 9.424 19.25 9.857 27.75 9.629 36.25 9.418 19.50 9.850 28.00 9.623 36.50 9.412 19.75 9.843 28.25 9.617 36.75 9.406 20.00 9.836 28.50 9.610 37.00 9.400 20.25 9.829 28.75 9.604 37.25 9.394 20.50 9.822 29.00 9.597 37.50 9.388 20.75 9.815 29.25 9.591 37.75 9.382 21.00 9.809 29.50 9.585 38.00 9.376 21.25 9.802 29.75 9.578 38.25 9.371 21.50 9.795 30.00 9.572 38.50 9.365 21.75 9.788 30.25 9.566 38.75 9.359 22.00 9.781 30.50 9.559 39.00 9.353 22.25 9.775 30.75 9.553 39.25 9.347 22.50 9.768 31.00 9.547 39.50 9.341 22.75 9.761 31.25 9.540 39.75 9.335 23.00 9.754 31.50 9.534 40.00 9.330 23.25 9.748 31.75 9.528 646 Schumann et al.: IFCC reference procedure for lactate dehydrogenase Measurement Procedure Equilibrate only an adequate volume (∼ 0.4 ml) of start reagent solution at 37°C in preparation of the measure- ment procedure. The remaining volume of the start reagent solution should be stored at 2°C–8°C. Pipette the volumes as listed in Table 4 one after an- other into the cuvette. Table 4 Analytical system for the measurement of the over- all rate of conversion. 2.000 ml Reaction solution Equilibrate to 37.0°C. 0.100 ml Sample Mix thoroughly and incubate for 180 s. At the end of the incubation time, the temperature of the solution in the cuvette shall have reached 37.0°C. 0.200 ml Start reagent solution Mix thoroughly, wait 90 s and monitor time and absorbance for additional 180 s. The expanded (k=2) combined uncertainty (normally distributed) of the kinetic photometric measurement shall not exceed 1%. (This uncertainty does not include the uncertainty of the wavelength adjustment.) The expanded (k=2) combined uncertainty (normally distributed) of the volume fraction of the sample shall be ≤ 1%. Reagent blank rate To determine the reagent blank rate, the specimen is re- placed by 9 g/l (154 mmol/l) sodium chloride solution. The measurement procedure is then carried out as de- scribed above. Sample blank rate Due to the lactate content in the sample, it is not possi- ble to determine the sample blank rate. Upper limit of the measurement range If the change of absorbance exceeds 0.00275 s-1 (0.165 min-1) in the measurement interval an analytical portion of the sample must be diluted with 9 g/l (154 mmol/l) sodium chloride solution and the measurement proce- dure must be repeated with the diluted specimen. The obtained value must then be multiplied by the corre- sponding factor of the dilution. Sources of error If alanine aminotransferase (ALT) or aspartate amino- transferase (AST) have been examined in the cuvette prior to the LDH determination, a possible interference of displaced LDH from AST/ALT test mixtures with the measurements must be taken into account. Calculation The temporal change of absorbance (s-1) is calculated with the analysis of regression (method of the least squares). After subtraction of the reagent blank rate the corrected change of absorbance is multiplied by the factor: F=3651 (measurement at 339 nm, ε339(NADH)= 630 m2/mol). The catalytic concentration of LDH is calculated in µkat/l. ∆A/∆tLDH: change of absorbance (in s-1) after correction of the reagent blank rate bLDH: catalytic concentration of LDH bLDH=3651 .∆A/∆tLDH Table 5 Comparison of the IFCC methods for the measurement temperatures of 30°C and 37°C. 37°C IFCC reference procedure 30°C Reference method Comment Specimen of investigation Calibration material, control specimens Human sera The reference procedure will be used and human sera primarily for the investigation calibration materials and control specimens. Uncertainty of the measurement temperature adjustment Uncertainty ≤ 0.1°C (k=2) Bias: less ± 0.05°C High quality spectrophotometer with devices Imprecision: less ± 0.1°C for temperature adjustment and control provide an uncertainty (k=2) of the temperature ≤ 0.1°C. Uncertainty of the pH value adjustment ∆pH ± 0.05 Not specified Delay time 90 s 30 s The reagent blank rate remains non-linear up to 90 s after the addition of the start reagent solution. lenovo 高亮 比色杯 Schumann et al.: IFCC reference procedure for lactate dehydrogenase 647 Table 5 Continued. 37°C IFCC Reference procedure Reference method 30°C Comment Measurement time 180 s At least 240 s The reaction rate is not linear and the non- linearity proportionally increases with the time and the catalytic LDH concentration. Shorter measurement time implies less non- linearity. Pipetting volumes and volume fractions Solution R: 2000 µl Solution R: 2700 µl Volumes better suited for conventional Serum: 100 µl Serum: 150 µl pipetting systems were used. Consequently, Start solution: 200 µl Start solution: 300 µl the concentrations of the reagent solutions were adapted to the new volume fractions (the volume fraction of sample changes from 1:21 to 1:23). Start reagent solution Aqueous mixture of NAD Aqueous solution of NAD The use of the free acid as start solution free acid and NAD lithium salt free acid changes the pH value of the reaction mixture. The mixture of free acid and lithium salt is more stable and the non-linearity of the reagent blank rate is reduced. Buffer stock solution No preparation of a buffer stock solution Preparation of a buffer stock The preparation of a buffer stock solution is solution not necessary. Temperature of the start reagent solution before use Start with substrate: before use the start No temperature equilibration of The use of start reagent solution with reagent solution should be at 37°C the start reagent solution is ambient temperature decreases the described temperature in the cuvette. Collection of data Number of readings ≥ 6 Monitoring of the increase in Modern spectrophotometers employ digital absorbance data processing. Several readings ≥ 6 should ensure a sufficient precision of the measure- ment results. Devices for a continuous monitoring are no longer in use. Determination of the slope (time versus absorbance) Regression analysis of the method of No information A well-defined statistical method is least squares necessary to ensure the reproducibility of the calculation of the slope. Reference range Women and men No reference values for the The reference values for women and men ≤ 4.12 µkat/l (≤ 245 U/l) method were investigated separately. The catalytic concentration in µkat/l can be converted to U/l by multiplication by the factor f=60. Preliminary Upper Reference Limits The preliminary upper reference limits for adults (≥ 17 years) were investigated separately for men (n=441) and women (n=438) (1). Gender Upper reference limit* (and 90% confidence interval) Women 4.12 µkat/l (4.07 µkat/l–4.25 µkat/l) Men 4.13 µkat/l (4.05 µkat/l–4.22 µkat/l) Gender Upper reference limit* (and 90% confidence interval) Women 247 U/l (244 U/l–255 U/l) Men 248 U/l (243 U/l–253 U/l) * The upper reference limits are the 97.5th percentiles of the reference collectives. Inside parentheses are the 90% confidence intervals of the 97.5th percentiles. 648 Schumann et al.: IFCC reference procedure for lactate dehydrogenase Appendix 1: Changes in the IFCC Reference Procedure for Measurements at 37°C Compared with the Reference Method for Measurements at 30°C as Described in the Original IFCC Document The primary reference procedure is deduced from the IFCC reference method (1) which provides optimised conditions for the measurement of catalytic activity concentrations of LDH. The measurement temperature of 37°C instead of 30°C requires only minimal changes of certain measurement parameters to retain the opti- mum measurement conditions. The modifications are listed and commented on in Table 5. Furthermore, if in comparison to the 30°C reference method a more ac- curate specification has become necessary for improv- ing the high standardisation of the measurements, it is also described here. References 1. Bais R, Philcox M. International Federation of Clinical Chem- istry (IFCC). Approved recommendation on IFCC methods for the measurement of catalytic concentrations of en- zymes. Part 8. IFCC method for lactate dehydrogenase. Eur J Clin Chem Clin Biochem 1994; 32:639–55.