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Measurement of [Na.sup.+]/[K.sup.+]-ATPase activity with an automated analyzer.

The enzyme [Na.sup.+]/[K.sup.+]-ATPase (EC 3.6.1.37) plays a central role in the regulation of intra- and extracellular cation homeostasis. Alteration of this transport enzyme is thought to be linked to several diseases (including cardiovascular disorders, hypertension, and diabetes mellitus) [1]. However, measurement of [Na.sup.+]/[K.sup.+]-ATPase activity is not widespread, partly because of the lack of a method with a low detection limit available for the general clinical laboratory. For this purpose we have applied the determination of [Na.sup.+]/[K.sup.+]-ATPase activity to a Hitachi 704 automated analyzer. Our method is based on an ATP-regenerating system (Fig. 1), where the linear rate of NADH oxidation correlates to the hydrolysis of ATP [2]. One unit (1 U) of ATPase represents 1 [micro]mol of NADH oxidation per minute.

For the estimation of precision and linearity, EDTA-[K.sub.2] anticoagulated blood samples of healthy volunteers were hemolyzed (1:15) in 10 mmol/L Tris-HCI (pH 7.6), 1 mmol/L EDTA, and washed four times at 12 000g (4[degrees]C) with the same solution. The protein content of hemoglobin-free pellets (ghosts) was determined according to Bradford [3], with bovine serum albumin as a calibrator. Ghosts were diluted to protein contents of 0.5, 1, 1.5, 2, and 2.5 g/L. The samples with protein contents of 0.5 g/L (low activity), 1.5 g/L (medium activity), and 2.5 g/L (high activity) were divided into aliquots and stored at -80[degrees]C until the measurements.

[FIGURE 1 OMITTED]

Samples (20 [micro]L) were added to 380 [micro]L of Reagent 1 (final concentration per liter: 100 mmol of NaCl, 20 mmol of KCl, 2.5 mmol of Mg[Cl.sub.2], 0.5 mmol of EGTA, 50 mmol of Tris-HCl, pH 7.4, 1.0 mmol of ATP, 1.0 mmol of phosphoenolpyruvate, 0.16 mmol of NADH, 5 kU of pyruvate kinase, 12 kU of lactate dehydrogenase; all from Sigma). After 300 s, 5 [micro]L of 10 mmol/L ouabain (Reagent 2) was added to inhibit the ouabain-sensitive ATPase activity. The change in absorbance was monitored at 340 ran (reference wavelength 415 rim) by a twin test (i.e., combination of two assays in one cuvette); Rate A (i.e., slope of total ATPase activity), 80-280 s; Rate B (i.e., slope of ouabain-resistant ATPase activity), 400-600 s. The difference between the two slopes is proportional to the [Na.sup.+]/[K.sup.+]-ATPase activity.

For the estimation of total, between-day, between-run, within-day, and within-run CVs, two measurements per specimen per assay and two assays per day from the aliquots were done for 20 days [4]. In the range of 1.7-41.5 mU, the curve of NADH oxidation was linear during the measured intervals (r = 0.98). The activities changed proportionally with increasing protein concentrations (y = 50.6x, r = 0.99). The calculated CVs are presented in Table 1. The detection limit (mean [+ or -] 3 SD of spontaneous NADH oxidation) was 0.16 mU.

For the determination of healthy reference intervals, ghosts were prepared from 100 [micro]L of heparinized blood samples taken from 53 neonates, 93 children of different ages (1-18 years), and 42 adults. The study was approved by the Institutional Ethical Committee.

The enzyme activities are lower in children (P <0.05) [median (95% confidence interval) 5.30 (5.07-5.52) U/g of protein] than in neonates [7.15 (6.52-7.70)] or in adults [7.35 (5.63-8.22)]. No fluctuation of enzyme activity is present during childhood. Our results agree with the findings of others [5], who also reported decreased enzyme activities in children. Moreover, in spite of the difference of the methods used, our data are in the same range, as described [5].

Our automated method has several advantages compared with the manual ones (e.g., low blood requirement, high precision, speed), so it might be a valuable tool for gathering data for the clinical importance of [Na.sup.+]/[K.sup.+]-ATPase.

This work was financially supported by Hungarian OTKA Grant T023845 and ETT Grant 182/97.

References

[1.] Rose AM, Valdes RJ. Understanding the sodium pump and its relevance to disease. Clin Chem 1994;40:1674-85.

[2.] Schwartz A, Allen JC, Harigaya S. Possible involvement of cardiac [Na.sup.+],[K.sup.+]adenosine triphosphatase in the mechanism of action of cardiac glycosides. J Pharmacol Exp Ther 1969;168:31-41.

[3.] Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein-binding. Anal Biochem 1976;72:248-54.

[4.] NCCLS. Tentative Guideline EP5-T. User evaluation of precision performance of clinical chemistry devices. Villanova, PA: National Committee for Clinical Laboratory Standards, June 1984.

[5.] Sigstrom L, Waldenstrom J, Karlberg P. Characteristics of active sodium and potassium transport in erythrocytes of healthy infants and children. Acta Paediatr Scand 1981;70:347-52.

Barna Vasarhelyi, (1,3) * Terez Szabo, (1) Agota Ver, (2) and Tivadar Tulassay (3) ((1) Central Lab., Pal Heim Pediatric Hosp., (2) Dept. of Med. Chem., Molec. Biol. and Pathobiochem., and (3) 1st Dept. of Paediatrics, Semmelweis Med. Univ., Budapest, Hungary; * address for correspondence: Pal Heim Pediatric Hosp., Central Lab., Budapest, Ulloi ut 86, H-1189 Hungary; fax 36-1-333-0167, e-mail barna@heim.sote.hu)
Table 1. Calculated CVs.

 Protein content, g/L

 0.5 1.5 2.5

Activity, mU
CV, % 4.2 13.1 25.7
 Within-run 5.8 4.3 5.0
 Between-run 11.0 8.5 8.5
 Within-day 9.0 7.8 7.8
 Between-day 5.3 6.0 4.9
 Total 9.1 7.2 7.6
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Title Annotation:Technical Briefs
Author:Vasarhelyi, Barna; Szabo, Terez; Ver, Agota; Tulassay, Tivadar
Publication:Clinical Chemistry
Date:Oct 1, 1997
Words:940
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