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Technical Briefs |
1
Central Lab., Pál Heim Pediatric Hosp.,
2
Dept. of Med. Chem., Molec. Biol. and Pathobiochem., and
3
1st Dept. of Paediatrics, Semmelweis Med. Univ., Budapest, Hungary;
a address for
correspondence: Pál Heim Pediatric Hosp., Central Lab., Budapest, Üllôi út 86, H-1189 Hungary, fax 36-1-333-0167
The enzyme Na+/K+-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+/K+-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+/K+-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 µmol of NADH oxidation per minute.
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For the estimation of precision and linearity, EDTA-K2 anticoagulated blood samples of healthy volunteers were hemolyzed (1:15) in 10 mmol/L Tris-HCl (pH 7.6), 1 mmol/L EDTA, and washed four times at 12 000g (4 °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 °C until the measurements.
Samples (20 µL) were added to 380 µL of Reagent 1 (final concentration per liter: 100 mmol of NaCl, 20 mmol of KCl, 2.5 mmol of MgCl2, 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 µL of 10 mmol/L ouabain (Reagent 2) was added to inhibit the ouabain-sensitive ATPase activity. The change in absorbance was monitored at 340 nm (reference wavelength 415 nm) 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+/K+-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 ± 3 SD of spontaneous NADH
oxidation) was 0.16 mU.
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For the determination of healthy reference intervals, ghosts were prepared from 100 µL 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+/K+-ATPase.
Acknowledgments
This work was financially supported by Hungarian OTKA Grant T023845 and ETT Grant 182/97.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Fekete, A. Vannay, A. Ver, B. Vasarhelyi, V. Muller, N. Ouyang, G. Reusz, T. Tulassay, and A. J. Szabo Sex differences in the alterations of Na+,K+-ATPase following ischaemia-reperfusion injury in the rat kidney J. Physiol., March 1, 2004; 555(2): 471 - 480. [Abstract] [Full Text] [PDF]
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 B. Vasarhelyi, T. Tulassay, A. Ver, M. Dobos, I. Kocsis, and I. Seri Developmental changes in erythrocyte Na+,K+-ATPase subunit abundance and enzyme activity in neonates Arch. Dis. Child. Fetal Neonatal Ed., September 1, 2000; 83(2): 135F - 138. [Abstract] [Full Text]
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