The erythrocyte Na+,K+-cotransport system and essential hypertension : in Vitro activity and reproducibility of measurement

The present study was initiated to determine whether erythrocyte Na⁺,K⁺- cotransport activity could be measured with enough reproducibility for use in a study of the effects of dietary constituents on this system. Two assay techniques were studied: the p-chloromercuribenzenesulfonate (PCMBS) and the nystatin techniques. Both were tested for reproducibility of Na⁺ and K⁺ loading and also for the rates of ouabain-insensitive, furosemide-sensitive (OIFS) Na⁺ and K⁺ efflux (indicators of Na⁺,K⁺-cotransport activity). Female subjects participated in blood pressure measurements and blood sampling after a 10-12 hour fast. In the PCMBS method, 5 subjects were each sampled on 2-10 different days (repetitions). In the nystatin experiment, 3 subjects were sampled on 2-3 different days. For each experiment, two sample aliquots comprised each repetition.

The reproducibility of loading erythrocytes with Na⁺ and depleting them of K⁺, as well as that of the OIFS efflux rates, was assessed by calculating and comparing the percent coefficient of variation (% CV) of intra-repetition, intra-individual, and inter-individual variability, for each of the parameters.

The nystatin method resulted in lower variability for Na⁺ and K⁺ loading than the PCMBS method. The PCMBS technique showed 4-9 times greater variability for depleting erythrocytes of K⁺ than the nystatin method. The high intra-repetition and intra-individual variability of K⁺ loading with the PCMBS method suggested that the low reproducibility of K⁺ loading was not due to technical error in itself, because Na⁺ loading showed low variability. Instead, the K⁺ variability was probably due to irreversible changes in the erythrocyte membrane permeability to K⁺ caused by PCMBS.

The variability in the rates of OIFS Na⁺ efflux was comparable for both methods. Again, the PCMBS technique resulted in high variability of K⁺ efflux. This non-reproducibility suggested that the PCMBS technique is not suitable for use in a dietary study.

Plots of the OIFS efflux rate versus the intracellular Na⁺ concentration from the literature studies reviewed indicated that normotensive and essential hypertensive subjects differ in the handling of Na⁺ and K⁺ at certain ranges of Na⁺ loading. The complex behavior of these curves suggests the need to assess subjects for OIFS Na⁺ and K⁺ efflux rates over a range of intracellular Na⁺ (and K⁺) concentrations.

Because of the higher reproducibility observed for Na⁺ and K⁺ loading and OIFS efflux from cells loaded by the nystatin method, this technique may be suitable for a dietary study. Before this is possible, however, the problem of selecting the proper intracel1ular cation concentrations for loading cells must be determined. Due to differences in the handling of Na⁺ and K⁺ between subjects, individuals must be assessed, both before and during a dietary study, for these characteristics. In addition, the question of whether a dietary intake study is sensitive enough for use in detecting differences in the activity of the erythrocyte Na⁺.K⁺-cotransport system between individuals, and within individuals on different days, must be resolved.