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

Author

Diana M. Kuklinski

Date of Award

6-1987

Degree Type

Thesis

Degree Name

Master of Science

Major

Nutrition

Major Professor

Nina L. Marable

Committee Members

Roy Beauchene, Frances Andrews

Abstract

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.

Recommended Citation

Kuklinski, Diana M., "The erythrocyte Na+,K+-cotransport system and essential hypertension : in Vitro activity and reproducibility of measurement. " Master's Thesis, University of Tennessee, 1987.
https://trace.tennessee.edu/utk_gradthes/13514