Journal of Human Hypertension (2003) 17, 313–318 & 2003 Nature Publishing Group All rights reserved 0950-9240/03 $25.00 www.nature.com/jhh ORIGINAL ARTICLE Red blood cell K+ could be a marker of K+ changes in other cells involved in blood pressure regulation

MC Delgado1 and A Delgado-Almeida2 1Division of Hypertension, University of Michigan, MI, USA; 2Clinical Research Unit, University of Carabobo, Venezuela

The aim of this study is to determine whether red blood DBP (r ¼À0.27, P ¼ 0.04) and in offspring of hyper- + cell K content (RBCKi) is associated with blood tensives between RBCKi and DBP (r ¼À0.43, P ¼ 0.02). pressure levels and, if so, could RBCKi be a marker of A significant correlation was found in hypertensives + potassium changes in other cells involved in blood between RBCKi and plasma K (r ¼ 0.3, P ¼ 0.02). pressure regulation. The study was performed on 50 A positive correlation with borderline significance was 2+ untreated hypertensives, 32 of their offspring and 50 found in hypertensives between RBCKi and ionized Ca age- and sex-matched controls. Systolic (SBP) and (r ¼ 0.2, P ¼ 0.1). In conclusion, our results support the diastolic (DBP) blood pressures, height, weight, plasma, hypothesis that RBCKi is associated with blood pressure urine and red blood cell electrolytes were measured levels and that the measurement of RBCKi levels may + in all subjects. RBCKi was significantly lower in hyper- represent a biochemical marker for K changes in other tensives than in offspring of hypertensives and normo- cells involved in blood pressure regulation. Further tensive controls. Offspring of hypertensives had studies are necessary to explain the exact mechanisms significantly lower RBCKi than normotensive controls. of reduced RBCKi levels in hypertensive patients and Plasma K+ was significantly lower both in hypertensives their offspring. and offspring of hypertensives when compared to Journal of Human Hypertension (2003) 17, 313–318. normotensive controls. A significant negative correla- doi:10.1038/sj.jhh.1001527 tion was found in hypertensives between RBCKi and

Keywords: potassium; erythrocyte; marker; hypertension; blood pressure

Introduction completely prevents the hyperpolarizing and vasodi-

+ lating action of endothelium-derived hyperpolarizing Potassium (K ) in vascular smooth muscle cells factor (EDHF) in the rat hepatic artery.4 This observa- (VSMC) and in the endothelium is a very important tion is evidence that K with the pharmacological factor in vasodilatation. In VSMC, K+ efflux through Ca 2+ + characteristics of small and intermediate conduc- Ca -activated K channels (KCa) causes membrane tance KCa are involved in the EDHF vasodilatation. potential hyperpolarization and closes voltage- + 2+ In red blood cells (RBC), K efflux is also affected dependent Ca channels, which leads to vasodila- by IK .5,6 Ciclazindol, which abolishes EDHF tation.1 In the endothelium, K+ efflux, through Ca relaxation in the presence of apamin, inhibits IKCa intermediate conductance KCa (IKCa), hyper- in RBC4 and cromakalim, a known K+ channel polarize VSMC via electrical coupling between endo- 2 activator with vasodilator properties, opens IKCa in thelium and VSMC, producing hyperpolarization 7 + 2+ RBC. Our data show that increased K efflux occurs and closing voltage-dependent Ca channels. in RBC treated with 8Br-cGMP (Delgado et al, 2002, Stimulation of endothelial cells results in an out- + 3 unpublished data) probably by activation of IKCa as wardly rectifying K current and the combination of 8 + it occurs in VSMC. Therefore, it is reasonable to the K channel inhibitors apamin and charybdotoxin suppose that K+ changes occurring in RBC are reflecting K+ changes in the VSMC or in the endothelium. Correspondence: Dr MC Delgado, Division of Hypertension, 3918, In the present study, we test the hypothesis that Taubman Center, Box 0356, University of Michigan, Ann Arbor, + red blood cell K content (RBCKi) is associated with MI 48109, USA. E-mail: [email protected] blood pressure levels and, if so, RBCKi could be a Received 29 March 2002; revised 5 November 2002; accepted 11 marker of potassium changes in other cells involved November 2002 in blood pressure regulation. Red blood cell K+ MC Delgado and A Delgado-Almeida

314 Materials and methods diastolic blood pressure was o90 mmHg. Height was measured using a wall-mounted tape measure Subjects and weight determined on a balance-beam scale. The study was performed on 50 patients (26 males and 24 females) with untreated essential hyper- tension, 32 of their offspring (13 males and 19 Biochemical measurements females) and 50 age- and sex-matched controls (26 On the day of the study, subjects provided a 12-h males and 24 females). All subjects had a normal urine sample after an overnight fast. Peripheral dietary salt intake and all subjects gave a written venous blood was drawn, immediately centrifuged informed consent. Patients were recruited at the and the buffy coat discarded. RBCKi and RBCNai were Clinical Research Unit, University of Carabobo, measured in the supernatants of lysed RBC. For Venezuela. Exclusion criteria were any secondary RBC 100 ml of packed RBC was lysed in 10 ml of form of hypertension, diabetes mellitus, renal Ki distilled water. For RBCNai, RBC were washed in insufficiency, gastrointestinal disorders, pregnancy isotonic choline solution  3 and then lysed in or other significant medical conditions. Patients 10 ml of distilled water. After appropriate dilutions, taking drugs that could affect blood pressure (BP) RBCKi and RBCNai were measured in duplicate were also excluded. In all, 41 patients were never in their respective hemolysates by flame photo- treated for hypertension. Six patients were treated metry (Corning 410C, Cambridge, MA, USA) using with ace inhibitors, two with b-blockers and one aK+/Na+ standard of 100/10 mmol/l. Values were with calcium channel blocker. All of them had a 2- expressed as mmol/l. The coefficients of variation week washout before starting the study and those for RBCKi and RBCNai were o3%. In 20 randomly receiving b-blockers had dosage tapering before the selected samples, the amount of trapped plasma was 2-week washout. All patients underwent physical measured with 3H inulin (0.8 7 0.07% for 100 mlof examination and routine biochemical tests before RBC). Na+ and K+, in both plasma and urine, were the study. measured by a flame photometer. Ionized Ca2+ was Offspring of hypertensives were recruited through measured directly in fresh plasma using a Ciba their parents. A total of 14 hypertensive parents (10 Corning 634 Ca2+/pH analyzer (Medfield, MA, USA). mothers and four fathers) representing 14 families The concentrations of fasting plasma glucose, were studied. All of the spouses were normotensive triglycerides and cholesterol were measured by and no laboratory studies were performed in them. standard methods in hypertensives and normo- Offspring 12 years old and older were invited to tensive controls. participate independent of their blood pressure level. The normotensive controls had systolic SBP less Statistical analysis than 140 mmHg and diastolic DBP less than 90 mmHg and were matched with hypertensive All statistical procedures were performed using the patients by sex and age. They had no history of SAS system for Windows release 6.12. Results are serious disease and had taken no medication for at expressed as mean 7 s.e. Statistical evaluations least 6 weeks. A subgroup of the normotensive was by analysis of variance. To reduce the proba- controls (n ¼ 10) that matched hypertensive patients bility of significant differences arising by chance, by age and body mass index (BMI) was selected to Bonferroni’s correction was applied following a exclude the influence of overweight. nalysis of variance. Differences were considered This study was reviewed and approved by the significant when Po0.05. The relation between Institutional Review Board of the University of RBCKi and other variables was evaluated using the Carabobo Medical School. Pearson partial correlation coefficient (age and gender as partial correlation variables). A subgroup of hypertensives and age- and BMI-matched normo- Blood pressure and other physical measures tensive controls was evaluated in order to exclude the effect of overweight. All subjects had their BP measured using a mercury sphygmomanometer on two separate occasions in the morning. Subjects rested seated for 5 min, after Results which blood pressure recordings were done in triplicate (each reading separated by 2 min using The characteristics of hypertensives, offspring of the appropriate cuff size based on the upper hypertensives and normotensive controls are shown midarm. Blood pressure values were the mean of in Table 1. There are no significant differences three recordings at the second visit. Subjects were among the three groups in gender distribution, + + + defined as having high BP if either SBP was RBCNai, plasma Na or 12-h urinary Na :K ratio. X140 mmHg or DBP (Korotkoff phase V) was SBP was significantly higher in hypertensives than X90 mmHg. Subjects were defined as normotensive in offspring of hypertensives and in normotensive if systolic blood pressure was o140 mmHg and controls. In spite of the fact that the offspring of

Journal of Human Hypertension Red blood cell K+ MC Delgado and A Delgado-Almeida

315 Table 1 Characteristics, blood pressure and laboratory findings in the three groups

Hypertensives Offspring Controls HT vs OF HT vs CO OF vs CO

Male/female 26/24 13/19 26/24 NS NS NS* Age (years) 45 7 1227 2457 1 Po0.001 NS Po0.001 Body mass index (kg/m2)277 1257 1257 1 Po0.05 Po0.01 NS Systolic BP (mmHg) 156 7 2 134 7 3 121 7 2 Po0.001 Po0.001 Po0.001 Diastolic BP (mmHg) 103 7 1767 2747 1 Po0.001 Po0.001 NS 7 7 7 RBCKi (mmol/l cell) 81.4 0.7 85.8 1.2 93.1 0.7 Po0.01 Po0.001 Po0.001 7 7 7 RBCNai (mmol/l cell) 6.75 0.16 7.25 0.31 6.79 0.17 NS NS NS Plasma K (mmol/l) 3.65 7 0.06 3.62 7 0.10 3.99 7 0.06 NS Po0.001 Po0.01 Plasma Na (mmol/l) 139.4 7 0.4 139 7 0.8 139.4 7 0.5 NS NS NS Ionized Ca2+ (mmol/l) 1.07 7 0.01 1.05 7 0.02 1.09 7 0.01 NS NS NS 12-h urinary Na:K ratio 3.65 7 0.31 3.28 7 0.60 3.72 7 0.38 NS NS NS

HT=hypertensives; OF=offspring; CO=controls; BP=blood pressure; RBCKi=red blood cell potassium; RBCNai=red blood cell sodium. Values represent the mean 7 s.e.m. Variables are adjusted for age and gender. *w2 analysis. hypertensives were younger than normotensive SBP (r ¼ 0.02, P40.05) or DBP (r ¼À0.14, P40.05); controls, their average SBP (but not DBP) was and between urinary Na+:K+ ratio and SBP (r ¼ 0.04, significantly higher. P40.05) or DBP (r ¼À0.03, P40.05). RBCKi was significantly lower in hypertensives Hypertensives had significantly higher glucose than in offspring of hypertensives and in normo- (6.21 7 0.11 vs 4.33 7 0.11 mmol/l, P ¼ 0.001), tri- tensive controls (Figure 1). Offspring of hyper- glycerides (2.10 7 0.68 vs 1.01 7 0.27 mmol/l, P ¼ tensives had significantly lower RBCKi than 0.001) and cholesterol levels (6.27 7 0.42 vs 4.79 7 normotensive controls. Plasma K+ was significantly 0.70 mmol/l, P ¼ 0.003) than age–sex-matched lower both in hypertensives and offspring of normotensive controls. hypertensives when compared to normotensive There were no significant differences in RBCKi controls. No difference was observed between (80.7 7 0.4 vs 81.1 7 0.4 mmol/l cell, P40.05) or 7 7 hypertensives and offspring of hypertensives. Io- RBCNai (6.73 0.14 vs 6.75 0.13, P40.05) between nized Ca2+ did not differ between hypertensives, never-treated hypertensives and those in whom the offspring of hypertensives and in normotensive antihypertensive treatment was stopped 2 weeks controls. prior to the study. A significant negative correlation was found in In the subgroup of hypertensives (n ¼ 10) and hypertensives between RBCKi and DBP (r ¼À0.27, normotensive controls (n ¼ 10) matched by age P ¼ 0.04) and in offspring of hypertensives between (48 7 2 vs 48 7 2 years, P40.05) and BMI (29 7 1 2 RBCKi and DBP (r ¼À0.43, P ¼ 0.02). A significant vs 29 7 1 kg/m , P40.05), significant difference was 7 7 correlation was found in hypertensives between observed in RBCKi (78.3 1.6 vs 91.3 1.6 mmol/l + RBCKi and plasma K (r ¼ 0.3, P ¼ 0.02). Hyper- cell, Po0.0001). No significant differences were 7 7 tensives exhibit a trend of higher RBCKi levels with observed in RBCNai, (7.31 0.42 vs 6.51 higher ionized Ca2+ (r ¼ 0.2, P ¼ 0.1). In the three 0.45 mmol/l cell, P40.05), plasma K+(3.69 7 0.19 groups, no significant correlations were observed vs 4.18 7 0.19 mmol/l, P40.05), plasma Na+ (139.5 7 7 + + between RBCNai and SBP (r ¼ 0.002, P40.05) or DBP 0.8 vs 138.1 0.8 mmol/l, P40.05), Na :K ratio (r ¼À0.29, P40.05); between Ca2+ and SBP (3.25 7 0.56 vs 2.98 7 0.64 mmol/l, P40.05) and (r ¼À0.17, P40.05) or DBP (r ¼À0.02, P40.05); ionized calcium (1.01 7 0.02 vs 1.01 7 0.02 mmol/l, between plasma K+ and SBP (r ¼À0.06, P40.05) or P40.05). DBP (r ¼ 0.01, P40.05); between plasma Na+ and

Discussion At the cellular level K+ is important in the regulation of arterial tone. K+ fluxes triggered by the opening of 1 KCa produce vasodilatation in the VSMC and underlie the hyperpolarization and relaxation due to EDHF.3,4 In the present study, we found that hypertensives and their offspring had significantly lower RBCKi compared to normotensives and that these levels were inversely related to DBP. Our data are consistent with previous studies showing a

Figure 1 Red blood cell potassium levels (RBCKi) in hyperten- decreased RBCKi in hypertensives and offspring of sives, offspring and controls. Data given as mean (s.e.m.). hypertensives9–11 and a significant inverse relation-

Journal of Human Hypertension Red blood cell K+ MC Delgado and A Delgado-Almeida

316 12 ship of RBCKi and blood pressure. Moreover, system and the sympathetic nervous system affect- offspring of hypertensives had significantly lower ing both Na+ and K+ homeostasis.23 In order to

RBCKi levels and higher SBP than normotensive exclude the effect of overweight on RBCKi levels, we controls, suggesting that RBCKi may decrease early compared a subgroup of overweight hypertensives in life in subjects susceptible to the development of with a subgroup of age–BMI-matched normotensive future hypertension.13 This observation is consistent controls. Our data showed that after controlling with earlier reports showing an independent inverse for overweight, the difference in RBCKi persists, relationship between childhood RBCKi and adoles- therefore demonstrating that a factor other than 14 cent blood pressure. Recently, it has been shown obesity is causing a decrease in RBCKi levels in that RBCKi measured by nuclear magnetic resonance hypertensives. is significantly decreased in hypertensives and that The mechanisms responsible for decreased RBCKi treatment of hypertension partially restores RBCKi in hypertensives remain unclear. Several studies levels.15 This observation confirms our previous have shown that the activity of Na+,K+-ATPase is report showing that hypertensive patients with low inhibited or decreased in hypertensive subjects.24,25 + + RBCKi who received antihypertensive therapy and Although inhibition of Na ,K-ATPase could ex- + K sparing drugs had a significant improvement in plain the decreased RBCKi levels, it would be 16 BP, RBCKi, and fasting glucose levels. The effect of expected to be accompanied by increased RBCNai K+ depletion on insulin sensitivity17 and on insulin- levels which we did not observe in our study. The + 18 stimulated cell K uptake could explain why fact that RBCNai is not increased in hypertensives is 26,9,10 hypertensives have decreased RBCKi and increased supported by several studies and opposed by glucose levels compared to hypertensives. others.27,28 These discrepancies may be caused by Ion transport alterations are often associated with differences in technique,29 the type of population abnormal lipid metabolism.19 In the present studies, studied, inadequate sample size or the use of hypertensives showed significant higher levels antihypertensive drugs.30 In this study there was of plasma total cholesterol and triglycerides asso- no difference in RBCKi levels between never-treated ciated with decreased RBCKi levels when com- hypertensives and those that were treated before the pared to normotensive controls. Future studies of washout period. This is an important factor to take K+ transport alterations with dyslipidaemia will into consideration since some antihypertensive require a careful analysis of cell membrane lipid drugs affect K+ homeostasis.31 composition and the determination of membrane While the activity of the outward RBC Na+,K+ microviscosity.20 cotransport has been shown to be increased in Potassium is an important regulator of aldosterone hypertensives,32 this mechanism does not appear levels.21 Preliminary data in a group of normoten- to explain our observation since only decreased sive offspring of hypertensives (n ¼ 15) showed that RBCKi levels were observed. plasma aldosterone levels exhibited a significant A possible mechanism explaining decreased correlation with 12 h Na/K ratio (r ¼ 0.5, P ¼ 0.04) RBCKi levels independent of RBCNai is an increased 5,6 and a trend of inverse relationship with RBCKi levels activity of RBC IKCa, which has been shown to (r ¼À0.46, P ¼ 0.08). Plasma K+ did not show any be increased in hypertensive patients.33 Red significant or trend association with aldosterone or blood cells, although lacking a nucleus, are useful renin (Delgado et al, 2002, unpublished data) This models for membrane transport studies34,35 could be due to the fact that plasma K+ represents and share membrane properties related to VSMC only a small fraction of total body K+, and significant and the endothelium. RBC, which are equipped + 36,37 changes in renal K excretion may occur without with IKCa and inducible nitric oxide synthase, + 22 7 detectable alterations in plasma K levels. There- respond to KCa activators producing vasodilatation fore, in subjects with no primary or secondary or to 8Br-cGMP (Delgado et al, 2002, unpublished hyperaldosteronism, very small changes in aldo- data) in a manner similar to the VSMC with the + + 8 sterone levels are better associated with renal K activation of KCa and the efflux of K . Consequently, excretion or cell K+ changes as opposed to estab- K+ changes in RBC could be a marker for K+ changes lished hyperaldosteronism where plasma K+ levels in other cells such as VSMC and the endothelium. are clearly associated with aldosterone levels. In the However, RBC IKCa were not measured in the present study, the decrease in RBCKi levels in present study and the contribution of this hypertensives and their offspring could be a channel in decreasing RBCKi levels remains spec- response to a relative hyperaldosteronism. However, ulative. the 12 h urinary N+:K+ ratio was not significantly In conclusion, our results support the hypothesis

different among hypertensives, offspring and nor- that RBCKi is associated with blood pressure levels motensive controls (see Table 1). Nevertheless, and that the measurement of RBCKi levels may future studies are necessary to characterize the represent a biochemical marker for K+ changes in neuroendocrine activity in these subjects and its other cells involved in blood pressure regulation. association with RBCKi. Further studies are necessary to explain the exact Several studies have shown that obesity leads to mechanisms of reduced RBCKi levels in hyper- an activation of the renin–angiotensin–aldosterone tensive patients.

Journal of Human Hypertension Red blood cell K+ MC Delgado and A Delgado-Almeida

317 Acknowledgements 14 Himmelmann A, Svensson A, Sigstro¨m L, Hansson L. Predictors of blood pressure and left ventricular mass We are indebted to Dr Alan B Weder, MD, University in the young: the hypertension in pregnancy offspring of Michigan, for his extraordinary support and his study. Am J Hypertens 1994; 7: 381–389. review of the manuscript. This work was supported 15 Resnick LM et al. Relation of cellular potassium to by grants (RP-C-IV-93, 93-023) from the CONICIT other mineral ions in hypertension and diabetes. (National Council of Scientific and Technological Hypertension 2001; 38: 709–712. Investigations) and CODECIHT (Council of Scienti- 16 Delgado-Almeida A, Delgado-Leon C, Delgado MC. The significance of a decreased erythrocyte potassium fic, Humanist and Technologic Development, Uni- in the management of severe essential hypertension. versity of Carabobo), Venezuela. Am J Hypertens 1999; 12: 64. 17 Norbiato G et al. 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