Association of sulfur content in erythrocytes with cardiovascular parameters and blood pressure

Abstract

OBJECTIVE:

The study aims at assessing the relationship between blood pressure, heart geometry parameters, and the erythrocyte content of sulfur, potassium, chlorine and phosphorus, in a group of patients with ambulatory systolic and diastolic blood pressure (SBP, DBP) below 140 or 90 mm Hg, respectively, who were otherwise healthy and untreated.

METHODS:

The study group consisted of 42 adults recruited in a primary care setting. The individuals were healthy, not undergoing any therapy and free from smoking. For each individual, data were obtained on: average 24-hour SBP and DBP, left ventricle geometry, complete blood count, lipids profile, fibrinogen, hs-CRP and the erythrocyte concentration of sulfur (S), potassium (K), chlorine (Cl) and phosphorus (P).

RESULTS:

Multivariate regression analysis showed statistically significant relationships of diastolic posterior wall thickness (PWTd) and relative wall thickness (RWT) with the concentration ratio of sulfur and potassium (S/K) in erythrocytes: PWTd and RWT increase as the S/K ratio increases. Also, SBP was found to be positively correlated with the S/K ratio.

CONCLUSIONS:

The increase in sulfur content in RBCs could be an indicator of the downregulation of nitric oxide (NO) erythrocyte bioavailability exerted by endogenously produced hydrogen sulfide (H₂S), and, in consequence, a marker of the development of hypertension and/or adverse changes in heart geometry.

Keywords

Blood pressure measurement/monitoring heart geometry elemental sulfur content of erythrocytes microelement concentration in erythrocytes

1 Introduction

Essential hypertension, being one of the main health problems of contemporary society, has no defined origin [1]. It is believed that this health abnormality is a consequence of an interplay between various genetic [2, 3] and environmental factors [4 –7], but none of these factors has been identified as the leading cause. High blood pressure (BP), if not properly treated, very often leads to severe fibrotic and inflammatory vessel and organ damage, thus largely contributing to morbidity and mortality [8 –10].

The detrimental effects of hypertension often relate to cardiovascular complications such as myocardial infarction, heart failure and stroke [11, 12]. Usually, more advanced stages of hypertension are preceded by a mild increase in blood pressure (ambulatory systolic blood pressure 130–139 mm Hg and/or ambulatory diastolic 80–89 mm Hg). It is of great importance to characterize and understand this initial stage of the disease as it may provide a better insight into the mechanism responsible for the development of hypertension and other accompanying disorders [13].

Great effort has been devoted to the study of human blood in hypertensive patients with the hope that this biological material, easily accessible for screening purposes, will show structural changes associated with the development of essential hypertension [14 –18]. The observation of each such association may shed additional light on the disease’s etiology. In particular, a lot of attention was paid to red blood cells and adverse changes were identified in these cells upon hypertension and heart disease development [19 –28]. More specifically, there was an indication that the membrane skeleton of RBCs displayed a honeycomb pattern in healthy individuals, whereas it showed a different structure in patients with essential hypertension [29]. Such morphological differentiation may be responsible for the impairment of hemoglobin oxygen transport, which was observed in hypertensive patients [30].

Another subject of investigations in the domain of hypertension etiology is related to the RBC’s concentration of some microelements such as phosphorus, chlorine, sulfur and potassium. Since these concentrations depend on many enzymatic processes which occur at the molecular level, they offer rich information on the body’s metabolic control. For example, it is well known that the decreased erythrocyte potassium content is associated with essential hypertension [31 –33] and could be a marker for potassium changes occurring in other cells involved in blood pressure regulation [34, 35]. On the contrary, not much is known about the relationship between sulfur concentration in erythrocytes and hypertension. A possible link in this case is offered by the metabolism of hydrogen sulfide H₂S, which is largely responsible for the erythrocyte content of sulfur.

The human body produces H₂S and uses it as a signaling molecule in several physiological processes [36]. The H₂S regulates cardiovascular functions [37 –39], plays the role of a messenger in the central nervous system [40 –42], as well as acts on pancreatic structure and functions [42 –45]. More specifically, studies on animal models have shown that H₂S induces vasorelaxation in arterial hypertension [46 –52] and is cardioprotective in myocardial ischemia [53 –57]. It has also been suggested that the mechanism of cardiac protection did not rely on blood pressure regulation only, but could have resulted from the antifibrotic and antioxidant properties of sulfide [58, 59]. Taking this all into account, H₂S appears to attenuate cardiovascular damage in hypertensive and nonhypertensive conditions, revealing its broad spectrum of protective mechanisms [60 –63].

According to the results of recent works, also RBCs play a very important role in H₂S hemostasis by participating in sulfide metabolism. H₂S may appear in RBCs as a result of two different pathways. It can be scavenged by RBCs during the microcirculation of H₂S-producing tissues or it can be produced endogenously.

In the first case, H₂S enters erythrocytes very rapidly, because the permeability of the erythrocyte membrane to H₂S is very high (this process does not require transporters) [64, 65]. After diffusing into RBCs, H₂S reduces metHb to oxyHb via formation of a metHb-SH intermediate complex; in the course of this reaction thiosulfate (HS₂O₃–) and polysulfides are produced [66 –68].

The second source of sulfide in RBCs, i.e., its endogenous production, is believed to be governed by the enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) [69]. This process takes place in two steps. First, 3-MST catalyzes the sulfur transfer from the 3-mercaptopyruvate (3-MP), an achiral a-keto acid which is a substrate of 3-MST, to an active site cysteine, giving pyruvate and 3-MST-bound persulfide. Then, the 3-MST-bound persulfide reacts with thiols or thioredoxin (Trx) to release H₂S –this is facilitated by the presence of the nicotinamide adenine dinucleotide phosphate (NADPH) and/or Trx reductase [69, 70].

The RBCs, which lack organelles, cannot remove H₂S via the mitochondrial sulfide oxidation pathway. Instead, it is oxidized by ferric hemoglobin to a mixture of thiosulfate and iron-bound polysulfides [68].

Another important finding, which is of special interest here, is the evidence that RBCs may also play an important role in the crosstalk between H₂S and another gasotransmitter, nitric oxide (NO), which is one of the well-known main regulators of cardiovascular homeostasis [69, 71]. While the RBC’s metabolic pathways of NO have been increasingly found to be associated with blood pressure regulation or cardiovascular pathophysiology, not much is known about such a link involving H₂S. In this context, the NO-H₂S crosstalk may provide a mechanism which links the elemental sulfur concentration in erythrocytes with blood pressure and heart geometry.

In the present study, we examined relations between elemental sulfur (S), chlorine (Cl), and phosphorus (P) concentration in erythrocytes measured with respect to the concentrations of potassium (K) on the one hand, and, on the other hand, parameters characterizing heart geometry and blood pressure in patients with SBP and DBP below 140 or 90 mm Hg, respectively. The patients were otherwise healthy and untreated. The observed relationships are discussed in the frame of RBC’s metabolic pathways involving mostly sulfur complexes.

2 Material and methods

2.1 Participants

The study group included 42 outpatients of the Primary Care Unit, who were selected with an objective to form a group of healthy individuals, characterized by a maximally wide distribution of arterial blood pressure (BP) parameters, but without involving patients with advanced stages of hypertension. To meet this requirement, we selected individuals whose average 24-hour systolic blood pressure (SBP) and average 24-hour diastolic blood pressure (DBP) were lower than 140 mmHg and 90 mmHg, respectively.

In addition, the patients did not use tobacco and were not on any therapy prior to entering the study.

For recruitment, all individuals underwent a full medical examination. The recruitment was conducted by one physician (M. F.) who collected thorough medical history and performed physical examination for each patient. Ambulatory blood pressure measurements (ABPM) were performed using the validated devices for 24-hour blood pressure monitoring (Spacelabs 90207 and 90217; Spacelabs Healthcare Inc., Washington, USA). The BP readings were taken at 20 and 30 minutes intervals during a daytime (6 AM to 10 PM) and nighttime (10 PM to 6 PM), respectively. ABPM data were included in the analysis if the number of successful BP readings exceeded 70%. In the analyses, averaged 24-hour BP values were taken for consideration. According to the latest ESC/ESH guidelines for the management of arterial hypertension [72], for 24-hour ABPM values, the diagnostic threshold for hypertension is SBP/DPB≥130/80 mm Hg.

The study was conducted in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans [73], and it was approved by the Bioethics Committee of the Jagiellonian University (KBET/255/B/2012). The informed consent was obtained from each patient enrolled in this study.

2.2 Clinical and laboratory data

For each individual enrolled in the study, clinical data were obtained on, in particular, the parameters of left ventricular geometry such as: left atrial diameter (LA), systolic and diastolic left ventricle diameter (LVs, LVd), systolic and diastolic posterior wall thickness (PWTs, PWTd), systolic and diastolic interventricular septal thickness (IVSTs and IVSTd), early-atrial wave ratio (E/A), and isovolumic relaxation time (IVRT) by using echocardiography (Vivid 4, General Electric). The relative wall thickness (RWT), defined as 2 times posterior wall end-diastolic thickness (PWTd) divided by the left ventricular diastolic diameter (LVd), was calculated as a measure of heart remodeling. All measurements were performed by the same dedicated person (J.K.).

Further laboratory data were collected on blood parameters such as hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and RBC distribution width (RDW). Also, the lipids profile, fibrinogen and hs-CRP were determined.

For the trace element concentration measurement in erythrocytes, peripheral whole blood was collected using sterile heparin tubes with a volume of 2.6 ml. The collection was done after overnight fasting of at least 12 hours at normal ambient temperature. Each sample was centrifuged at 1200 g for 10 minutes, after which the plasma and erythrocytes were drawn off and decanted into a pre-cleaned, pre-weighed, labeled container while the buffy coat layer was discarded. These were frozen at –80 °C and placed into a freeze drier for 24 hours initially and then a further 24 hours until a constant weight was achieved.

Elemental analyses of samples were carried out using the PIXE (Proton Induced X-ray Emission) technique, employing the proton microprobe [74] at the Van de Graaff accelerator at the Institute of Nuclear Physics PAN in Krakow. A 2 MeV proton beam with a diameter of about 30μm was used and all samples were analyzed under vacuum using a typical beam current of 200–300 pA, for a collection time of 30 minutes per sample. The size of the X-ray Amptek SDD detector was 70 mm², internally collimated to 50 mm². The silicon crystal (500μm thick) was covered with a 12.5μm beryllium window, allowing quantitative elemental analysis for elements from sodium upwards, with an energy resolution of 125–135 eV. The detector was placed at l35° with respect to the incident proton beam, at a sample to crystal distance of 30 mm.

For the elemental analysis, the 30μl droplets of erythrocytes were deposited on carbon tape and dried. After drying, sample formed a shape resembling the top of a volcano, with a flat center and circular edges. The proton beam was penetrating the thick part of the sample material, thicker than the range of protons. The X-ray spectra were collected using the proprietary software and analyzed using the GUPIX code [75], which allowed quantification of phosphorus (P), chlorine (Cl), sulfur (S) and potassium (K) elemental concentrations at the ppm (weight) level.

To avoid any systematic uncertainties, only the ratios between mutual microelement erythrocyte concentrations (all pairs out of P, Cl, S and K) were considered in the analyses.

2.3 Statistical analysis

Normal distribution of continuous variables was verified by using the Shapiro-Wilk test. Demographic and clinical characteristics are presented as mean±standard deviation. Correlation analysis was used to estimate unadjusted relationships between variables associated with parameters of heart geometry and arterial blood pressure on the one hand, and the ratios of microelements concentration in erythrocytes, P/Cl, S/P, P/K, S/Cl, Cl/K, S/K on the other hand. Also, correlations with some selected laboratory parameters, Ht, RDW and MCHC were considered. The relationships which showed high statistical significance were adjusted to the subjects’ age, gender and Body Mass Index (BMI) (the adjusted models) using standard regression analysis technique. Since the S/K ratio was the only independent variable which, in the analyses described above, showed statistically significant relationships with the parameters of heart geometry RWT, PWTd and systolic blood pressure SBP, the final multiple regression models were built by individually taking RWT, PWTd and SBP as dependent parameters, and the S/K ratio, together with age, gender, BMI, and laboratory blood-related quantities Ht, RDW and MCHC, as independent variables.

The level of significance was set at the alpha level≤0.05, two-tailed. The database management and statistical analysis were performed using the SAS software, version 9.3 (SAS Institute Inc, Cary, NC), licensed for the Jagiellonian University Medical College, in Krakow.

3 Results

The average values of measured parameters for the study group are shown in Table 1. The group included 15 men and 27 women. The average age and its standard deviation in the group was 47.2 and 10.5 years, respectively. The average value of the BMI was 25.8 kg/m² with the standard deviation of 3.1 kg/m². These main patients’ characteristics played a crucial role in data analysis, since special emphasis was put on the adjustment for age, gender and BMI.

Table 1
Characteristics of the study group. Data are presented as mean±standard deviation

Variable Mean±SD (n = 42) Normal range

General

Age [years] 47.2±10.5

Male vs. female (%) 35.7 vs. 64.3

Body Mass Index (kg/m²) 25.8±3.1 18.5–24.9

Blood pressure

24-hour systolic BP [mm Hg] 119.1±11.9 < 130

24-hour diastolic BP [mm Hg] 76.0±7.8 < 80

Heart geometry

Left atrial diameter (LA) [mm] 39.5±4.9 20–40

Left ventricle end-systolic (LVs) [mm] 29.1±5.0 22–37

Left ventricle end-diastolic (LVd) [mm] 47.7±4.5 33–56

Posterior wall end-systolic thickness (PWTs) [mm] 15.6±1.6 10–15

Posterior wall end-diastolic thickness (PWTd) [mm] 9.2±1.4 6–11

Interventricular septum end-systolic thickness (IVSTs) [mm] 13.7±1.6 -

Interventricular septum end-diastolic thickness (IVSTd) [mm] 9.5±1.4 6–11

Left ventricular mass index (LVMI) [g/m²] 85.0±20.1 Men 49–115 g/m² Women 43–95 g/m²

Relative wall thickness (RWT) 0.39±0.05 0.22–0.42

Complete blood count

Erythrocyte count [million/mm³] 4.67±0.42 4.00–5.00×10⁶

Hemoglobin [g/dl] 13.8±1.4 12–16

Hematocrit [%] 41.0±3.3 37–47

MCV [fl] 87.9±3.6 80–96

MCHC [g/dl] 33.8±1.3 31–36

Red blood cell distribution width (RDW) [%] 13.00±0.69 11.6–14.4

Laboratory blood parameters

Total cholesterol [mmol/l] 5.16±0.86 3.2–5.2

Low-density lipoprotein (LDL) [mmol/l] 3.13±0.78 < 3.4

Triglycerides (TG) [mmol/l] 1.07±0.53 < 2.26

Fibrinogen [g/l] 2.82±0.86 1.8–3.5

Glucose [mmol/l] 4.86±0.49 3.3–5.6

Glomerular filtration rate (eGFR) [ml/min/1.73m²] 105.6±24.6 > 60

High-sensitivity C-reactive protein (hs-CRP) [mg/l] 1.05±0.62 < 3

RBC microelement concentration ratio

S/K 0.85±0.18

S/P 4.97±1.07

S/Cl 2.10±0.55

P/K 0.18±0.06

Cl/K 0.43±0.13

P/Cl 0.43±0.10

Variable	Mean±SD (n = 42)	Normal range
General
Age [years]	47.2±10.5
Male vs. female (%)	35.7 vs. 64.3
Body Mass Index (kg/m²)	25.8±3.1	18.5–24.9
Blood pressure
24-hour systolic BP [mm Hg]	119.1±11.9	< 130
24-hour diastolic BP [mm Hg]	76.0±7.8	< 80
Heart geometry
Left atrial diameter (LA) [mm]	39.5±4.9	20–40
Left ventricle end-systolic (LVs) [mm]	29.1±5.0	22–37
Left ventricle end-diastolic (LVd) [mm]	47.7±4.5	33–56
Posterior wall end-systolic thickness (PWTs) [mm]	15.6±1.6	10–15
Posterior wall end-diastolic thickness (PWTd) [mm]	9.2±1.4	6–11
Interventricular septum end-systolic thickness (IVSTs) [mm]	13.7±1.6	-
Interventricular septum end-diastolic thickness (IVSTd) [mm]	9.5±1.4	6–11
Left ventricular mass index (LVMI) [g/m²]	85.0±20.1	Men 49–115 g/m² Women 43–95 g/m²
Relative wall thickness (RWT)	0.39±0.05	0.22–0.42
Complete blood count
Erythrocyte count [million/mm³]	4.67±0.42	4.00–5.00×10⁶
Hemoglobin [g/dl]	13.8±1.4	12–16
Hematocrit [%]	41.0±3.3	37–47
MCV [fl]	87.9±3.6	80–96
MCHC [g/dl]	33.8±1.3	31–36
Red blood cell distribution width (RDW) [%]	13.00±0.69	11.6–14.4
Laboratory blood parameters
Total cholesterol [mmol/l]	5.16±0.86	3.2–5.2
Low-density lipoprotein (LDL) [mmol/l]	3.13±0.78	< 3.4
Triglycerides (TG) [mmol/l]	1.07±0.53	< 2.26
Fibrinogen [g/l]	2.82±0.86	1.8–3.5
Glucose [mmol/l]	4.86±0.49	3.3–5.6
Glomerular filtration rate (eGFR) [ml/min/1.73m²]	105.6±24.6	> 60
High-sensitivity C-reactive protein (hs-CRP) [mg/l]	1.05±0.62	< 3
RBC microelement concentration ratio
S/K	0.85±0.18
S/P	4.97±1.07
S/Cl	2.10±0.55
P/K	0.18±0.06
Cl/K	0.43±0.13
P/Cl	0.43±0.10

Data are presented as mean±standard deviation. BP: blood pressure; S/K: sulfur to potassium erythrocyte concentration ratio; S/P: sulfur to phosphorus erythrocyte concentration ratio; S/Cl: sulfur to chlorine erythrocyte concentration ratio; P/K: phosphorus to potassium erythrocyte concentration ratio; Cl/K: chlorine to potassium erythrocyte concentration ratio; P/Cl: phosphorus to chlorine erythrocyte concentration ratio.

The first part of the remaining analysis regarded the relationships between variables related to blood pressure and heart function parameters in all patients participating in the study on the one hand, and P/Cl, S/P, P/K, S/Cl, Cl/K, S/K ratios on the other hand. Unadjusted linear regression analysis, the results of which are presented in Table 2 and Table 3, showed statistically significant positive correlations between: (i) RWT and S/K (Fig. 1a), (ii) LVMI and S/K, (iii) SBP and S/K, (iv) SBP and P/K, (v) PWTd and S/K (Fig. 1b), (vi) PWTd and P/K, (vii) IVSTd and S/K, (viii) IVSTd and P/K. However, the regression analyses in which the above relationships were adjusted to patients’ age, gender and BMI, confirmed the high statistical significance only for the cases of RWT vs. S/K (p < 0.014) and PWTd vs. S/K (p < 0.004) (Table 2 and 3). In addition, of importance are the correlations RWT vs. SBP and PWTd vs. SBP (Fig. 1c and Fig. 1d), which also display high statistical significance in age-gender-BMI-adjusted regression analysis: p < 0.036 and p < 0.009, respectively.

Table 2

Results of the unadjusted and age-gender-BMI-adjusted analyses with RWT, LVMI, SBP as dependent variables, and age, gender, BMI, S/K, S/P, S/Cl, P/K, Cl/K, P/Cl, SBP, DBP, Ht, RDW-CV and MCHC as independent variables. The age-gender-BMI-adjusted regression analysis was performed only when the unadjusted approach showed statistically significant dependence

	Relative wall thickness (RWT)					Left ventricular mass index (LVMI) [g/m²]					24-hour systolic blood pressure (SBP) [mm Hg]
	Unadjusted		Age-gender-BMI adjusted			Unadjusted		Age-gender-BMI adjusted			Unadjusted		Age-gender-BMI adjusted
	r	p	β (SE)	p	R²	r	p	β (SE)	p	R²	r	p	β (SE)	p	R²
Gender [F = 1/M = 2]	0.377	0.014	–	–	–	0.417	0.006	–	–	–	0.531	0.0003	–	–	–

Age [years]	0.141	0.374	–	–	–	0.223	0.155	–	–	–	0.056	0.724	–	–	–
Body Mass Index	0.101	0.524	–	–	–	0.265	0.090	–	–	–	0.333	0.031	–	–	–
S/K	0.501	0.0007	0.119 (46)	0.014	0.295	0.430	0.0045	28.5 (17.3)	0.108	0.314	0.480	0.0013	17.9 (9.5)	0.067	0.408
S/P	0.108	0.498	–	–	–	–0.135	0.393	–	–	–	–0.037	0.814	–	–	–
S/Cl	0.220	0.162	–	–	–	0.105	0.508	–	–	–	0.158	0.317	–	–	–
P/K	0.220	0.161	–	–	–	0.335	0.030	62.5 (53.4)	0.249	0.290	0.305	0.050	12.3 (30.1)	0.685	0.354
Cl/K	0.085	0.591	–	–	–	0.126	0.427	–	–	–	0.217	0.167	–	–	–
P/Cl	0.146	0.355	–	–	–	0.248	0.113	–	–	–	0.148	0.348	–	–	–
SBP [mm Hg]	0.467	0.002	0.0017 (8)	0.036	0.263	0.444	0.003	0.414 (288)	0.159	0.303	–	–	–	–	–
DBP [mm Hg]	0.308	0.047	0.0016 (12)	0.209	0.203	0.201	0.201	–	–	–	–	–	–	–	–
Ht [%]	0.220	0.162	–	–	–	0.080	0.613	–	–	–	0.474	0.002	0.775 (656)	0.245	0.375
RDW-CV [%]	–0.172	0.275	–	–	–	–0.118	0.455	–	–	–	–0.228	0.147	–	–	–
MCHC [g/dl]	0.162	0.304	–	–	–	0.045	0.778	-	-	-	0.163	0.302	–	–	–

r: Pearson correlation coefficient; β (SE): regression coefficient [standard error]; R²: adjusted R² for the regression models; S/K: sulfur to potassium erythrocyte concentration ratio; S/P: sulfur to phosphorus erythrocyte concentration ratio; S/Cl: sulfur to chlorine erythrocyte concentration ratio; P/K: phosphorus to potassium erythrocyte concentration ratio; Cl/K: chlorine to potassium erythrocyte concentration ratio; P/Cl: phosphorus to chlorine erythrocyte concentration ratio.

Table 3

Results of the unadjusted and age-gender-BMI-adjusted analyses with LA, PWTd, IVSTd as dependent variables, and age, gender, BMI, S/K, S/P, S/Cl, P/K, Cl/K, P/Cl, SBP, DBP, Ht, RDW-CV and MCHC as independent variables. The age-gender-BMI adjusted regression analysis was performed only when the unadjusted approach showed statistically significant dependence

	Left atrial diameter (LA) [mm]					Posterior wall end-diastolic thickness (PWTd) [mm]					Interventricular septum end-diastolic thickness (IVSTd) [mm]
	Unadjusted		Age-gender-BMI adjusted			Unadjusted		Age-gender-BMI adjusted			Unadjusted		Age-gender-BMI adjusted
	r	p	β (SE)	p	R²	r	p	β (SE)	p	R²	r	p	β (SE)	p	R²
Gender [F = 1/M = 2]	0,340	0.028	–	–	–	0.608	0.0002	–	–	–	0.553	0.0001	–	–	–
Age [years]	0.262	0.094	–	–	–	0.154	0.329	–	–	–	0110	0.486	–	–	–
Body Mass Index [kg/m²]	0.360	0.019	–	–	–	0.329	0.033	–	–	–	0.270	0.083	–	–	–
S/K	0.213	0.175	–	–	–	0.606	< 0.0001	2.93 (95)	0.004	0.563	0.409	0.007	1.31 (1.13)	0.253	0.378
S/P	–0.284	0.068	–	–	–	–0.068	0.667	–	–	–	–0.101	0.524	–	–	–
S/Cl	–0.051	0.749	–	–	–	0.187	0.235	–	–	–	0.182	0.248	–	–	–
P/K	0.253	0.105	–	–	–	0.406	0.008	3.80 (3.19)	0.241	0.472	0.335	0.030	2.52 (3.48)	0.473	0.365
Cl/K	0.157	0.321	–	–	–	0.184	0.244	–	–	–	0.072	0.649	–	–	–
P/Cl	0.152	0.337	–	–	–	0.278	0.075	–	–	–	0.298	0.056	–	–	–
SBP [mm Hg]	0.436	0.004	0.102 (69)	0.148	0.312	0.641	< 0.0001	0.045 (16)	0.009	0.547	0.524	0.0004	0.032 (18)	0.094	0.403
DBP [mm Hg]	0.234	0.135	–	–	–	0.402	0.008	0.019 (26)	0.480	0.460	0.403	0.008	0.030 (29)	0.293	0.375
Ht [%]	0.327	0.035	0.265 (287)	0.362	0.288	0.279	0.073	–	–	–	0.298	0.056	–	–	–
RDW-CV [%]	–0.072	0.650	–	–	–	–0.085	0.591	–	–	–	–0.402	0.008	–0.604 (255)	0.023	0.440
MCHC [g/dl]	0.116	0.463	–	–	–	0.188	0.233	–	–	–	0.179	0.256	–	–	–

Fig. 1

Dependence of the relative wall thickness (RWT) and posterior wall end-diastolic thickness (PWTd) on the sulfur to potassium erythrocyte concentration ratio (S/K) and 24-hour systolic blood pressure (SBP): (a) RWT vs. S/K, (b) PWTd vs. S/K, (c) RWT vs. SBP, (d) PWTd vs. SBP, for all patients.

The results of the final multivariate regression models, which were constructed to check the high statistical significance of correlations observed in the age-gender-BMI-adjusted approach (Table 4), confirmed the above findings: RWT and PWTd increase in a highly statistically significant manner with increasing S/K concentration ratio: p < 0.019 and p < 0.018, respectively. Similarly, positive correlations with high statistical significance were found between SBP and S/K: p < 0.0035. The results are presented in Table 4.

Table 4

Results of the full multivariate regression analyses with RWT, PWTd, SBP as dependent variables, and age, gender, BMI, S/K, Ht, RDW-CV and MCHC as independent variables. Only those dependent variables were considered, for which the age-gender-BMI-adjusted regression analysis showed high statistical significance with the elemental erythrocyte concentration ratio

	Multivariate standard regression analyses
	RWT model R² = 0.310		PWTd [mm] model R² = 0.573		SBP [mm Hg] model R² = 0.525
	β [SE]	p-value	β [SE]	p-value	β [SE]	p-value
Gender [F = 1/M=2]	0.009 [27]	0.737	1.49 [56]	0.013	0.04 [510]	0.994
Age (years)	0.0004 [8]	0.653	0.011 [17]	0.532	–0.125 [156]	0.428
BMI [kg/m2]	–0.0003 [26]	0.920	0.083 [52]	0.126	0.945 [475]	0.055
S/K	0.138 [56]	0.019	2.89 [116]	0.018	32.8 [105]	0.0035
Ht [%]	0.0022 [37]	0.544	–0.034 [75]	0.651	1.65 [68]	0.020
RDW-CV [%]	–0.007 [12]	0.598	0.046 [251]	0.855	–2.21 [226]	0.334
MCHC [g/dl]	–0.0025 [78]	0.754	–0.092 [161]	0.571	–2.07 [145]	0.162

Model R²: adjusted R² for the regression models.

4 Discussion

The data presented in this work show that in the carefully selected group of healthy patients, who are nonsmokers and did not undergo any therapy, and whose ambulatory blood pressure values SBP and DBP are below 140 mm Hg or 90 mm Hg, respectively, the erythrocyte S/K concentration ratio demonstrates positive correlation with both blood pressure and the degree of adverse changes in heart geometry. Upon a first glance, one could argue that such correlation could result entirely from the changes of the RBC potassium concentration: decreasing erythrocyte potassium content leads to adverse changes in blood pressure and heart geometry. However, since none of the remaining RBC microelement ratios involving potassium, i.e., P/K and Cl/K, were related to SBP, DBP and heart parameters in any statistically significant manner, it was hypothesized that also sulfur concentration in erythrocytes can be a marker of the blood pressure regulation and cardiac remodeling. This triggered a discussion on the potential mechanism responsible for such a process.

Not much is known about the association of the sulfur concentration level in erythrocytes with metabolic processes. A possible link in this case is offered by the metabolism of hydrogen sulfide H₂S, which, to a large extent, is responsible for the erythrocyte content of sulfur.

As mentioned earlier, H₂S can be synthetized in RBCs with the help of the enzyme 3-MST, but these cells lack organelles and cannot dispose of H₂S via the mitochondrial sulfide oxidation pathway. Instead, it is possible that sulfide generated in the RBCs, before being oxidized by hemoglobin (in noncanonical pathways generating metabolites, which substantially contribute to the pool of elemental sulfur in erythrocytes [66, 67]), may affect the regulation of NO signaling in RBCs and, consequently, the RBC’s NO bioavailability in the circulatory system.

In the work of B. Geng et al. [76] it was shown that H₂S downregulates the aortic L-Arg/eNOS/NO pathway in vitro and in vivo, and that the K_ATP channel could be involved in this regulatory process. It was also recently demonstrated that RBCs carry a functional endothelial nitric oxide synthase (eNOS) [77 –79] and they are able to synthesize, transport and release NO metabolic products and ATP, thereby substantially contributing to the systemic NO bioavailability and vascular tone. It is very likely that the mechanism of the H₂S-induced downregulation of the NO bioavailability, which was described above in case of the aortic L-arginine/nitric oxide pathway [76], will also be active in erythrocytes. In consequence, the amount of the NO metabolic products would be inversely proportional to the amount of the sulfur-containing complexes resulting from this metabolic pathway. Thus, larger quantity of sulfur accumulated in RBCs would indicate the lowering of the RBC’s NO bioavailability and the circulating eNOS deficiency, which could be the cause of the less effective RBCs pleiotropic activity, leading to the increase of blood pressure and adverse changes in circulatory and cardiovascular systems. Support for this scenario comes from the paper by M. Zheng et al. [80] who showed that the increase of endogenous H₂S production in erythrocytes in vitro is strongly correlated with both the increase of blood pressure and the lowering of the serum NO level.

Of course, one should be aware that, in addition to the mechanism considered above, other metabolic processes may also contribute to the relationship observed between the sulfur concentration in RBCs on the one hand and both blood pressure and adverse changes in heart geometry on the other hand, and the need to identify these processes requires further research.

There are several advantages and limitations of the presented study. First of all, one should notice that, to our knowledge, investigations aiming at relating the sulfur erythrocyte concentration with the parameters of blood pressure and heart geometry has, thus far, not been reported in the literature. What contributes to the strength of the work is also the high precision of the PIXE method in establishing the ratio between the erythrocyte elemental concentrations. There are, however, several limitations to this study, which have to be considered when planning next series of measurements. The assessment of microelement concentration in erythrocytes using the PIXE method are very time consuming which makes it impossible to perform measurements for large groups of patients. In our case this necessitated, for example, a small sample size in gender subgroups and we were unable to perform the analysis separately for men and women.

5 Conclusions

The presented results evidence an association of heart geometry as well as the blood pressure with the sulfur and potassium concentration ratio (S/K) in erythrocytes in a group of healthy patients, carefully selected to be free of confounding factors such as smoking or undergoing any treatment. The observed relationships indicate the presence of a mechanism which relates the increase of erythrocyte sulfur concentration with the increase of blood pressure and/or development of adverse changes in heart geometry. Such mechanism may be associated with the downregulating role of the H₂S, endogenously produced in erythrocytes, on the RBC’s eNOS activity, which can lead to the lowering of the RBC’s NO bioavailability and the reduction of RBC’s carried eNOS activity and, further, to worsening the circulatory and cardiovascular conditions. The increase of H₂S-induced downregulation would be associated with a larger deposition of sulfur-containing products in erythrocytes. To understand the reason behind the increased crosstalk between endogenously produced H₂S and eNOS in RBCs in patients who show a tendency of hypertension development and heart remodeling, one needs further studies which take into account clinical observables together with a detailed analysis of the processes occurring in erythrocytes at the molecular level.

Footnotes

Acknowledgments

The authors would like to thank prof. Wojciech M. Kwiatek for his comments on the manuscript. The work was supported by the Collegium Medicum of the Jagiellonian University project K/ZDS/005668.

Conflicts of interest

The authors declare no conflict of interest.

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