Effect of magnesium supplementation on blood rheology in NOS inhibition-induced hypertension model

Abstract

This study investigated the effects of magnesium on blood rheological properties and blood pressure in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension was induced by oral administration of the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/day) for 6 weeks and systolic blood pressure was measured by the tail-cuff method. The groups receiving magnesium supplementation were fed with rat chow containing 0.8% magnesium oxide during the experiment. At the end of experiment, blood samples were obtained from abdominal aorta, using ether anesthesia. Plasma and erythrocyte magnesium levels were determined by the atomic absorption spectrometer. RBC deformability and aggregation were determined by rotational ektacytometry. Plasma fibrinogen concentration was evaluated by ELISA. Whole blood and plasma viscosities were determined by viscometer and intracellular free Ca⁺⁺ level was measured by using spectroflurometric method. Blood pressure was elevated in hypertensive groups and suppressed by magnesium therapy. Plasma viscosity and RBC aggregation were found to be higher in hypertensive rats than control animals and these parameters significantly decreased in magnesium supplemented hypertensive animals. Other measurements were not different between experimental groups. These results confirm that blood pressure, plasma viscosity and RBC aggregation increased in NOS inhibition-induced hypertension model and oral magnesium supplementation improved these parameters.

Keywords

Hypertension magnesium supplementation blood rheology

1 Introduction

Essential hypertension is a multi-factorial disorder and associated with increased total peripheral vascular resistance (TPVR). Many determinants have been shown to involve in the pathogenesis of essential hypertension such as sympathetic nervous system hyperactivation, renin-angiotensin-aldosteron system and endothelial dysfunction [27, 37]. On the other hand clinical and experimental studies support the role of magnesium in hypertension and most of the studies suggest that there is an inverse relationship between intake of magnesium and high blood pressure [1, 35]. Decreased serum and tissue magnesium concentrations have also been reported in various experimental models of hypertension including nitric oxide synthase (NOS) inhibition-induced hypertension [4 , 28].

It has been suggested that magnesium supplementation might be used as an antihypertensive agent in human hypertension despite the fact that there is no definite consensus on this issue. Several studies proposed that magnesium supplementation has a lowering effect on high blood pressure and this effect is more prominent in patients using antihypertensive medications [15, 36] while, some other studies have reported the opposite [38]. The effect of magnesium therapy on high blood pressure has also been studied on some experimental animal models of hypertension. Magnesium has been found to have lowering effect on high blood pressure in DOCA-salt and cyclosporine-induced hypertension models [5, 20].

Although the interpretation between hypertension and blood rheology is not clear, it is strongly supported that variations in the rheological properties of blood contribute to hypertension pathogenesis. Increased hematocrit (Htc), whole blood and plasma viscosity and red blood cell (RBC) aggregability have been presented by several investigations in hypertensive patients [18, 24]. On the other hand, several experimental investigations also demonstrated altered RBC rheological properties in hypertensive animals [6, 10]. It has been suggested that these rheological variations might contribute to inadequate microcirculation and tissue damage that is seen in hypertension [9 , 24]. However, potential blood rheology improving effects of anti-hypertensive treatments remain uncertain.

The aim of the present study was to investigate the effect of oral magnesium supplementation on blood rheological parameters in NOS inhibition-induced hypertension model.

2 Materials and methods

2.1 Animals

Forty Wistar male rats (8 weeks old) were used in this study. The rats were housed in stainless steel cages with a constant room temperature at 23 ± 2°C and on a 12:12-h light-dark cycle and had free access to rat chow and drinking water. The experimental protocol was approved by the Animal Care and Usage Committee of Akdeniz University and followed the guidelines for using animals in experimental research.

2.2 Groups

The animals were divided randomly into four groups: control (C; n = 10), magnesium (M; n = 10), hypertensive (HT; n = 10) and hypertensive+magnesium (HT+M; n = 10). In HT and HT+M groups, hypertension was induced by oral administration of the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME; 25 mg/kg/day) which dissolved in drinking water, for 6 weeks. The C and H groups were fed with standard rat chow, meanwhile the M and HT+M groups fed with rat chow containing 0.8% magnesium oxide (MgO). L-NAME and magnesium supplementation were simultaneously given to the HT+M group. The body weights were assessed at the beginning and at the end of the experiment, fluid and food consumption was monitorized regularly.

2.3 Blood pressure measurement

The systolic blood pressure of rats was measured using a non-invasive tail-cuff method. The measurements were performed at the beginning of the experiment and once a week during the experiment. Measurement data was obtained with a MAY-BPHR 9610-PC unit and MP 150 data-acquisition system (BIOPAC Systems; Santa Barbara, CA). The systolic blood pressure value for each rat was calculated by using the mean of at least 3 clear measurements.

2.4 Preparation of blood samples

At the end of the experiment, the rats were anesthetized with ether. Blood samples were collected from abdominal aorta of rats and anticoagulated with heparin. Eight mL of blood was layered on top of 16 mL of a polysucrose (60 g/L) and sodium diatrizoate (167 g/L) solution (Histopaque1119, Sigma Chemical Co., St. Louis,MO,USA) in a 50 mL polypropylene tube and centrifuged at 700×g for 30 minutes to remove leukocytes. The RBC pellet was washed three times and re-suspended to a Htc of 0.4 L/L with autologous plasma. Some of the blood and plasma samples were kept at –80°C until the time of measurement of magnesium levels.

2.5 Biochemical parameters

Plasma fibrinogen level was measured by ELISA (Assaymax, catalog no: ERF1040-1, Assaypro LLC, St. Charles, MO 63304). Htc, mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were detected by an oto-analyzer system (Micros, ABX Co, France).

2.6 RBC defomability measurements

RBC deformability was measured at 0.3–50 Pascal (Pa) shear stresses by laser diffraction analysis by using an ektacytometer (LORCA, RR Mechatronics, Hoorn, The Netherlands) as described before [33]. Briefly a low Htc suspension samples of RBC in an isotonic viscous medium (4% polyvinylpyrrolidone 360 solution, MW = 360 kDa) is sheared in a Coutte system consisted of a glass cup and fitting bob, with a gap of 0.36 mm between the cylinders. A laser beam is directed through the sheared sample and the diffraction pattern produced by the deformed cells is analyzed by a desktop personal computer, which also controls the stepper motor that generates the pre-determined shear stresses. All measurements were done at 37°C. Based upon the geometry of the elliptical diffraction pattern, an elongation index (EI) is calculated as: EI = (L-W)/(L + W), where L and W are the length and width of the diffraction pattern. EI values, determined for nine shear stresses between 0.3–50 Pa, were used to calculate the shear stress required for half-maximal deformation (SS1/2) by applying a Lineweaver-Burk analysis procedure. Impaired RBC deformability leads to increased SS1/2 values; SS1/2 values are used herein since the presentation and comparison of data via this approach are more convenient than via merely displaying shear stress-EI curves [2].

2.7 Determination of RBC aggregation

RBC aggregation was also evaluated using an ektacytometer (LORCA, RR Mechatronics, Hoorn, The Netherlands). Red blood cell aggregation was determined by placing 1 ml of blood sample into the space between two glass cylinders. Blood sample was rotated at a shear rate of 800 sn^–1 for 10 seconds. This kind of movement of blood decays red blood cell aggregates which is called disaggregation and following the disaggregation procedure, the reflection of light bundles emitted to blood samples by a diot placed inside of the cylinder was recorded to the computer for 120 seconds. The alterations in the reflection of light during the aggregation process of the erythrocyte suspension placed in the cylindrical system were monitored and several parameters which present RBC aggregation were simultaneously calculated by a computerized system. These parameters include the rank of RBC aggregation (average erythrocyte numbers in the aggregates) besides the progression of aggregation due to time course. All measurements were conducted at 37°C.

2.8 Determination of plasma and whole blood viscosity

Plasma and whole blood viscosity measurements were carried out by using rotational viscometer (Brookfield, DVII-Pro viscometer, Massachusetts, USA) at 37°C, at 10 rpm.

2.9 Intracellular free calcium measurement

Intracellular free calcium concentration of erythrocytes was measured spectrofluorometrically using Fluo-4 AM calcium indicator [4]. After erythrocytes were washed 2 times with HEPES buffer solution consisted of HEPES 10 mM; NaCl 151 mM; KCl 4.7 mM; CaCl2 2 mM; MgCl2 1.2 mM; glucose 7.8 mM, cells have been resuspended in 1 ml vials containing 15×10⁶ cells. Then the vial was incubated with 1 μM Fluo-4AM for 30 minutes in dark room following 2 times washing steps by centrifugation (2500 rpm, 5 min) to remove excess dye and then equilibrated for deesterification for 15 min. Indicator loaded cells were measured via Shimadzu RF-5500, Kyoto, Japan spectrofluorometer using 485 nm excitation and 515 nm emission wavelength. Each vial was measured successively 3 times for 200 second. In order to determine maximum (F_max) and minimum (F_min) fluoresence intensity 1% Triton-X and 200 mM EDTA were respectively added into suspension. Intracellular calcium concentration was calculated by following equation;

[Ca]_i = K_d×(F-F_min) / (F_max-F)

Dissociation constant (K_d) value selected as 345 nM.

2.10 Measurements of erythrocyte and plasma magnesium levels

The plasma and erythrocyte magnesium levels were measured using the atomic absorption spectrometer (Varian AA280FS, USA).

2.11 Statistical analysis

All results were presented as means ± SE. The statistical significance between the weekly blood pressure measurements were tested using repeated measures ANOVA followed by the Bonferoni post-hoc test. The one-way ANOVA supported by the Newman Keuls post-hoc test was used for the statistically assessment of other parameters. A P value <0.05 was considered significant.

3 Results

Body weight, fluid and food consumption were not different between the groups (data not shown).

The initial levels of systolic blood pressure were not different between the groups. Blood pressure was found to be significantly higher after the first week of L-NAME supplementation in the HT group compared to C group, and remained higher during the experimental period (Fig. 1, p < 0.001). Blood pressure values of HT+M group were found to be similar with HT group during the first 4 weeks of the experiment. In the 5th week, a slight but not statistically significant fall in the systolic blood pressure was detected in the HT+M group while this decrement became significant at the 6th week of the study (p < 0.01).

Plasma and erythrocyte magnesium levels were shown in Table 1. Plasma magnesium levels increased in M group compared to C group (p < 0.05). Intracellular magnesium concentration were lower in HT group compared to C group (p < 0.001) while there was no difference in terms of plasma magnesium concentration in these groups. On the other hand, magnesium supplementation caused a significant increment in magnesium levels of both plasma and erythrocyte in HT+M rats compared with H animals (p < 0.05).

Htc, MCV and MCHC values were shown in Table 2. MCV significantly increased in HT group compared to control rats (p < 0.01). However magnesium supplementation did not affect this parameter in HT+M group. Htc and MCHC values were similar among all experimental groups.

Blood and plasma viscosity were illustrated in Fig. 2. Whole blood viscosity did not differ among experimental groups (Fig. 2A). However, the plasma viscosity increased in HT animals compared with controls (Fig. 2B, p < 0.001). Magnesium supplementation caused a significant decrement in plasma viscosity in HT+M group compared with H group (p < 0.01).

RBC aggregation increased in HT rats compared with controls and magnesium supplementation caused a significant decrement in RBC aggregation in HT group (Fig. 3, p < 0.01). Plasma fibrinogen levels did not show any significant difference among all four groups (Fig. 4).

RBC deformability and cytosolic calcium levels were found similar in all experimental groups (Figs. 5 and 6, respectively).

4 Discussion

The present study demonstrated that systolic blood pressure, RBC aggregation and plasma viscosity increased in NOS inhibition-induced hypertensive rats and magnesium supplementation caused significant decreases in all of these parameters.

The relationship between magnesium insufficiency and hypertension was clearly demonstrated both in experimental hypertension models [5, 20] and epidemiological human studies [12, 15]. Since plasma magnesium level alone may be incapable at evaluation of total magnesium content [8] we measured both plasma and red blood cell magnesium levels in present study. Although plasma magnesium level in hypertensive rats was not changed red blood cell total magnesium level in the same group was lower compared with controls in our experiment. It was already demonstrated that plasma magnesium level did not change while the magnesium content of red blood cells and various tissues reduced in L-NAME hypertension model generated by NOS inhibition [28]. Both plasma and red blood cell magnesium concentrations were significantly increased in hypertensive rats receiving oral magnesium therapy. This increase in plasma and erythrocyte magnesium concentrations in HT+M group compared with HT group was in accordance with the previous studies conducted with other experimental hypertension models [3 , 34].

Since there are some studies which have conflicting results on the efficiency of magnesium in the therapy of hypertension in human subjects, it is prevalently accepted that magnesium intake may be helpful in preventing the progression of hypertension [30, 33]. The results of the studies which investigate the effects of magnesium therapy in experimental hypertension models are much more compatible. The blood pressure lowering effect of magnesium therapy was demonstrated in DOCA-Salt hypertension model, cyclosporine induced hypertension model and spontaneous hypertensive rats [3 , 20]. Magnesium is an important regulator of vascular tonus and its blood pressure reducing effects may result from various mechanisms. The probable mechanisms include intracellular calcium reducing effects of magnesium in vascular smooth muscle cells [23, 25] and secretion regulating effects of magnesium for endothelium derived constructive and dilative agents [26, 31]. In our study, we demonstrated that oral magnesium therapy reduced the increment of blood pressure in L-NAME hypertension model and this effect was statistically significant at sixth week of the therapy. The therapeutically effective dose of magnesium in various hypertension models varies between 0.6% to 1% of rat chow mass [3 , 20]. The magnesium dose we used in our study was 0.8% which remains in the dose range mentioned in previous studies.

On the other hand, the rheological properties of red blood cells that constitute the main part of the cellular elements of whole blood and plasma viscosity contribute to occurrence of resistance to blood flow and determination of TPVR [7 , 21]. It has previously been demonstrated that alteration in rheological properties of blood may contribute to increment of peripheral resistance and hence elevated blood pressure in hypertensive patients [18, 24]. The increase in plasma and blood viscosity, decrease in red blood cell deformability and increase in erythrocyte aggregation are the hemorheological alterations observed in hypertensive patients [18, 24]. However, it is not clear whether the hemorheological alterations observed in hypertensive patients are the cause or the result of hypertension.

The alterations of rheological properties of blood were investigated in various experimental hypertension models. Red blood cell aggregation index was shown to be increased in 2K-1C and DOCA-salt induced hypertension models in rats and increment in plasma fibrinogen concentration accompanied to the elevation of aggregation index in these hypertension models [10]. On the other hand, besides increase in erythrocyte aggregation index, red blood cell deformability was reported to be decreased in L-NAME hypertension model and elevation of intra-erythrocyte free calcium concentration accompanied to reduction in red blood cell deformability [6]. We observed a significant increase in plasma viscosity and red blood cell aggregation index in HT group which received 25 mg/kg/day L-NAME for 6 weeks compared with C group. These findings are in accordance with the previous studies’ results [6, 10]. Additionally both plasma viscosity and aggregation index were significantly lower in HT+M group which supplied simultaneously magnesium therapy with L-NAME, compared with H group in our study. However, there was no difference in terms of whole blood viscosity and RBC deformability between those groups.

Plasma protein content is well accepted to be the most important factor in determination of plasma viscosity. Acute phase reactants such as fibrinogen make significant contribution to plasma viscosity during illness [22]. In our study, despite an elevation in plasma fibrinogen concentration in HT group, this increment was not statistically significant. The reason of this finding may originate from different effects of L-NAME on plasma fibrinogen levels in dose and time dependent manner. Further plasma fibrinogen concentration increases with acute exposure of L-NAME and reverses to basal values in chronic usage [32]. Therefore, increased plasma viscosity detected in hypertensive animals may result from acute phase reactants other than fibrinogen.

Plasma proteins as well have influence on RBC aggregation. Although, fibrinogen is known to be the major plasma protein which facilitates RBC aggregation, changes in plasma fractions of other acute phase reactants and plasma proteins are reported to have similar effects. Besides, Htc level, plasma osmolarity, pH and red blood cell surface charges are other variables which alter RBC aggregation [19, 29]. In our study, RBC aggregation index was found to be significantly increased in HT group compared with C group and magnesium therapy significantly inhibited this increment. Because there is no statistically changes in plasma fibrinogen levels and Htc value in hypertensive animals, the increased aggregation index in our study may result from possible changes in proteins other than fibrinogen and in red blood cell surface charges. Therefore lowering effect of magnesium therapy on these parameters may also be related to these factors.

There was no difference between groups in terms of red blood cell deformability in our study. Moreover, this assumption is supported with the finding that there was no difference in terms of intra-erythrocyte free calcium levels between groups. Although these findings were not correlated with the results of previous studies, this discrepancy may result from usage of 2-3 fold higher doses of L-NAME at these studies [6, 10]. Thereby, the dose of 25 mg/kg/day which we used in our study may be below the range of effective dose that cause alteration in erythrocyte deformability.

In conclusion, RBC aggregation index and plasma viscosity increased in NOS inhibition induced hypertension model. Oral magnesium supplementation caused significant decrease in elevated plasma viscosity and red blood cell aggregation index, as well as increased blood pressure. The blood pressure lowering effects of magnesium may be contributed, even if partly, by its improving impacts on rheological features of blood.

Footnotes

Acknowledgments

This study was supported by the Akdeniz University Research Projects Unit (project no. 2011.03.0122.001).

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