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Keywords:

  • adiponectin;
  • nitric oxide;
  • membrane fluidity;
  • erythrocytes;
  • hypertension

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective: Abnormalities in physicochemical properties of the cell membranes may underlie the defects that are strongly linked to hypertension. Recent evidence indicates that adiponectin may have protective effects against cardiovascular diseases. The purpose of the present study was to assess the possible link between plasma adiponectin and membrane fluidity in normotensive (NT) and hypertensive (HT) men.

Research Methods and Procedures: We measured the membrane fluidity (a reciprocal value of membrane microviscosity) of erythrocytes in NT and HT men by using an electron paramagnetic resonance and spin-labeling method.

Results: The order parameter (S) for the spin label agent (5-nitroxide stearate) and the peak height ratio (h0/h−1) for 16-nitroxide stearate in the electron paramagnetic resonance spectra of erythrocytes were significantly higher in HT men than in NT men, indicating that membrane fluidity of erythrocytes was decreased in HT men compared with NT men. Both of plasma adiponectin and nitric oxide (NO) metabolite levels were significantly lower in HT men than in NT men. The plasma adiponectin levels were correlated with plasma NO metabolites. The S and the h0/h−1 of erythrocytes were inversely correlated with the plasma adiponectin and NO metabolite levels, indicating that the decreased membrane fluidity of erythrocytes was associated with hypoadiponectinemia and reduced plasma NO metabolites.

Discussion: The results of the present study demonstrated that plasma adiponectin levels were lower in HT men than in NT men and that hypoadiponectinemia was associated with decreased membrane fluidity of erythrocytes. The finding suggests that adiponectin may be linked to the rheologic behavior of the erythrocytes and the microcirculation in men, at least in part, by the NO-dependent mechanism.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

There is evidence that dysregulation of adipocytokines may be accompanied by obesity, type 2 diabetes, dyslipidemia, and hypertension and finally result in atherosclerotic vascular diseases (1,2,3,4). Adiponectin, the most abundant secretory protein of adipose tissue in human plasma, has been suggested to actively participate in the regulation of cardiovascular functions in humans because hypoadiponectinemia might be observed in subjects with hypertension and other cardiovascular diseases (1,2,3,4). It has also been demonstrated that plasma adiponectin levels increased during weight reduction or blockade of the renin-angiotensin system (5), indicating that adiponectin might be beneficial for preventing the development of atherosclerotic changes.

Many studies have focused on the cardioprotective effects attributable to nitric oxide (NO)1 and have shown that hypertension and other circulatory disorders may be associated with insufficient NO production and availability (6,7). Recently, Chen et al. (8) demonstrated that adiponectin may stimulate production of NO in vascular endothelial cells. It has been shown that plasma adiponectin was correlated with endothelium-dependent vasodilation of the brachial artery in humans (2,9). However, the precise relationships between adiponectin and NO and their roles in the pathophysiology of human hypertension remain to be elucidated.

It has been proposed that abnormalities in physicochemical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases (10,11,12). An electron paramagnetic resonance (EPR) and spin-labeling method have been developed to evaluate the membrane fluidity and perturbations of the membrane function by external agents (12,13). The membrane fluidity is a reciprocal value of membrane microviscosity and is an important factor in modulating the cell rheological behavior (12,13). We have shown previously that the membrane fluidity of erythrocytes was significantly lower in both spontaneously hypertensive (HT) rats and patients with essential hypertension than in the normotensive (NT) controls (14,15,16), and we proposed that abnormal membrane fluidity of erythrocytes might contribute to the pathogenesis of hypertension. Recently, it has been shown that NO may be involved in the regulation of cell membrane fluidity (17). Our previous in vitro study demonstrated that the NO donor improved membrane fluidity of erythrocytes in subjects with essential hypertension (16), indicating that NO could have a beneficial effect on the rheologic behavior of erythrocytes and the microcirculation in hypertension. The present study was undertaken to assess the relationships among plasma adiponectin, NO, and membrane fluidity of erythrocytes in NT and HT men by using the EPR and spin-labeling method.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Study Subjects

A total of 27 men with mild essential hypertension were studied and compared with 18 age-matched NT men (Table 1). No subjects had received or were receiving any drugs before the study. Written informed consent was obtained from all participants after they were informed about the nature and objective of the study. All subjects had similar life styles and dietary habits and were instructed to avoid smoking and any changes in dietary habits at least 12 weeks before the study.

Table 1.  Clinical characteristics and laboratory findings of normotensive (NT) and hypertensive (HT) men
 NTHT
  • Values are means ± standard error.

  • *

    p <0.05 between NT and HT.

Number of subjects1827
Age (years)64 ± 362 ± 2
BMI (kg/m2)24.1 ± 0.724.2 ± 0.5
Systolic blood pressure (mm Hg)125 ± 2147 ± 1*
Diastolic blood pressure (mm Hg)69 ± 288 ± 1*
Heart rate (beats/min)74 ± 273 ± 1
Erythrocyte counts (104 cells/μl)450 ± 11477 ± 8
Hemoglobin (g/dL)14.0 ± 0.414.2 ± 0.2
Hematocrit (%)43.0 ± 1.143.1 ± 0.4
Leucocyte counts (103 cells/μL)5.5 ± 0.35.4 ± 0.2
Platelets (104 cells/μL)21 ± 122 ± 1
Total cholesterol (mg/dL)208 ± 7211 ± 7
High density lipoprotein (mg/dL)51 ± 252 ± 3
Low density lipoprotein (mg/dL)131 ± 6128 ± 6
Triglycerides (mg/dL)129 ± 16140 ± 14
Serum sodium (mM)140.5 ± 0.4140.1 ± 0.3
Serum potassium (mM)4.0 ± 0.14.0 ± 0.1
Serum creatinine (mg/dL)0.8 ± 0.10.9 ± 0.1
Fasting plasma glucose (mg/dL)107 ± 3119 ± 7

EPR Measurements of Membrane Fluidity of Erythrocytes

All blood pressure monitoring and blood sampling were performed early in the morning after subjects had fasted overnight. After a minimum of 30 minutes of bed rest, venous blood samples were collected. We used heparin as the anticoagulant (10 U heparin/10 mL blood). The procedure of erythrocyte preparation was shown previously (14,15,16). The erythrocyte suspension (100 μL erythrocytes and 200:300 μL Tris-HCl buffer) was incubated for 2 hours at 37 °C with 100 μL of the solution containing fatty acid spin label agents [5-nitroxide stearate (5-NS) or 16-nitroxide stearate (16-NS); 5 × 10−5 M].

The EPR measurements were then performed using an EPR spectrometer (Model Jeol JES-FE2XG; Nihon Denshi, Tokyo, Japan) with a microwave unit (Model Jeol ES-SCXA; Nihon Denshi) (14,15,16). The microwave power was 5 mW, and the modulation amplitude was 2.0 gauss. The temperature of the measurement was controlled at 30 °C. The receiver scan width was 3280 ± 50 gauss, with a sweep time of 8 minutes, and receiver gain was 4 × 103, with a response time of 1.0 second.

The fatty acid spin label agents are believed to be anchored at the lipid aqueous interface of the cell membranes by their carboxyl ends, whereas the nitroxide group moves rapidly through a restricted angle around the point of attachment (18). Therefore, the EPR spectra of the fatty acid spin label agents are used to detect an alteration in the freedom of motion in biological membranes and to provide an indication of membrane fluidity (18). In addition, 5-NS could be an example of the properties of superficial membrane layers, whereas 16-NS could be an indicator referring to more hydrophobic core of the lipid membranes. For indices of membrane fluidity of erythrocytes, we have evaluated the values of outer and inner hyperfine splitting (2T′, ∥; 2T′, ⊥; in gauss, respectively) in the EPR spectra for 5-NS and calculated the order parameter (S) from 2T′ ∥ and 2T′ ⊥ (14,15,16,18) (Figure 1). In the EPR spectra for 16-NS, we used the peak height ratio (h0/h−1) value for an index of the membrane fluidity (14,15,16,18) (Figure 1). The greater the values of S and h0/h−1, the lesser the freedom of motion of the spin labels in the biomembrane bilayers, which indicates lower membrane fluidity of erythrocytes (14,15,16,18).

image

Figure 1. Typical EPR spectra of erythrocytes for the fatty acid spin label agents (upper, 5-NS; bottom, 16-NS). (2T′ ∥) Outer hyperfine splitting. (2T′ ⊥) Inner hyperfine splitting. (Tzz and Txx) Hyperfine constants. (an/a′n) Isotropic coupling constant. Greater values of S and h0/h−1 indicate lower membrane fluidity of erythrocytes.

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Plasma Adiponectin Analysis

The plasma adiponectin was measured by using an ELISA assay kit (Otsuka, Tokushima, Japan) (1).

Plasma NO Metabolite (Nitrite and Nitrate) Analysis

The plasma level of the NO metabolites (nitrite and nitrate) was measured according to the method described previously (19).

Drugs

The spin label agents, 5-NS and 16-NS, were purchased from Aldrich Co., Ltd. (Milwaukee, WI). All other drugs were standard laboratory reagents of analytical grade.

Statistics

Values are expressed as mean ± standard error. The differences between NT and HT men were analyzed using an unpaired Student's t test. Linear regression analysis was performed to assess the relationship between membrane fluidity (S) of erythrocytes and plasma adiponectin or NO metabolite levels.

Multivariate regression analysis with membrane fluidity (S or h0/h−1) of erythrocytes as a dependent variable and plasma adiponectin levels, age, BMI, blood pressure (systolic), total cholesterol, and fasting plasma glucose as independent variables was also performed. p < 0.05 was accepted as the level of significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Membrane Fluidity of Erythrocytes in NT and HT Men

The values of the S for 5-NS and the h0/h−1 in the EPR spectra of erythrocytes were significantly higher in HT men than in NT men (S for HT: mean ± standard error, 0.729 ± 0.002, n = 27; S for NT: 0.719 ± 0.002, n = 18, p < 0.01; h0/h−1 for HT, 5.28 ± 0.02, n = 27; h0/h−1 for NT, 5.14 ± 0.03, n = 18, p < 0.01). The finding indicated that membrane fluidity of erythrocytes was decreased in HT men compared with NT men.

Plasma Adiponectin and NO Metabolite Levels in NT and HT Men

The age, BMI, and other routine laboratory findings were not different between NT and HT groups (Table 1). There was an inverse correlation between plasma adiponectin levels and BMI in the overall analysis of NT and HT men (r = −0.402, n = 45, p < 0.01). The plasma adiponectin levels were significantly lower in HT men than in NT men (HT, 6.8 ± 0.3 μg/mL, n = 27; NT, 8.2 ± 0.5 μg/mL, n = 18, p < 0.05). The plasma NO metabolites were also lower in HT men than in NT men (HT, 34.8 ± 2.2 μM, n = 27; NT, 54.3 ± 5.8 μM, n = 18, p < 0.01). In the overall analysis of NT and HT men, plasma adiponectin levels were significantly correlated with plasma NO metabolites (r = 0.386, n = 45, p < 0.01) (Figure 2).

image

Figure 2. Correlation between plasma adiponectin and NO metabolite levels.

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Relationships between Plasma Adiponectin or NO Metabolite Levels and Membrane Fluidity of Erythrocytes in NT and HT Men

The S of erythrocytes was inversely correlated with the plasma adiponectin (r = −0.342, n = 45, p < 0.05) (Figure 3) and NO metabolite levels (r = −0.325, n = 45, p < 0.05) (Figure 4), indicating that the decreased membrane fluidity of erythrocytes was associated with hypoadiponectinemia and reduced plasma NO metabolites. On multivariate regression analysis, plasma adiponectin (standard regression coefficient = −0.355, p = 0.0212) was found to be an independent factor influencing the S of erythrocytes together with systolic blood pressure and fasting plasma glucose (Table 2).

image

Figure 3. Inverse correlation between plasma adiponectin and the S of erythrocytes.

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image

Figure 4. Inverse correlation between plasma NO metabolites and the S of erythrocytes.

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Table 2.  Multivariate regression analysis for predicting order parameter of erythrocytes
 SRCp value
  1. SRC, standard regression coefficient.

  2. R2 = 0.353; n = 45; F = 3.455; p = 0.0080.

Age (years)−0.0640.7287
BMI (kg/m2)−0.1340.4301
Blood pressure (systolic; mm Hg)0.2770.0488
Total cholesterol (mg/dL)−0.2740.0943
Fasting plasma glucose (mg/dL)0.2900.0455
Plasma adiponectin (μg/mL)−0.3550.0212

Similarly, the h0/h−1 of erythrocytes was inversely correlated with the plasma adiponectin (r = −0.343, n = 45, p < 0.05) (Figure 5) and NO metabolite levels (r = −0.448, n = 45, p < 0.01). Multivariate regression analysis also showed that plasma adiponectin (standard regression coefficient = −0.318, p = 0.0399) was an independent factor predicting the h0/h−1 of erythrocytes together with systolic blood pressure (Table 3).

image

Figure 5. Inverse correlation between plasma adiponectin and the h0/h−1 of erythrocytes. Data were obtained in the overall analysis of HT and NT men. Greater values of S and h0/h−1 indicate lower membrane fluidity of erythrocytes.

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Table 3.  Multivariate regression analysis for predicting peak height ratio of erythrocytes
 SRCp value
  1. SRC, standard regression coefficient.

  2. R2 = 0.336; n = 45; F = 3.207; p = 0.0120.

Age (years)−0.0610.7416
BMI (kg/m2)−0.2470.1553
Blood pressure (systolic; mm Hg)0.3400.0181
Total cholesterol (mg/dL)−0.2360.1523
Fasting plasma glucose (mg/dL)−0.0150.9154
Plasma adiponectin (μg/mL)−0.3180.0399

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Recently, evidence for a causal relationship between decreased levels of plasma adiponectin and risk of cardiovascular diseases has been demonstrated (1,2,3,4,5). However, it has been demonstrated that abnormalities in physical properties of the cell membranes may be an etiologic factor of hypertension (10,11,12,13). The present study was performed to evaluate the possible link between plasma adiponectin and membrane fluidity (a reciprocal value of membrane microviscosity) of erythrocytes in NT and HT men by using the EPR method.

The values of S and h0/h−1 obtained from the EPR spectra of erythrocyte membranes were significantly greater in HT men than in NT men. These results suggest that the membrane fluidity of erythrocytes was decreased in HT men compared with NT men and confirm our previous reports showing that the cell membranes were stiffer and less fluid in primary hypertension (14,15,16). Plasma adiponectin levels were significantly lower in HT men than in NT men and inversely correlated with the S and the h0/h−1 of erythrocytes, indicating that hypoadiponectinemia might be associated with decreased membrane fluidity of erythrocytes. To our knowledge, this is the first report demonstrating that plasma adiponectin may have a close correlation with membrane fluidity of erythrocytes in humans. Multivariate regression analysis also showed that plasma adiponectin was an independent determinant of membrane fluidity of erythrocytes. Because the deformability of erythrocytes is highly dependent on the membrane fluidity (12,13), the reduction in membrane fluidity associated with hypoadiponectinemia could cause a disturbance in the blood rheologic behavior and the microcirculation.

Recently, it was demonstrated that adiponectin may stimulate production of NO in vascular endothelial cells in vitro (8). It has also been shown that plasma adiponectin was correlated with endothelium-dependent vasodilation of the brachial artery, suggesting that plasma adiponectin is considered as a useful marker of endothelial function in HT subjects (2,9). The present study demonstrated that the plasma levels of the NO metabolites were significantly lower in HT men than in NT men. In addition, we showed that the plasma adiponectin levels were correlated with plasma NO metabolite levels in the overall analysis of NT and HT men. It is speculated that hypoadiponectinemia could be associated with the reduced NO production and endothelial dysfunction.

It was also shown that S and h0/h−1 in the EPR spectra of erythrocyte membranes were inversely correlated with the plasma NO metabolites, indicating that decreased membrane fluidity of erythrocytes was correlated with reduced plasma NO metabolites. We demonstrated that the NO donor might improve membrane fluidity of erythrocytes in HT subjects (16). The finding might propose that NO could have a crucial role in the regulation of membrane fluidity of erythrocytes and further support the hypothesis that adiponectin might be associated with alterations in membrane fluidity of erythrocytes, at least in part, by the NO-dependent mechanism. However, the influence of adiponectin on the membrane lipid-protein interactions (20,21) cannot be fully excluded.

The precise mechanisms of NO in regulating membrane fluidity of erythrocytes are still uncertain. Jubelin and Gierman (22) showed that erythrocytes of rats and humans are positive for NO synthase, which indicated that erythrocytes possess all of the cellular machinery to synthesize their own NO. Chen and Mehta (23) provided direct evidence that human erythrocytes possess endothelium-type NO synthase in the cytosol. It would be possible that the membrane action of NO could be one of the mechanisms responsible for its beneficial effects in improving the rheological behavior of erythrocyte membranes and the microcirculation, although it is unclear whether the effect of adiponectin on erythrocyte membrane fluidity might depend on the direct stimulation of NO synthase in the erythrocytes. Further studies should be performed to assess more precisely the relationships among adiponectin, NO, and membrane functions and their contribution to the pathophysiology of hypertension.

In summary, the results of the present study demonstrated that plasma adiponectin levels were lower in HT men than in NT men and that hypoadiponectinemia was associated with decreased membrane fluidity of erythrocytes. The finding suggests that adiponectin may be linked to the rheologic behavior of the erythrocytes and the microcirculation in men, at least in part, by the NO-dependent mechanism.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported in part by the grants-in-aid for scientific research from the Ministry of Education, Science, Sports, Culture and Technology of Japan (No. 15590604, 18590658), the Uehara Memorial Foundation (1999, 2005), the Mitsui Foundation (1999, 2006), and the Takeda Science Foundation (2002, 2006).

Footnotes
  • 1

    Nonstandard abbreviations: NO, nitric oxide; EPR, electron paramagnetic resonance; HT, hypertensive; NT, normotensive; 5-NS, 5-nitroxide stearate; 16-NS, 16-nitroxide stearate; S, order parameter; h0/h−1, peak height ratio.

  • The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References