Red blood cell deformability: Effect of age and smoking

The effects of the aging process of healthy subjects on red blood cell membrane deformability and how this parameter changes with smoking were investigated using the micropipette aspiration technique. In this method, a portion of the cell membrane is aspirated into a micropipette via a step negative pressure and the membrane modulus of elasticity is calculated using the viscoelastic models. Red blood cells were obtained from 18 non-smoking (8 females and 10 males) and 18 smoking (10 females and 8 males) subjects. All smoking subjects were chronic smokers (smoked for at least 2 years and smoked more than 1 pack/day). The age of the subjects ranged from 20–54 years for nonsmokers [average 39.5±11.5 (SD) years] and 23–61 for smokers [average 39.5±11.7 (SD) years]. The results showed a significant increase in elasticity modulus with age. An increase in elasticity modulus indicates a decrease in cell deformability. There was no significant difference in the rate of increase with age in smokers as compared to non-smokers. Further studies are warranted to determine the exact membrane properties responsible for decrease in deformability during the aging process.     

Effects of cigarette smoking or nicotine replacement on cardiovascular risk factors and parameters of haemorheology

OBJECTIVES: Cigarette smoking causes cardiovascular (CV) disease, but the relative roles of nicotine and other components of tobacco smoke remain unclear. We investigated the effect of stopping smoking, by using nicotine replacement therapy (NRT), on haemorheology parameters. DESIGN: Open, parallel-group trial (intervention group and control smokers). SETTING: Clinic within university department of pharmacology. SUBJECTS: One hundred and ninety-seven males, aged 25-45 years, smoking >20 cigarettes per day. INTERVENTIONS: One hundred and sixty-four subjects were instructed to stop smoking and received NRT for 12 weeks and 33 acted as controls. After 12 weeks, NRT was discontinued, and all subjects were followed-up at 26 weeks. At the end of the study, the NRT group was divided into abstainers (self-reported, verified by exhaled carbon monoxide <10 ppm) and relapsers, who were unable to remain abstinent. MAIN OUTCOME MEASURES: Plasma viscosity, fibrinogen, erythrocyte deformability, reactive capillary blood flow, transcutaneous partial oxygen tension (tcpO2) and haematocrit, assessed at 4, 8, 12, and 26 weeks. Results. After 6 months, plasma fibrinogen (9.95 vs. 8.24 micromol x L(-1) at baseline; P < 0.003), reactive capillary flow (t-pmax: 9.3 vs. 11.2 s at baseline; P < 0.005), and tcpO2 (50.4 vs. 34.9 mmHg at baseline; P < 0.0001) were significantly improved in abstainers, but changes in plasma viscosity and erythrocyte deformability were inconclusive. Other CV risk factors, such as haematocrit and white blood cell count, decreased to a greater extent in abstainers than in relapsers. Expired carbon monoxide concentrations reflected the changes in smoking and decreased in abstainers from 30.4 ppm at baseline to 4.2 ppm; P < 0.0001). Conclusions. Smoking cessation improved CV parameters, and use of nicotine medications did not negate these improvements.     

Effects of cigarette smoking on morphology and aggregation of erythrocytes

Erythrocyte deformability and aggregation are important parameters related to blood flow in cardiovascular system. These are generally determined by various techniques. The present work is an attempt to implement a computerized procedure based on microscopic images of erythrocytes, obtained from cigarette smokers, to calculate these and related parameters. The subjects who have been smoking for more than ten years (Group 2) are selected and the calculated parameters are compared with that of non-smoking subjects (Group 1). The shape parameters area, perimeter and form factor of erythrocytes show significant change in Group 2 compared to that of Group 1. The erythrocyte aggregation, as measured by area occupied by aggregates, shows significant reduction in smokers, attributed to enhanced cell-cell interaction in Group 2. The ratio of crenated to normal erythrocytes increases immediately after smoking but reverts back to initial level after a gap of more than one hour, which is attributed to the change in metabolic activity of erythrocytes.     

Direct measurement of erythrocyte deformability in diabetes mellitus with a transparent microchannel capillary model and high-speed video camera system

To measure erythrocyte deformability in vitro, we made transparent microchannels on a crystal substrate as a capillary model. We observed axisymmetrically deformed erythrocytes and defined a deformation index directly from individual flowing erythrocytes. By appropriate choice of channel width and erythrocyte velocity, we could observe erythrocytes deforming to a parachute-like shape similar to that occurring in capillaries. The flowing erythrocytes magnified 200-fold through microscopy were recorded with an image-intensified high-speed video camera system. The sensitivity of deformability measurement was confirmed by comparing the deformation index in healthy controls with erythrocytes whose membranes were hardened by glutaraldehyde. We confirmed that the crystal microchannel system is a valuable tool for erythrocyte deformability measurement. Microangiopathy is a characteristic complication of diabetes mellitus. A decrease in erythrocyte deformability may be part of the cause of this complication. In order to identify the difference in erythrocyte deformability between control and diabetic erythrocytes, we measured erythrocyte deformability using transparent crystal microchannels and a high-speed video camera system. The deformability of diabetic erythrocytes was indeed measurably lower than that of erythrocytes in healthy controls. This result suggests that impaired deformability in diabetic erythrocytes can cause altered viscosity and increase the shear stress on the microvessel wall.     

Red cell membrane stiffness in iron deficiency

The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron- deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.     

New insights provided by a comparison of impaired deformability with erythrocyte oxidative stress for sickle cell disease

Sickle cell disease (SCD) is associated with increase in oxidative stress and irreversible membrane changes that originates from the instability and polymerization of deoxygenated hemoglobin S (HbS). The relationship between erythrocyte membrane changes as assessed by a decrease in deformability and oxidative stress as assessed by an increase in heme degradation was investigated. The erythrocyte deformability and heme degradation for 27 subjects with SCD and 7 with sickle trait were compared with normal healthy adults. Changes in both deformability and heme degradation increased in the order of control to trait to non-crisis SCD to crisis SCD resulting in a very significantly negative correlation between deformability and heme degradation. However, a quantitative analysis of the changes in deformability and heme degradation for these different groups of subjects indicated that sickle trait had a much smaller effect on deformability than on heme degradation, while crisis affects deformability to a greater extent than heme degradation. These findings provide insights into the relative contributions of erythrocyte oxidative stress and membrane damage during the progression of SCD providing a better understanding of the pathophysiology of SCD.     

Alpha- and beta-haemoglobin chain induced changes in normal erythrocyte deformability: comparison to beta thalassaemia intermedia and Hb H disease.

The alpha- and beta-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess alpha- and beta-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-containing alpha- and beta-chains were entrapped within normal erythrocytes. Entrapment of purified alpha-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in beta-thalassaemia intermedia. The decreased deformability was correlated with alpha-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane alpha-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the alpha-chain loaded cells. While both beta-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the alpha-chain loaded and beta-thalassaemic cells and may arise from either the increased intracellular viscosity of the beta-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.     

Hemorheological disturbances in patients with chronic cerebrovascular diseases

Hemorheological disturbances may occur in more than 40% of patients with ischemic cerebrovascular diseases. In this study the changes of rheological factors--hematocrit, plasma fibrinogen concentration, whole blood and plasma viscosity, red blood cell aggregation and deformability were investigated in 297 patients (173 males, 124 females, mean age 60 +/- 11 years) with transient ischemic attack or chronic phase (> 3 months after onset) ischemic stroke, and in 73 healthy volunteers (35 males, 38 females, mean age 38 +/- 7 years). Hematocrit, plasma and whole blood viscosity were significantly (p < 0.0001) elevated in cerebrovascular patients compared to controls. Plasma fibrinogen concentration (p < 0.001), red blood cell aggregation (p < 0.05) and deformability (p < 0.01) were also impaired in stroke patients. Hemorheological disturbances were dominant in stroke patients with diabetes, hyperlipidemia and smoking habits. Hematocrit, plasma viscosity and red blood cell aggregation showed a significant (p < 0.025-0.001) correlation with the severity of carotid artery stenosis. We could not find any characteristic distribution of rheological parameters among the three subtypes of brain ischemia. Our results show that all of the measured rheological parameters are significantly impaired in chronic ischemic cerebrovascular disorders, especially in diabetic, smoking and alcoholic patients. They correlate with the severity of the carotid artery stenosis, but there is no association with the type of ischemic stroke.     

α- and β-haemoglobin chain induced changes in normal erythrocyte deformability: comparison to β thalassaemia intermedia and Hb H disease

Summary The α and β-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess α and β-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-con-taining α and β-chains were entrapped within normal erythrocytes. Entrapment of purified α-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in β-thalassaemia intermedia. The decreased deformability was correlated with α-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane α-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the α-chain loaded cells. While both β-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the α-chain loaded and β-thalassaemic cells and may arise from either the increased intracellular viscosity of the β-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.     

Red blood cell deformability as a predictor of anemia in severe falciparum malaria.

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.     

Role of erythrocyte deformability in the acute hypoxic pressor response in the pulmonary vasculature

To assess the importance of erythrocyte deformability in the pulmonary hypoxic pressor response (HPR) we examined whether alterations in erythrocyte deformability are related to the differences between the brisk HPR in rats vs the small HPR in hamsters, and between the HPR in low altitude rats vs high altitude rats (10 days in 10% oxygen). Deformability of the erythrocytes (RBC) was assessed by filtering equal volume of RBC suspension through Nucleopore filters (4.7 micron) using the same pressure head across the filter. The results show that during hypoxia, rat RBC become relatively nondeformable compared to hamster's RBC. This finding is consistent with a large HPR in rats but a small HPR in hamsters. Furthermore, the deformability of RBC from high altitude rats became unaffected by hypoxia and was associated with blunting in the HPR in isolated lungs from high altitude rats. The HPR in isolated lungs from low altitude rats was larger when they were perfused with blood from normal rats (= 86% increase in resistance) than when perfused with blood from high altitude rats (= 36% increase in resistance). This finding further supports the possible role of RBC deformability in HPR. Inconsistent with the importance of deformability, however, was the finding that high altitude rat lungs had a blunted HPR whether they were perfused with normal rat blood or high altitude rat blood. This may be due to restructuring of the pulmonary microvascular bed in the lung from high altitude rats. The results favor the idea that changes in erythrocyte deformability may be responsible for the difference between the HPR in low altitude rats and hamsters, and between the HPR in low and high altitude rats. We suggest that 'obstruction' of the capillaries by less deformable erythrocyte is another factor, besides smooth muscle contraction, responsible for the hypoxic pressor response in the pulmonary vasculature.     

In vitro exposure to hydroxyurea reduces sickle red blood cell deformability

Hydroxyurea is a drug that is used to treat some patients with sickle cell disease. We have measured the deformability of sickle erythrocytes incubated in hydroxyurea in vitro and found that hydroxyurea acts to decrease the deformability of these cells. The deformability of normal erythrocytes was not significantly affected by hydroxyurea except at very high concentrations. Hydroxyurea also did not consistently reduce the deformability of sickle erythrocyte ghosts. We propose that the decreased deformability, observed in vitro, is due to the formation of methemoglobin and other oxidative processes resulting from the reaction of hydroxyurea and oxyhemoglobin. Although the reaction with normal hemoglobin is similar to that of sickle hemoglobin, the sickle erythrocytes are affected more. We propose that the sickle erythrocyte membrane is more susceptible to the reaction products of the reaction of hemoglobin and hydroxyurea. An earlier report has shown that hydroxyurea increases the deformability of erythrocytes in patients on hydroxyurea. Taken together, these data suggest that the improved rheological properties of sickle erythrocytes in vivo are due to the elevated numbers of F cells [cells with fetal hemoglobin]. The presence of the nitrosyl hemoglobin or methemoglobin from the reaction with hydroxyurea may also benefit patients in vivo by reducing sickling.     

Splenectomy prolongs in vivo survival of erythrocytes differently in spectrin/ankyrin- and band 3-deficient hereditary spherocytosis

Red cell (RBC) deformability and membrane-bound immunoglobulin G (IgG) were studied to better understand premature clearance of erythrocytes in hereditary spherocytosis. Averaged deformability profiles from cells having comparable cell age revealed that splenectomy was more beneficial for spectrin/ankyrin-deficient than for band 3-deficient RBCs. Splenectomy prevented an early loss of young cells in both types of deficiencies. It had an additional beneficial effect on spectrin/ankyrin-deficient but not band 3-deficient RBCs. It prolonged the survival of mature spectrin/ankyrin-deficient RBCs such that they lost their deformability more slowly than RBCs from patients who had not undergone splenectomy. Band 3-deficient RBCs lost their deformability at the same rate before and after splenectomy. In HS patients with band 3 deficiency who underwent splenectomy, RBC deformability inversely correlated with the number of RBC-bound IgG (up to 140 molecules per cell). In spectrin/ankyrin deficiency, RBC-bound IgG remained at control levels (60 IgG or less per cell). It appears that spectrin/ankyrin-deficient RBCs escaped opsonization by releasing band 3-containing vesicles because their band 3 content and deformability dropped in parallel with increasing cell age. Band 3-deficient RBCs did not lose band 3 with increasing cell age. Hence, it is possible that band 3 clusters required for bivalent binding of low-affinity-IgG, naturally occurring antibodies were retained in band 3-deficient RBCs with a relative excess of skeletal proteins but were released from spectrin/ankyrin-deficient RBCs, in which vesicle budding was facilitated by an impaired skeleton.     

Red blood cells deformability and oxidative stress in acute pancreatitis

The aim of this study was to evaluate the RBC deformability and oxidative stress parameters during acute pancreatitis. Healthy volunteers and patients with mild or severe acute pancreatitis were evaluated. There were no changes in erythrocyte's deformability in patients with mild acute pancreatitis. In severe acute pancreatitis loss of deformability of erythrocytes was observed. Serum lipofuscin level increased both in mild and severe form of the disease. The superoxide dismutase (SOD) activity was increased in erythrocytes from mild and severe form without systemic complications and was positively correlated with erythrocyte's deformability in a severe form of acute pancreatitis. Significant positive correlation between serum total antioxidant status and deformability of erythrocytes in healthy humans and negative correlation in mild pancreatitis were found.     

Decreased erythrocyte deformability in Behçet's disease

Beh?et's disease (BD) is a chronic recurrent systemic vasculitis of unknown cause. Thrombotic tendency is another characteristics of BD. While hemorheological changes play crucial role in thrombosis, erythrocyte deformability (ED), which is one of the main determinants of microcirculation and blood viscosity, has not been studied before in BD. ED was measured by the filtration method in 13 active BD patients diagnosed according to the criteria proposed by International Study Group for BD (ISG-BD) and in 13 age-, sex-, weight-, smoking habit-, and blood pressure-matched healthy controls. ED was decreased in active BD patients compared to the healthy control subjects. The median deformability indices (DIs) in BD patients and healthy controls were 3.19 and 2.08 (p<0.001), respectively. The decrease in ED may be one of the reasons for increased thrombotic tendency in BD. Alteration in ED possibly plays a crucial role in the etiopathogenesis of thrombotic complications in BD and thus may be a target for treatment. Drugs increasing ED, such as pentoxifylline, have already been shown to be beneficial in the treatment of BD. Therefore, we propose that the therapeutic mechanism underlying the beneficial effect of this drug in BD is likely to be the correction of impaired ED.     

Hyperviscosity as a possible risk factor for cerebral ischemic complications in atrial fibrillation patients

Recent studies have suggested that hyperviscosity is frequent in patients with atrial fibrillation (AF). The aims of this study were to evaluate if hemorheologic alterations play a role in the occurrence of cerebral ischemic events in patients with AF and to explore a possible association between inflammation and hyperviscosity in these patients. Sixty-two patients with AF with a history of >or=1 cerebral ischemic event and 94 patients with AF without cerebral ischemic events were studied. A control population included 130 age- and gender-matched healthy volunteers. Hemorheologic variables (whole-blood viscosity, plasma viscosity, the erythrocyte deformability index, and hematocrit), fibrinogen, and high-sensitivity C-reactive protein levels were assayed. An alteration in whole blood viscosity at 94.5 seconds(-1) and the erythrocyte deformability index were found more frequently in patients with previous ischemic events on univariate and multivariate analyses (odds ratio 3.19, p=0.023 and odds ratio 4.26, p=0.002, respectively) adjusted for age, gender, hypertension, diabetes, history of coronary artery disease, left ventricular dysfunction, smoking habit, dyslipidemia, hematocrit, fibrinogen, high-sensitivity C-reactive protein, and hemorheologic parameters. These results should stimulate prospective studies on the role of hemorheologic alterations in the occurrence of cerebral ischemic complications in patients with AF.     

Asymmetric dimethylarginine reduced erythrocyte deformability in streptozotocin-induced diabetic rats

To investigate the effect of asymmetric dimethylarginine on erythrocyte deformability in streptozotocin-induced diabetic rats, a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) in male Sprague-Dawley rats was carried out to induce diabetes and normal erythrocytes were incubated with asymmetric dimethylarginine or aortic rings from diabetic rats in the presence of L-arginine or vitamin E. We found that erythrocyte deformability was significantly decreased in diabetic rats. The levels of asymmetric dimethylarginine in plasma and erythrocytes of diabetic rats were elevated significantly from 2-week diabetic duration to 8-week diabetic duration. Nitric oxide in erythrocytes was decreased at 8-week diabetic duration while plasma nitric oxide remained unchanged all along. The content of malondialdehyde in erythrocytes of diabetic rats was increased. After incubation of erythrocytes with asymmetric dimethylarginine (10(-6) M) for 30 min, erythrocyte deformability and nitric oxide level in erythrocytes were decreased markedly. Reactive oxygen species and malondialdehyde production in erythrocytes were promoted by asymmetric dimethylarginine. Both L-arginine and vitamin E reversed the effects of asymmetric dimethylarginine. After incubation of erythrocytes with aortic rings from diabetic rats, erythrocyte deformability was decreased, which was attenuated by L-arginine. These results indicated that reduction of erythrocyte deformability in diabetic rats was associated with promoted oxidant stress as well as impaired nitric oxide synthesis by elevation of asymmetric dimethylarginine.     

Impaired erythrocytes deformability in H(2)O(2)-induced oxidative stress: protective effect of L-carnosine

Impaired red blood cell deformability is a hemorheological perturbation induced by many kinds of diseases. An increase in free radicals causes a reduction in erythrocyte flexibility and deformability. Carnosine is a dipeptide abundant in skeletal muscle and brain of humans. One of the main function of carnosine is its antioxidant and free-radical scavenger effect. In this study our aim is to investigate the protective effect of L-carnosine on RBCs in H(2)O(2)-induced oxidative stress in vitro conditions.Twenty male wistar albino rats, 10 were 3 months old, 10 were 12 months old used. The blood from each rat were divided into ten tubes and these blood samples divided into two groups. The first tube of the first group was the control and the rest 4 tubes were treated with different concentrations of L-carnosine. All tubes in the second group were incubated with H(2)O(2) additively. The deformability indexes of the erythrocytes were measured by a laser diffractometer (Myrenne Rheodyne SSD).L-carnosine has improved the RBC deformability significantly which is impaired by H(2)O(2) treatment (p<0.05). Increase in deformability is more significant in young rat group when compared to old rat group.L-carnosine, as an antioxidant molecules, has a dose dependent positive effect on RBC deformability and has improved or protect the deformability of erythrocytes, especially in young rat group which impaired by H(2)O(2)-induced oxidative stress in vitro conditions. The results of this study first suggest that L-carnosine supplemention can be used to improve the RBC quality or to protect them from oxidative damage in survival of RBC in the circulation.     

Erythrocyte cellular and membrane deformability in hereditary spherocytosis

In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.     

Nitric oxide pathway activation and impaired red blood cell deformability with hypercholesterolemia

The pathophysiological effects of the activation or inhibition of the nitric oxide (NO)-mediated pathway on the deformability of red blood cells (RBC) were evaluated in the presence of hypercholesterolemia induced in rabbits fed a cholesterol-rich diet. RBC deformability was assessed using a microchannel array flow analyzer system. The maximum passage time (MPT) by flowing a suspension of RBC through the microchannels was used as an index of RBC deformability. During cholesterol feeding for 12 weeks, MPT gradually increased with no significant elevation in the serum asymmetric dimethylarginine (ADMA) and arginine/ADMA ratio. The reduction in RBC deformability associated with hypercholesterolemia was significantly improved during incubation with each of three different NO pathway activators: a NO donor, 8-bromo-cyclic GMP, and arginine; however, no additional reduction was observed with ADMA administration. The inhibition of NO synthase due to ADMA caused a significant reduction in the deformability of normal RBC, which was reversed with NO pathway activation. These results suggest that impaired RBC deformability may be associated with a dysfunction in the NO pathway that is partially dependent upon the accumulation of ADMA in RBC, and exogenous NO pathway activators may improve the microcirculation by restoring RBC deformability in the presence of hypercholesterolemia.