Haemorheological Variables Research Articles

Functional and structural adaptations of the coronary macro- and microvasculature to regular aerobic exercise by activation of physiological, cellular, and molecular mechanisms: ESC Working Group on Coronary Pathophysiology and Microcirculation position paper.

Regular aerobic exercise (RAEX) elicits several positive adaptations in all organs and tissues of the body, culminating in improved health and well-being. Indeed, in over half a century, many studies have shown the benefit of RAEX on cardiovascular outcome in terms of morbidity and mortality. RAEX elicits a wide range of functional and structural adaptations in the heart and its coronary circulation, all of which are to maintain optimal myocardial oxygen and nutritional supply during increased demand. Although there is no evidence suggesting that oxidative metabolism is limited by coronary blood flow (CBF) rate in the normal heart even during maximal exercise, increased CBF and capillary exchange capacities have been reported. Adaptations of coronary macro- and microvessels include outward remodelling of epicardial coronary arteries, increased coronary arteriolar size and density, and increased capillary surface area. In addition, there are adjustments in the neural and endothelial regulation of coronary macrovascular tone. Similarly, there are several adaptations at the level of microcirculation, including enhanced (such as nitric oxide mediated) smooth muscle-dependent pressure-induced myogenic constriction and upregulated endothelium-dependent/shear-stress-induced dilation, increasing the range of diameter change. Alterations in the signalling interaction between coronary vessels and cardiac metabolism have also been described. At the molecular and cellular level, ion channels are key players in the local coronary vascular adaptations to RAEX, with enhanced activation of influx of Ca2+ contributing to the increased myogenic tone (via voltage-gated Ca2+ channels) as well as the enhanced endothelium-dependent dilation (via TRPV4 channels). Finally, RAEX elicits a number of beneficial effects on several haemorheological variables that may further improve CBF and myocardial oxygen delivery and nutrient exchange in the microcirculation by stabilizing and extending the range and further optimizing the regulation of myocardial blood flow during exercise. These adaptations also act to prevent and/or delay the development of coronary and cardiac diseases.

Red blood cell deformability is reduced inhomozygous sickle cell disease patients with leg ulcers.

Previous reports differ as to whether a decreased elongation index (EI), a proxy for red blood cell (RBC) deformability, is associated with leg ulcers (LU) in people with homozygous sickle cell disease (SCD). We sought to determine whether erythrocyte deformability (ED) and haematological indices were associated with the presence of LU in patients with SCD. The study design was cross-sectional. Twenty-seven patients with LU and 23 with no history of ulceration were recruited into the study. A laser assisted rotational red cell analyzer was used in the determination of the EI. Haematological indices were determined using a CELL-DYN Ruby haematology analyzer. Data were normally distributed and presented as means±SD. Two-sample t-test was used to test for associations between haemorheological variables in SCD patients with and without LU. Statistical significance was taken as p < 0.05. The EI was significantly lower in the group with ulcers (0.30±0.07 vs. 0.35±0.07, p = 0.02). Haematological indices were comparable in patients with and without LU. Erythrocyte deformability, but not haematological indices, was associated with LU in patients with SCD.

Effect of changes in the intravascular volume during hemodialysis on blood viscoelasticity

Adoption of high rate of ultrafiltration (UF) during hemodialysis (HD) may affect the hemorhelogical blood profile, by changing Hematocrit (Hct) and the concentration of plasma proteins, which may in turn interfere with tissue perfusion. The aim of this work is to examine the effect of acute volume change during dialysis on the hemorheological variables. The study included 21 hemodialysis patients. Hematocrit (Hct) and percent decrease in blood volume (BV) were recorded by blood volume monitor. Blood samples were taken before and at the end of dialysis, for measuring plasma fibrinogen and haemorheological variables, which included blood viscosity, plasma viscosity, red cells elasticity and aggregation. The UF volume was 3.52±1.54 L. Hct increased from 34.2±6.1 to 42.1±7.3% (P<0.001), and blood volume (BV) decreased to 85.5±6.4% (P<0.001). Blood and plasma viscosity significantly increased from 3.28±0.69 to 5.48±0.85 mPa.s (P<0.001), and from 1.24 ± 0.16 to 1.65±0.24 mPa.s (P<0.001), respectively. Changes in plasma viscosity were correlated to changes in plasma fibrinogen (r=0.63, P<0.05), while the increase in blood viscosity was correlated to the percent reduction in blood volume (r=0.85, P<0.005). Red cells elasticity increased from 0.26±0.12 to 0.48±0.18 mPa.s (P<0.05), and the aggregation index rose from 0.86±0.31 to 1.25±0.26 (P<0.01). This combination of increased plasma viscosity and red cell aggregability may lower the velocity of erythrocyte transfer inside the tissue capillaries after HD, which may affect tissue perfusion. Moreover, increased elasticity may require more energy from the heart to disaggregate the cells, and this may induce problems in the patients with cardiac dysfunction. In conclusion, the hemorheological variables change after dialysis in the direction which may impede the flow inside the microvessels.

Metabolic alterations in morbid obesity. Influence on the haemorheological profile

There are few studies on haemorheological disturbances in morbidly obese patients. The role played by the metabolic syndrome on the rheological profile of morbidly obese subjects has not yet been established, and it is not clear whether morbidly obese, but "metabolically healthy", show rheological alterations. We aimed to determine the whole rheological profile in 136 morbidly obese patients and 136 normo-weight volunteers, along with plasma lipids, inflammatory and insulin resistance parameters. Patients had statistically higher glucose, triglycerides, HbA1c, leptin, insulin, HOMA, CRP, leucocytes, fibrinogen, plasma viscosity (p < 0.001, respectively), erythrocyte aggregation at 3 s-1 (p = 0.011) and lower erythrocyte elongation index 60 Pa (p = 0.015). In the multivariate regression analysis, the anthropometric, lipidic, insulin resistance and inflammatory parameters predicted haemorheological variables (p < 0.001). No differences were observed for the rheological parameters when morbidly obese subjects with (n = 75) and without (n = 61) the metabolic syndrome were compared (p > 0.05), indicating that the altered rheological profile not only related to the metabolic syndrome, but to obesity itself. When further patients were classified as "metabolically healthy" obese (n = 23) and "metabolically unhealthy" obese (n = 113), the latter presented higher insulin resistance (insulin p < 0.01, HOMA p < 0.05, glucose p < 0.001, triglycerides p < 0.05 and HbA1c p < 0.01) than the former, but no differences in the rheological parameters (p > 0.05) were observed. When "metabolically healthy" obese (n = 23) were compared with "metabolically healthy" controls (n = 81), the former still showed higher HOMA (p < 0.001), triglycerides (p < 0.05), CRP (p < 0.001) and HbA1c (p < 0.05), higher fibrinogen (p < 0.001), plasma viscosity (p < 0.001), erythrocyte aggregation at 3 s-1 (p < 0.05), but a lower erythrocyte elongation index 60 Pa (p < 0.05). Morbidly obese subjects present a more pronounced altered rheological profile in those with metabolic alterations, although the "metabolically healthy" obese also displayed rheological alterations if compared with "metabolically healthy" non-obese controls. These rheological alterations relate to both insulin resistance and inflammation.

Functional Effects of Endothelial Nitric Oxide Synthase Genetic Polymorphisms on Haemorheological Parameters in Healthy Human Individuals

The constitutive endothelial nitric oxide synthase (eNOS) plays a major role in circulatory homoeostasis and shows genetic polymorphism. eNOS is expressed and functional in blood cells, including erythrocytes. There is limited knowledge about the consequences of eNOS genetic variability in haemorheological parameters and erythrocyte functioning. The purpose of this study was to investigate the effects of three eNOS genetic polymorphisms, namely exonic G894T (Glu298Asp), intronic VNTR (27-bp repeat) and 5'-flanking T(-786)C polymorphisms on haemorheological variables, such as erythrocyte deformability and erythrocyte aggregation (rouleaux formation) in healthy non-smoking volunteers. Sixty subjects (19 women, 41 men) were examined for genotypes and haemorheological variables. Genotypes were determined by polymerase chain reaction and restriction analysis. Haemorheological variables were measured by means of a laser-assisted optical rotational cell analyser (LORCA). Erythrocyte aggregation was significantly decreased in individuals with 894TT genotype when compared to subjects with the (G) allele. Aggregation indices (AI) were 54.7±3.2% versus 61.0±0.9% (p=0.026), and the half-lives (t(1/2) ) for aggregation formation were 3.43±0.43 versus 2.55±0.12 sec. (p=0.024), respectively. Similarly, VNTR-bb genotype significantly altered erythrocyte aggregability. AI values were 58.7±1.1% in subjects with VNTR-a allele versus 63.7±1.2% in subjects with bb genotype (p=0.011); t(1/2) values were 2.86±0.16 versus 2.20±0.13 sec., respectively (p=0.016). T(-786)C polymorphism did not change any haemorheological parameters. These findings suggest that eNOS 894TT genotype is associated with decreased erythrocyte aggregation, while VNTR-bb genotype increases aggregability in healthy human individuals. eNOS genetic variants may contribute in the pathogenesis of microvascular disorders by altering erythrocyte functions in human beings.

Association among low whole blood viscosity, haematocrit, haemoglobin and diabetic retinopathy in subjects with type 2 diabetes

ObjectivesHaemorheological variables influence endothelial function through the release of several factors. Clinical studies have described an association among blood viscosity, haematocrit, haemoglobin and macro-angiopathy. Few data are reported about the...

Interaction effects of time of day and sub-maximal treadmill exercise on the main determinants of blood fluidity

The purpose of this study was to investigate the effects of time of day on responses of the main determinants of blood rheology to acute endurance exercise. Ten healthy male subjects (age, 26.9 +/- 5.5 yr) performed two bouts of running at 65% of VO2peak for 45 min on a motorised treadmill in the morning (08:00 h) and evening (20:00 h), which were followed by 30 min recovery. The two exercise trials were performed in two separate days with 7 days intervening. Haemorheological variables were measured before, immediately after exercise and after recovery. Haematocrit, haemoglobin and RBC count were increased significantly (p < 0.01) after 45 min running in both morning and evening trials and normalised following recovery, irrespective of time of day. Plasma viscosity increased significantly (F2,18 = 12.4, p < 0.01) following sub-maximal exercise in both trials and returned to pre-exercise level at the end of recovery. Baseline values (p < 0.05) but not responses of plasma viscosity to exercise were significantly affected by time of day. Neither a significant main effect of exercise nor a significant (p > 0.05) time-of-day effect was found for plasma proteins. It was concluded that sub-maximal running at 08:00 or 20:00 h does not induce different responses in the main determinant of blood rheology.

Effects of a dairy product (pecorino cheese) naturally rich in cis-9, trans-11 conjugated linoleic acid on lipid, inflammatory and haemorheological variables: A dietary intervention study

Background and aim Some studies recently reported a favourable effect for cis-9, trans-11 conjugated linoleic acid (CLA) on plasma lipoprotein profile of healthy subjects. Aim of this crossover intervention study was to evaluate the influence of a short-term dietary intake of a cheese derived from sheep's milk naturally rich in CLA on several atherosclerotic biomarkers, in comparison with a commercially available cheese. Methods and results Ten subjects (6 F; 4 M) with a median age of 51.5 followed for 10 weeks a diet containing 200 g/week of cheese naturally rich in CLA ( intervention period) and for the same period a diet containing a commercially available cheese of the same quantity ( placebo period). Consumption of the dairy product naturally rich in cis-9, trans-11 CLA determined a significant ( p < 0.05) reduction in inflammatory parameters such as interleukin-6 (pre: 8.08 ± 1.57 vs. post: 4.58 ± 0.94 pg/mL), interleukin-8 (pre: 45.02 ± 5.82 vs. post: 28.59 ± 2.64 pg/mL), and tumour necrosis factor-α (pre: 53.58 ± 25.67 vs. post: 32.09 ± 17.42 pg/mL) whereas no significant differences in the placebo period were observed. With regard to haemorheological parameters, the test period significantly ameliorated erythrocytes' filtration rate (pre: 7.61 ± 0.71% vs. post: 9.12 ± 0.97%; p = 0.03) with respect to the placebo period. Moreover, a reduction in the extent of platelet aggregation, induced by arachidonic acid [pre: 87.8 ± 1.76% vs. post: 77.7 ± 3.56%; p = 0.04] was observed during the test period in comparison with the placebo period. Conclusions Dietary short-term intake of the tested dairy product naturally rich in cis-9, trans-11 CLA appeared to cause favourable biochemical changes of atherosclerotic markers.

Lipid, inflammatory and haemorheological profiles are significantly affected by farmed fish eating: an intervention study

Aim The objective of the present study was to evaluate the influence of short-term dietary intake of farmed fish on biomarkers related to the atherosclerotic process.Methods and results Lipid, inflammatory, and haemorheological variables before (T0) and after a dietary intervention with about 800 g Orbetello farmed sea bass per week for 10 weeks (T1) were evaluated in nine dyslipidemic subjects. Fish intake significantly decreased triacylglycerols (T1, 140.2±20.3 mg/dl versus T0, 183.3±29.2 mg/dl; P =0.04), whereas no significant changes for the other lipid variables have been observed. Moreover, dietary intervention significantly (P <0.05) decreased all of the inflammatory parameters investigated; namely, high-sensitivity C-reactive protein, interleukin-6, and interleukin-8. Furthermore, a significant (P =0.04) improvement in erythrocytes’ deformability index was reported after 10 weeks of fish dietary intake (9.0±0.7% versus 5.4±1.0% for T1 and T0, respectively).Conclusion Dietary short-term intake of farmed fish seems to impose favourable biochemical changes in dyslipidemic subjects.

Effect of acute folic acid ingestion on blood pressure, haemorheological and plasma lipid variables in healthy young men

The increased viscosity of blood has been associated with increased incidence of cardiovascular diseases and blood rheology has been shown to be influenced by nutrition. This study was designed to elucidate whether acute folic acid ingestion has any effect on blood pressure (systolic and diastolic), haemorheological variables (haematocrit, relative plasma viscosity, and total plasma proteins), plasma cholestrol, triglyceride, LDL-and HDL-cholesterol in twenty normotensive healthy young men. Ingestion of folic acid (10mg) after 60min was found to cause significant (p Keywords : acute ingestion, folic acid, plasma viscosity, blood pressure, cholesterol, triglycerides, lipoproteins Nigerian Journal of Health and Biomedical Sciences Vol. 4(2) 2005: 117-120

Association of hyperviscosity and subclinical atherosclerosis in obese schoolchildren

Since December 2001, a total of 52 obese schoolchildren and 38 non-obese controls have undergone vascular ultrasound at our institution. From this cohort, we enrolled 40 obese children (16 girls, median age 12 years, mean body mass index (BMI) 27.5+/-4.6 kg/m2) and 26 non-obese peers (12 girls, median age 12 years, mean BMI 16.1+/-2.4 kg/m2) for the present study. We analysed the association between obesity, subclinical atherosclerosis and haemorheological variables, including whole blood viscosity (WBV) at four different shear rates (200 s-1, 30 s-1, 5 s-1 and 1 s-1), plasma viscosity, and serum lipid concentrations. Blood lipids and apolipoproteins were also determined and correlated with carotid intima-media thickness (IMT) and flow-mediated dilatation (FMD) of the brachial artery, measured by high-resolution ultrasonography. WBV at 200 s-1, 30 s-1 and 5 s-1, as well as plasma viscosity, were significantly higher in the obesity group (all P <0.01). No difference was seen for WBV at 1 s-1, erythrocyte aggregation and haematocrit. WBV at high shear rates was significantly associated with BMI, increased IMT and decreased FMD ( P <0.05). After controlling for BMI on stepwise linear regression, WBV at 30 s-1 was the strongest predictor of IMT ( P < 0.01) and FMD ( P < 0.05) among our study variables. Hyperviscosity is related to subclinical atherosclerosis in obese schoolchildren. Viscosity parameters contribute new information in addition to body mass index, blood pressure and blood lipids and may help to facilitate risk stratification in obese paediatric patients.

Haemorheological variables as risk factors of ischaemic heart diseases

Haemorheological variables as risk factors of ischaemic heart diseases

Haemorheological variables as risk factors of ischaemic heart diseases: Reply

Haemorheological variables as risk factors of ischaemic heart diseases: Reply

Effects of training period on haemorheological variables in regularly trained footballers

Objective: To investigate the effects of one football training period on haemorheological variables in regularly trained footballers. Method: Ten subjects were randomly selected from the reserve team of a football...

Blood Rheology, Cardiovascular Risk Factors, and Cardiovascular Disease: The West of Scotland Coronary Prevention Study

The West of Scotland Coronary Prevention Study (WOSCOPS) showed that pravastatin reduced the risk of coronary heart disease (CHD) events in 6,595 middle-aged hypercholesterolaemic men aged 45-64 years without prior myocardial infarction followed for an average of 4.9 years. We hypothesised prospectively (a) that baseline levels of haemorheological variables were related to baseline and incident CHD and to mortality; and (b) that reduction in lipoproteins by pravastatin would lower plasma and blood viscosity, a potential contributory mechanism to CHD events. We therefore studied plasma and blood viscosity, fibrinogen, haematocrit, and blood cell counts at baseline and 1 year. At baseline, plasma and blood viscosity were related to risk factors, CHD measures, and claudication. On univariate analysis, baseline levels of all rheological variables (except platelet count) were related to incident CHD; CHD mortality; and total mortality. On multivariate analysis including baseline CHD and risk factors, plasma and blood viscosity, haematocrit and white cell count each remained significantly associated with incident CHD; while fibrinogen remained an independent predictor of mortality (all p < 0.03). After one year, lipoprotein reduction by pravastatin was associated with significant reductions (about one quarter of a standard deviation) in plasma viscosity (mean difference 0.02 mPa.s, p <0.001) and in blood viscosity (mean difference 0.06 mPa.s, p<0.001), but was not associated with significant changes in other rheological variables. We therefore suggest that pravastatin therapy, which reduces elevated lipoproteins in hypercholesterolaemic men, may lower risks of CHD and mortality partly by lowering plasma and blood viscosity. Further studies are required to test this hypothesis.

Anaesthesia for vascular surgery of the lower limb

The patient with peripheral vascular disease is usually at a considerably increased operative risk owing to coexisting, generalized, cardiovascular atherosclerotic involvement; hypertension; chronic bronchitis and diabetes. Regional (most often epidural) as well as general anaesthesia is commonly used for vascular surgical procedures of the lower limb. Although regional anaesthesia is sometimes considered associated with fewer overall perioperative complications than general anaesthesia, there is a lack of convincing evidence clearly demonstrating a major difference in overall outcome related to the choice of anaesthetic approach. The sympathetic nerve block induced by epidural anaesthesia may, however, be advantageous, since leg blood flow is enhanced, which may be of importance for postoperative graft function. The risk of local complications to central blocks must always be considered, especially in vascular surgery patients who receive anticoagulants during the revascularization procedure. Since haemorheological abnormalities play an important role in the pathophysiology of circulatory disease, it seems essential to use perioperative fluid regimens which reduce blood viscosity and enhance blood flow. Colloid-containing fluids are preferred to crystalloids owing to better intravascular volume supporting capacity and specific colloid-associated pharmacological effects on haemorheological variables. Hypertonic saline (7.5% NaCl) in combination with colloid may be the future intraoperative fluid regimen of choice for peripheral vascular reconstructive surgery.

Altered flow properties of blood and increased plasma fibrinogen in cyclosporin-treated renal allograft recipients.

Abnormalities in blood rheology may be factors contributing to cardiovascular complications and the progression of renal failure in kidney allograft recipients. The haemorheological variables haematocrit, fibrinogen, whole blood viscosity, plasma viscosity, erythrocyte aggregation tendency and fluidity were measured in 27 cyclosporin A (CyA)-treated patients who had received a renal graft at least 6 months previously. Their creatinine clearance was in the range of 12-92 ml/min/1.73 m2 (mean 55+/-19). The values were compared with those obtained from a control group comprising 20 healthy subjects matched according to age, sex and smoking habits. The haematocrit, plasma fibrinogen, whole blood viscosity, plasma viscosity, erythrocyte aggregation tendency, body mass index (BMI), mean arterial pressure (MAP) and serum triglycerides were increased in the transplanted patients, and the serum high density lipoprotein (HDL)-cholesterol and erythrocyte fluidity decreased. The haemorheological variables were used as dependent variables in a stepwise regression analysis with age, MAP, BMI, urinary albumin excretion rate, blood CyA concentration, creatinine clearance, and serum triglycerides, cholesterol and HDL-cholesterol as independent variables. Plasma fibrinogen was positively correlated with BMI and blood CyA. The whole blood viscosity was positively correlated with blood CyA and negatively with serum HDL-cholesterol. Only serum triglycerides remained correlated with erythrocyte aggregation tendency. All variables with a known impact on blood viscosity were altered in the present group of renal transplant recipients. Inappropriate regulation of erythrocyte formation, overweight, the use of CyA, high triglycerides and low HDL-cholesterol levels may be factors contributing to this. The importance of impaired flow properties of blood for the development of cardiovascular diseases and transplant glomerulosclerosis needs to be examined.

Associations of blood rheology and interleukin-6 with cardiovascular risk factors and prevalent cardiovascular disease.

Haemorheological variables (whole-blood, plasma and relative blood viscosity, haematocrit, red cell aggregation, white cell count and fibrinogen) were measured in 753 men and 821 women aged 25-74 years, and related to cardiovascular risk factors and prevalent cardiovascular disease (CVD). Men had higher levels than women of blood viscosity, haematocrit, corrected viscosity and relative viscosity. Post-menopausal women had higher levels than pre-menopausal women of blood viscosity, haematocrit, corrected blood viscosity, plasma viscosity and fibrinogen: each of these differences was completely or partly abolished by use of hormone replacement therapy. Serum total cholesterol, triglycerides, diastolic blood pressure, body mass index and smoking markers showed positive associations with most rheological variables, whereas HDL-cholesterol, plasma vitamin C and social class showed inverse associations. Rheological variables were associated with prevalent CVD after age-adjustment. However, after multiple risk factor adjustment only plasma viscosity and red cell aggregation showed significant (P<0.04) associations in both men and women (comparing top to bottom quarters). Plasma interleukin-6 (measured in a 25% subsample of 196 men and 221 women) correlated significantly with age, fibrinogen, white cell count, plasma and blood viscosity, current smoking, and (in men) with low serum vitamin C levels; but not with other major risk factors or with prevalent cardiovascular disease.

Foot microcirculation and blood rheology in diabetes

Diabetes mellitus is associated with circulatory abnormalities. The blood flow in the skin of the dorsum of the foot and haemorheological variables were measured in 36 subjects. They were divided into three equal groups of diabetic patients: those with neuropathy, and both age and sex matched non-diabetic subjects; all were characterized by age, duration of diabetes and blood biochemistry. High and low shear rate blood viscosities were measured; aggregation was characterized using a Myrerene Aggregometer. The microcirculation in the skin of the dorsum of the foot was measured using a laser Doppler flowmeter. Measurements were made at room temperature with the subjects supine with the leg horizontal, and then with the lower leg vertical; the measurements were repeated at 42° C. Both diabetic groups had significantly increased low shear whole blood viscosity compared with normal subjects. The aggregation index was significantly greater in diabetic neuropaths than normal subjects. There were significant differences in the depth of vasomotor activity between the three groups, with the diabetic neuropaths commonly showing no motor activity at room temperature. The only significant correlations were between equilibrium laser Doppler values with the limb horizontal and both the low and high shear whole blood viscosities.

Haemorheological components in the pre-geriatric and geriatric age range in a randomly selected Western Sicily population sample (Casteldaccia study)

{Received 24.8.1992; accepted 7.12.1992} On a randomly selected western Sicily population sample (Casteldac­ cia Study: 600 subjects; 300 males and 300 females) of an age range from 40 to 79 years, were determined: Haematocrit (Htc) , blood vi­ scosity, plasma viscosity, filterability of whole blood and fibrinogen. Par­ ticipation was 97%, being 582 subjects (M = 287, F = 295). All the parameters measured showed a progressive increase with age and si­ gnificant differences were found between male and female subjects: Htc and blood viscosity were higher in male subjects (p < 0.01), while plasma viscosity, whole blood filterability and fibrinogen were higher in female subjects (p < 0.05). In subjects who smoke, compared to non­ smokers, all the haemorheological variables had significantly higher values (p < 0.005). Analysis of simple linear regressions showed a posi­ tive correlation between Htc and blood viscosity (r = 0.80), between plasma viscosity and fibrinogen (r = 0.81), between filterability and fibrinogen (r = 0.59) and between plasma viscosity and filterability (r = 0.64). Fibrinogen, plasma viscosity and filterability of whole blood were positively correlated with age (r = 0.37, r = 0.46, r = 0.41 respec­ tively). The authors conclude that the measurement of haemorheologi­ cal variables is a necessary for the assessment of cardiovascular risk, especially in subjects in age range 40-79 years.