Coagulation, fibrinolysis and haemorheology in premenopausal obese women with different body fat distribution

Thrombosis Research.Vol. 75, No. 3, pp. 223-231, 1994 Copyright8 1994 Elsevier Science Ltd Printedin the USA. All rights reserved 0049-3848/94 $6.00 + .oO

Pergamon

COAGULATION, FIBRINOLYSIS AND HAEMORHEOLOGY IN PREMENOPAUSAL OBESE WOMEN WlTH DIFFERENT BODY FAT DISTRIBUTION Gino Avellone, Vincenzo Di Garbo, Rosamaria Cordova, Gilia Raneli, Rosa De Simone, GianDomenico Bompiani From Institute of Clinical Medicine, University of Palermo Piazza Cliniche 2, 90127 Palermo, Italy

(Received

12 January 7994 by Editor G.G. Neri Semeri; revi.sa&‘accepted 6 May 1994)

ABSTRACT

Recently waist/hip ratio (WHR), a marker of body fat distribution, has been described as a risk factor for cardiovascular disease (CVD). The aim of the present study was to evaluate the influence of body fat distribution on metabolic, haemostatic and haemorheological pattern in premenopausal obese women with different WHR. Fourty premenopausal obese women were subdivided into two groups, matched for age and body mass index (BMI): 20 women with abdominal obesity (WHR= 0.94 f 0.02) and 20 women with peripheral obesity (WHR= 0.77 f 0.03). Twenty nonobese women were recruited as control group. The abdominal obesity group had significantly higher blood glucose, triglycerides, total cholesterol, Apolipoprotein B and plasma insulin levels and lower high density lipoprotein (HDL) cholesterol and Apolipoprotein Al levels than the control group. All the haemostatic (fibrinogen, Factor VII, plasminogen activator inhibitor (PAI) activity and tissue plasminogen activator (t-PA) antigen (Ag) pre venous occlusion (VO)) and haemorheological parameters (haematocrit, whole blood filterability, blood and plasma viscosity) were significantly higher in the abdominal obesity group as compared to the control group. In contrast, mean values of t-PA (Ag) post VO were significantly lower in abdominal obese women. Moreover positive correlations between WHR and plasma insulin (r= 0.66, p< 0.05), between WHR and fibrinogen (r= 0.63, p< 0.05) and between WHR and PAl pre VO (r= 0.71, p< 0.05) and a negative correlation between WHR and tPA (Ag) post VO (r= - 0.55, p< 0.05) were found. Our findings suggest that the lipid, haemostatic and haemorheological alterations may contribute to the high rate of cardiovascular events reported in abdominal obesity.

Key words: abdominal obesity, coagulation, fibrinolysis, haemorheology. Corresponding author: Prof. Gino Avellone, via De Gasperi 203, 90146 Palermo, Italy. 223

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Several prospective epidemiological studies have reported that obesity is a significant and independent risk factor for cardiovascular disease (CVD) and related mortality (l-3). Recent reports indicate that body fat distribution is more predictive of metabolic and cardiovascular diseases than the extent of body fat (4-6). Obesity with abdominal type of body fat distribution is a component of the so called *Poiymetabolic Syndromem or *Syndrome Xm;in fact, it has been shown to be closely related to cardiovascular risk factors such as hyperlipidemia, hypertension and both impaired glucose tolerance and non-insulin dependent diabetes mellitus, even if the mechanism responsible for this relationship has yet to be established (7-9). Body fat distribution can be evaluated by waist/hip ratio (WHR)that permits to distinguish abdominal (with high WHR) or peripheral (with low WHR) obesity. Abdominal obesity, characterized by high WHR, seems to be associated with an increased androgenic activity that may play an important role in the localization of body fat in the abdominal region and its accompanying metabolic disturbances (10-12). Many authors suggest that abdominal obese women (as well as normal men) have an exceedingly sensitive system for the mobilization of free fatty acids (FFA) due to a preponderance of beta-adrenergic receptors in deep abdominal adipose tissues. Exposure of the liver to elevated concentration of portal FFA might be the key event to several important consequences (12,13). It has been known for a long time that FFA stimulate hepatic gluconeogenesis and the synthesis and secretion of very-low-density lipoproteins (VLDL) and apolipoprotein BlOO, which are all established risk factors for atherosclerotic vascular disease. An additional important effect of FFA seems to be the inhibition of hepatic clearance of insulin, creating peripheral hyperinsulinemia and insulin resistance (5,13). Although it is possible that the accentuated metabolic risk profile may lead to the increased incidence of atherosclerotic manifestations, other abnormalities may also play an important role. The association between raised serum FFA and platelet activation is well documented and recently an increase in the concentration of FFA generated by lipoiysis of VLDL has been shown to cause an activation of coagulation pathway (14). Moreover many studies have indicated that obesity is associated with an activation of the haemostatic system, an impaired fibrinolytic activity and pathological haemorheological behaviour (1519). The aim of the present study was to evaluate the influence of body fat distribution on metabolic, haemostatic and haemorheological pattern in premenopausal obese women with different WHR. MATERIALS AND METHODS Patients.

Fourty premenopausal obese women, were included in the study among patients attending our Institute of Clinical Medicine. All obese women (with a body mass index (BMI) greater than 30) on the basis of a high or low WHR were subdivided into two groups, matched for age and BMI: 20 women (mean age: 36 f 4.5 years; BMI: 37.1 f 2.9) with abdominal (or android) obesity and 20 women (mean age: 37 f 3.5 years; BMI: 37.6 f 3.0) with peripheral (or gynoid-gluteal-femoral)obesity. WHR was calculated using waist circumference measured at midlevel between processus xiphoideus and the umbilicus and widest hip circumference. The abdominal obesity group showed a mean WHR =

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0.94 f 0.02. The peripheral obesity group had a mean WHR= 0.77 f 0.03 (p < 0.01). The two groups were comparable in terms of ratio of smokers to non smokers (5/20 and 4/20 respectively) and mean duration of obesity (14 f 5 years and 16 f 7 years respectively). Twenty nonobese women (showing a BMI value of 22 f 1.6 and aged 35 f 4 years) were recruited as control group.

Experimental procedure. All subjects gave informed consent before the investigation and were asked not to take any drugs for at least 10 days before the study. All subjects were apparently healthy, did not use oral contraceptives and had regular menstrual cycles of 24-30 days (median, 26 days). None of them had history or clinical signs of endocrine disorder, atherosclerotic vascular disease, hyperlipidaemia, diabetes mellitus or impaired glucose tolerance. Body weight was stable for at least 4 weeks before admission and during the testing period all subjects were asked to keep an isocaloric diet (20% protein, 30% fat and 50% carbohydrates). Blood collection was invariably performed during the early follicular phase (days 5 to 7) after an 6 hr abstinence from smoking and physical exercise. Blood was drawn from an antecubital vein (between 6:00 and 9:00 AM) after overnight fasting using a 1.2 mm siliconized needle without or with minimal stasis. Methods. We determined in serum using conventional enzymatic methods (Boehringer Mannheim, Milano, Italy): blood glucose (Glucose Oxidase), triglycerides (Glycerol Phoshate Oxidase), total cholesterol (Cholesterol Oxidase) and high density lipoprotein (HDL) cholesterol (after precipitation by dextran-magnesium chloride). The apolipoproteins Al and B were measured by radial immunodiffusion (R.I.D., Behring plates, Behring Institute, Scoppito, Italy) and plasma insulin was determined with a radioimmunoassay (Sorin Biomedica, Saluggia, Italy). Blood samples for the measurements of the haemorheological tests were collected in polypropylene tubes, using the following anticoagulants: for the determination of blood and plasma viscosity and haematocrit, sodium heparin 40 pL (from a solution containing 5000 lU/ml = 150 lU/mg) for 5 ml of blood to give a final concentration of 0.6%; for blood filterability, EDTA-K, (2 drops of solution at 10%) for 5 ml of blood to give a final concentration of 1%. All the haemorheological determinations were made within 2 hours after the drawing of the samples. We measured: haematocrit (by Wintrobe macromethod), blood and plasma viscosity (according to Rand (20) by a cone-on-plate micro-viscosimeter (model DV-II, Wells-Brookfield,Stoughton, Massachusetts, USA) estimated at different shear rates) and blood filterability (according to Reid’s method (21) by Nucleopore polycarbonate filters (Costar Corporation, Cambridge, USA) with pore diameter 5 u). Blood samples for haemostatic and fibrinolytic variables were drawn in polypropylene tubes containing one-tenth final volume of 3.8% sodium citrate, kept on crushed ice until centrifugation (4”C, 2,500 x g, 15 min) and plasma was stored in small aliquots at 70°C until use. A venous occlusion (VO) test was also performed in all subjects. A sphygmomanometer cuff was applied to the controlateral arm and inflated midway between systolic and diastolic pressure for 10 min. A further blood sample’was obtained before deflating the cuff from the occluded arm and separated as previously described. We determined:tissue plasminogenactivator (t-PA)antigen (Ag) pre and post VO by enzyme-linked immunosorbentassay (EUSA) with a commerciallyavailable kit (Innogenetics NY, Antewerp, Belgium);plasminogenactivator inhibitor (PAl) activity pre and post VO by a chromogenic substrate assay with reagents obtained from Behring (using an automated device, Behring Chromo Time System), Factor VII by a chromogenic substrate assay (BehringChromo Tie System)and fibrinogenby R.I.D. (Behringplates).

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IN OBESE

WOMEN

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Analysis was performed in duplicate following the manufacturer’s instructions and, in one series for each participant, within 8 months after sampling. Statistical analysis. All the values determined were expressed as mean f standard deviation (SD). The significance of differences between groups was determined by Student’s t-test for unpaired data after an analysis of variance. Linear regression analysis was used to study relationship between the variables. RESULTS Table 1 shows the metabolic characteristics of abdominal and peripheral obese women. The abdominal obesity group had significantly higher blood glucose, triglycerides, total cholesterol, Apolipoprotein B and plasma insulin levels and lower HDL-cholesterol and Apolipoprotein Al levels than the control group. In contrast, there were no significantly differences between the peripheral obesity group and the control group with the exception of HDL-cholesterol levels. All the haemorheological parameters (haematocrit, whole blood filterability, plasma viscosity and blood viscosity at both low (11.5-l) and high (230-t) shear rates) were significantly higher in the abdominal obesity group as compared to the control group (Table 2). In the peripheral obesity group only haematocrit and plasma viscosity were significantly higher than in the control group. Differences between the two groups of obese women and controls for haemostatic and fibrinolytic parameters are reported in Table 3. In the peripheral obesity group only fibrinogen levels were significantly higher than in the control group. In android obese women baseline levels of all the parameters were significantly higher as compared to the control group. Instead, after VO test, t-PA(Ag) was much lower and PAI higher in the abdominal obesity group than in the control group.

Table 1 Metabolic pattern In pmmenopausel obese and nonobeee women (mean f SD) Abdominal obesity

QmP

(n = 20) Blood glucose Total Chdestercl Triglycerides HDL-Chdesterol Apolipoprotein Al Apolipoprotein B Plasma insulin

(mddl)

81.6 f 4.8. 238.4 f 17.1’

(mgldl) (mgkil) (mgldl) (mgldl) (mW

228.7 30.2 120.8 159.9 18.2

(mgld0

?? p
f 46.8*’ f 4.8.’ .k 14.4.’ f 22.2” f 5.7’

Petiphetal obesity QrooP

(n = 20) 87.5 222.4 175.2 38.1 134.3 136.7 12.1

f f f f f f f

4.8 14.2 23.8 4.0” 13.6 16.4 4.3

contmi QrouP (n = 20) 86.4 218.8 138.3 46.2 138.7 132.5 Il.6

f f f f f f f

5.0 12.3 18.5 3.2 11.6 14.8 4.0

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Table 2

Haemorheological pattern in premenopausal obese and nonobese women (mean f SD) Abdominal obesity srmp (n = 20)

Peripheral obesity group (n = 20)

Control group (n = 20)

Haematocrit

(%)

47.2

f

1S’

44.1

f

2.2.

42.5

f

1.8

Whole blood

(set/ml)

32.5

f

5.3**

26.8

f

2.5

24.5

f

2.6

(cps)

5.23

f 0.38*’

4.61

f

0.40

4.37

f

0.35

Blood viscosity (11.5-I)

(cps)

11.66

*0.78-

9.98

f

0.55

9.85

f

0.50

Plasma viscosity

(cps)

1.84

f 0.15”

1.69

f

0.11’

1.52 f

filterability Blood viscosity (230.0’)

?? p
0.10

p < 0.01 vs control group

Table3 Haemostatic and fibrinolytic pattern in premenopausal obese and nonobese women (mean f SD) Abdominal obesity

Factor VII Fibrinogen PAIpra VO PAIpostVO t-PA pm VO t-PA postVO *p
(Q/o) (mgldf) (U/ml) (U/ml) (n&f0 (ns/df)

Petipheral obesity

group

QmP

group

(n = 20)

(n = 20)

121.5 562.5 7.2 8.8 8.5 11.9

f f f f f f

12.2” 102.5 f 151.1** 409.3 f 2.4. 5.4 f 2.9** 5.6 f 2.4’ 6.8 f 2.3** 15.9 f

Control (n =

20)

10.4 100.3 f 9.0 85.4’ 324 f 44.3 1.9 4.9 f 2.0 2.0 4.8 f 2.4 1.4 6.6 f 1.9 2.6 16.4 f 3.0

p < 0.01 vs control group

The correlations between metabolic, haemostatic and haemorheological data and WHR were also calculated. Data from the three groups of women were pooled for these calculations. There was no significant correlation between WHR and haemorheological and metabolic parameters with the exception of a significantly positive correlation between WHR and plasma insulin (r= 0.68, p < 0.05). Instead, as concerns haemostatic and fibrinolytic parameters, significantly positive correlations were reported between WHR and fibrinogen (r= 0.63, peO.05) and between WHR and PAI pre VO (r= 0.71, pgO.05). Furthermore, a significantly negative correlation was observed between WHR and t-PA post VO (r= - 0.55, p
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DISCUSSION

Our results show that body fat distribution is related to metabolic, haemorheological and haemostatic abnormalities in premenopausalobese women. A number of epidemiological studies have demonstrated that abdominal obesity, indicated by a high WHR, strongly correlates with established risk factors for cardiovascular disease being independent of the degree of obesity (3,Q).Accompanying risk factors of obesity include hypertriglyceridemia, hypercholesterolemia, low HDL-cholesterol levels and an impaired glucose tolerance combined with hyperinsulinemia; this has even been regarded as a link between obesity and hypertension (13). Together with a significant change in metabolic parameters, the present study shows in abdominal obese women a high blood and plasma viscosity and impaired erythrocyte deformability. We also observed in the android obesity group a significantly increased haematocrit, that could be a factor responsible for elevated whole blood viscosity (22). The elevated haematocrit reflects an increased erythropoietic activity present in obese patients possibly related to ventilation disturbances. Impaired pulmonary function should be more pronunced in obese patients with abdominal distribution of the body fat (19). Whole blood hyperviscosity in abdominal obesity is also influenced by changes in erytrocyte membrane structure and function, involving disturbances in membrane lipid composition and in the cation transport and alterations in insulin receptors (23,24). Instead, it is known that plasma viscosity is strongly influenced by the concentration of macromolecules and therefore by lipoproteins and fibrinogen. Because abdominal obese women have significantly higher fibrinogen levels, blood and plasma hyperviscosity, as well as increased erythrocyte aggregability,are at least in part a consequence of hyperfibrinogenemia. The increase in Factor VII activity in abdominal obese women needs to be emphasized together with the increase in fibrinogen levels. In fact high fibrinogen and Factor VII levels are expression of an elevated turnover of the coagulation pathway and may be involved in the pathogenesis of ischaemic heart disease (25,26). In previous studies a positive correlation between coagulation factors and BMI or WHR was reported (17,27). Moreover recent findings have shown that the increase in Factor VII activity is related to the triglyceride content of lipoproteins (26-30). In agreement with others (31), baseline concentrations of t-PA (Ag) were higher in abdominal obese women and a positive correlation was observed between WHR and PAI, the major inhibitor of fibrinolysis. These observations are of particular interest, because an increase in baseline levels of t-PA and PAI has been proposed as a predictor of CVD (32-34). However, baseline fibrinolytic activity is very low and cannot be commonly used as a good marker for hypofibrinolysis. More recently, an increase of PAI activity after VO test has been proposed as a marker of a deficient t-PA (Ag) release by endothelial cells with an alteration of t-PA/PA1balance which could predispose to thrombosis (35). Our findings seem to suggest that the impaired fibrinolytic activity in obesity is dependent on the regional distribution of the adipose tissue. Abdominal obese women showed an impaired fibrinolytic response after 10 min of VO linked to higher levels of plasma PAI activity and to lower release of t-PA (Ag) by endothelium as compared to peripheral obese women.

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Hyperinsulinemia may play an important role in the impaired fibrinolytic activity reported in obese patients (1536). Vague found a negative correlation between plasma insulin levels and fibrinolytic activity, possibly due to an increased PAI level (16). PAI level also seems to be influenced by other metabolic risk factors, such as the triglyceride levels, suggesting the existence in the dismetabolic states of an abnormality of endothelial cell function that contributes to a poor fibrinolytic response to VO (13,32,36,37). Consequently, the increased risk for CVD associated with an high WHR may be due to a reduced fibrinolytic activity reflected by the elevated baseline levels of PAI activity and fibrinogen and deficient release of t-PA (Ag) after VO. This risk is further enhanced by the haemorheological abnormalities associated with high WHR in obese women. These are known not only to influence the fluidity of blood, but also to contribuite to the pathogenesis of atherosclerotic vascular disease and to the high rate of thromboembolic events (16,38). In conclusion, the synergic effects of metabolic, haemorheological and haemostatic abnormalities could explain the pathophysiological interrelationships between obesity, diabetes mellitus, hyperlipidaemia, hypertension, atherosclerotic vascular disease and the increased morbility and mortality reported in abdominal obesity. Acknowledgements This research (N092.00010.PF41)was supported by Italian National Research Council (CNR): Targeted Project #Prevention and Control Disease Factorsm,Subprojects SP8 *Control Cardiovascular Pathologys. REFERENCES 1. HUBERT, H.B., FEINLEIB, M., McNAMARA, PM., CASTELLI, W.P. Obesity as independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham Heart Study. Circulafion 67,668-977, 1983. 2. SHAPER, A.G., POCOCK, S.J. Risk factors for ischaemic heart disease in British men. BrHeati J57,11-16, 1987. 3. COLEMAN, M.P., KEY, T.J.A., WANG, D.Y., HERMON, C., FENTIMAN, I.S., ALLEN, D.S., JARVIS, M., PIKE, M.C., SANDERS, T.A.B. A prospective study of obesity, lipids, apolipoproteins and ischaemic heart disease in women. Atherosclerosis 92, 177-185, 1992. 4. LARSSON, B., SVARDSUDD, K., WEUN, L., WILHELMSEN, L., BJORNTORP, P., TIBBLIN, G. Abdominal adipose tissue distribution, obesity and risk of cardiovascular disease and death: a 13-year follow up of partrcrpantsin the study of men born in 1913. Br Med J Exp Med Biol189,277-297,1985. 5. STERN, M.P., HAFFNER, S.M. Body fat distribution and hyperinsulinemia as risk factors for diabetes and cardiovascular disease. Arteriosclerosis 6, 123 130, 1986. 6. HAUNER, H., STANGL, K., SCHMATZ, C., BURGER, K., BLOMER, H., PFEIFFER, E.F. Body fat distribution in men with angiographically confirmed coronary artery disease. Ath8fOSd8mSiS 85,203-210, 1990. 7. AVOGARO, P., CREPALDI, G. Essential hyperlipemia, obesity and diabetes. In: European Association for the Study of Diabetes, First Annual Meeting, p.53, Montecatini Terme, (1965). 8. REAVEN, G.M. Role of insulin resistance in human disease. Diabetes 37, 15951607,1988.

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