Prolonged very low calorie diet in obese subjects reduces factor VII and PAI but not fibrinogen levels

Fibrinolwis ( 1994) 8, l&2 I 01994 Longman Group UK Ltd

Prolonged Very Low Calorie Diet in Obese Subjects Reduces Factor VII and PA1 But Not Fibrinogen Levels

G. Palareti, C. Legnani, M. Poggi, M. Parenti, A. C. Babini, R. Biagi, L. Baraldi, A. Luchi, M. Capelli, S. Coccheri

SUMMARY! Fibrinogen, factor VII and plasminogen activator inhibitor (PAI) are now considered hematologic risk factors for cardiovascular disease and it is well known that their levels are increased in obesity. 52 obese healthy subjects (31 women), with BMI90, were investigated to assess: (a) fibrinogen, factor VII and PA1 levels in relation to the different types of body fat distribution, and (b) the effect of a prolonged period (3 months) of very low calorie diet (VLCD, 514 and 470 K&/day in women and men respectively) on the above mentioned variables. Only significantly higher factor VII and insulin levels were found in the subjects with the highest waist/hip ratio (WHR) values and no significant correlations were found between test results and WHR levels. After 3 months VLCD, body weight and BMI decreased markedly, and factor VII and PA1 levels dropped significantly in 3W34 individuals who were compliant with the diet; changes in factor VII and PA1 were correlated with decreases in BMI and apolipoprotein Al, but not with changes in insulin values or their sum after glucose load. Fibrinogen did not change significantly after diet. In conclusion, the present study in a group of highly obese subjects shows that a severe dietary restriction associated with marked weight loss consistently lowers factor VII and PAI, but not fibrinogen.

Several epidemiological studies have recently shown that the levels of three hemostatic factors, namely fibrinogen (for a review see l), factor VII2 and plasminogen activator inhibitor (PAI), have predictive value for cardiovascular disease (CVD). It seems therefore reasonable to investigate the mechanisms by which the levels of these factors can be lowered in groups of patients at high cardiovascular risk. Obesity is a well known predisposing condition for CVDti and seems closely related with changes of the said clotting and fibrinolytic factors. In fact, increased levels of one or more of the above mentioned proteins have consistently been reported in obese individuals both adults and children.7 Moreover, the abdominal type of obesity, characterized by a high waist/hip ratio (WHR), is reportedly associated with a higher risk of CVD and death than the gluteo-femoral type,*T9 and with higher values of fibrinogen and PAI. This study aimed at investigating whether: (a) levels of fibrinogen, factor VII and PAI in obese healthy subjects differ according to the different types of body fat distribution, and (b) prolonged administration of a very low

calorie diet, associated with substantial weight loss, affects levels of hemostatic risk factor for CVD.

CLINICAL MATERIAL AND METHODS Clinical Material From a series of obese subjects attending the outpatient service of the Institute of Clinical Medicine and Gastroenterology (University Hospital S. Orsola, Bologna, Italy) 52 individuals (31 women) were selected on the basis of the following criteria: body mass index (BMI=W/H2; kg/m2) >30 (median 36.5, range 32-55); absence of diabetes (according to the WHO criteria,“); no history or symptoms of metabolic, cardiovascular, respiratory, hematologic or endocrine disease. The women were not in menopause or on the pill and ovarian polycystosis was excluded. These individuals, who lived within 80 Km of our Hospital, agreed to take part in a clinical study protocol which included: a 3 months’ period of very low calorie diet with follow-up visits at the outpatient service every 2 weeks for physical examination, diet compliance control, weight and arterial pressure measurement, assessment of intercurrent illness and routine laboratory testing. The characteristics of the investigated obese subjects are summarized in Table 1.

G. P&ret& C. Legnani, M. Poggi, R. Bii, S. Coccberi, Depattment of Angiology and Blood Coagulation, Institute of Clinical Medicine and Gastroenterology, M. Capelli, M. Parenti, A. C. Babini, L. Baraldi, A. Luchi, Central Laboratory, University Hospital S. Orsola, Bologna, Italy. I6

Fibrinolysis

Table 1 Characteristics

of the investigated

obese subjects.

17

Results are given as median and

range Women n. 31 Age Weight BMI WHR Smokers Fibrinogen Factor VII:C PA1 Glucose Insulin Zinsulin Cholesterol HDL Cholesterol Triglycerides Apolipoprotein AI Apolipoprotein B

(Y) (Kg) (Kg/m*)

(mg/dl) (lo) (AU/ml) (mmolll) (NJ/ml) CPU/ml) (mmolll) (mmolll) (mmol/l) (g/l) (g/l)

35 ( 18-50) 92 (76-145) 35 (32-55) 0.84 (0.73-0.98) n.I I 296 (2 15462) I IO (74-165) 27(l-38) 5.60(4.16-7.21) 23 ( 12-60) 242 (92-893) 5.39 (3.73-7.41) I .06 (0.65-I .68) I .22 (0.55-2.00) I .46 (I .03-2.28) I. I4 (0.55-I .55)

*=p
Type of Fat Distribution Type of obesity was evaluated in the whole group of 52 subjects by waist to hip circumference ratios (WHR). Circumferences were measured according to Cigolini et al” at waist (midlevel between lower rib margin and iliac crest), and hip (widest circumference over the great trochanters) levels, in subjects in standing position and at end of gentle expiration. The distribution in tertiles (for women and men separately) of the investigated obese subjects in relation to WHR values is shown in Figure 1. 8 women and 5 men investigated at baseline did not present at outpatient clinic for periodic examinations and were excluded from further study. Of the remaining 39 obese subjects who completed the study period, 4 women and 1 man were excluded from final evaluation because of poor compliance with diet, confirmed by the criteria specified in the Method section. The final study group consisted of 34 obese subjects (median age 33 years, range 18-56), of whom 19 were women (median age 32 years, range 18-50) and 15 males (median age 4 1 years, range 23-56). They met the study protocol criteria in full, and were compliant with the very low calorie diet.

Men n. 21 31 (18-56) 120(103-163) 40 (32-54) 0.99 (0.92-I I) n.6 314(216492) 104 (69-128) 33 (740) 5.05 (4.22-9.32) 26 (2-149) 245 (126-1043) 5.28 (3.94-7.12) 0.96 (0.78-I .22) I .5 I (0.56-5.59) 1.30(1.18-1.54) 1.27(1.17-1.38)

*** ** ***

* * **

test); WHR=waist

to hip ratio

850 ml water and preferably consumed at 5 times during the day. Both VLCDs comprised 56% protein (derived from milk and eggs), 40% lipids (lecitins), 4% glucides (maltodextrins); in both cases diet was supplemented with vitamins and mineral salts (Supradyn-R 1 cp/d, La Roche, Switzerland) and at least 2 litres water per day, or other drinks if not sugared, were recommended. The diet period lasted 3 months. Poor compliance with diet

Obese

.

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? ? ? ? ? ? ?? ? ? ?..i.”

I

1st

0.9

men

095

.

1

2nd

.

term

3rd

1

.

.

1.05

WHR

”

Obese women

Very Low Calorie Diet The very low calorie

diet (VLCD) adopted by the women supplied 514 Kcal/day. This formula diet was based on: (a) 1 preparation/day of ‘Precision N’ (Sandoz Nutrition Company, USA), diluted with at least half litre water and divided in 4 daily administrations; (b) once a day 200g chicken or veal (or 300g of fish) cooked without any fat or salt. In men the VLCD supplied 470KcaYday, comprising a daily diet pack (by Gazzoni S.p.A., Italy), to be diluted with not less than

07

0 75

08

0 85

09

0 95

WliR’

Fig. 1 Distribution of WHR values at baseline in the investigated obese subjects (women and men separately). The dotted vertical lines indicate the tertile limits.

18

Prolonged Very Low Calorie Diet in Obese Subjects

mg,dl

FIBRINOGEN

FACTOR VII

PAI

AU/ml 50

550 500 450 400 -

B

VLCD

B

VLCD

B

VLCD

Fig. 2 Individual changes in fibrinogen, factor VII and PAI levels recorded in the 34 compliant obese subjects examined at baseline (B) and after 3 months of very low calorie diet (VLCD). The shaded areas represent the normal values.

was inferred by the following objective criteria: recurrent high urinary sodium elimination in relation to scheduled dietary intake, absence of acetonuria, and unexpectedly low weight reduction after 3 months (less than 6 Kg). Blood Sampling Blood sampling was performed before (baseline) and at the end of the 3 month diet period. Blood was sampled at 07:30 to 09:OOafter overnight fasting and 10 min resting in supine position. Venous blood was anticoagulated with trisodium citrate (0.129 M, l/10) for fibrinogen and factor VII; for PA1 measurements, Diatube Htm tubes (sodium citrate, citric acid, theophylline, adenosine and dipirydamole, Stago) were used. After 30min centrifugation at 3000xg at room temperature (4°C for PA1 determination tubes), plasma samples were immediately snap frozen in aliquots and stored at -7OOC until further processing.

thromboplastin (Behring Institute, Scoppito, Italy) and the KC-10 coagulometer (CV: 4.9% intra- and 5.8% interassay). PA1 was measured as activity (Coatest PAI, Kabi Vitrum, CV: 4.5% intra- and 7.1% interassay). Routine laboratory tests were performed at beginning and end of diet period and at least once monthly throughout. They included: blood lipids, liver function tests, whole blood cell count, total and fractionated plasma proteins, sideraemia, plasma and urinary Na, K, Mg electrolytes, blood glucose, creatinine, uric acid, ketonuria, insulinaemia (immuno-radiometric method) and Apo Al and Apo B (immunochemical method). At the beginning and end of the study period an oral glucose (75g as Curvosio, Sclavo, Italy) tolerance test (OGTT) was performed. Samples for capillary blood were taken at 0,30,60,90, 120 and 180 min for glucose determination, whereas venous blood samples were drawn at hourly intervals after baseline for insulin measurement; the sum of insulin values was also calculated @insulin).

Laboratory Methods Fibrinogen was measured by the Clauss method,‘* carried out with a semiautomatic KC- 10 Amelung coagulometer (Mascia Brunelli, Milan, Italy) and liquid bovine thrombin (Fibrinogen-Test, Diamed, Murten, Switzerland). A standard curve was prepared to transform the clotting times into fibrinogen concentrations by l/5, l/10 and l/20 dilutions (in Owrens’ buffer, pH 7.35) of a reference plasma pool, whose fibrinogen content had previously been measured by gravimetric method.i3 The coefftcients of variation (CV) of the method were: 3.4% intra- and 4.8% interassay. Factor VII levels were determined by means of onestage clotting assay using a commercial deficient plasma (Baxter-Dade, Rome, Italy), the Thromborel-S

Statistical Methods Statistical analysis was performed by two-tailed Wilcoxon test or Mann-Whitney U-test on paired or unpaired data as appropriate. The results were expressed as median values and ranges. The correlations between data were assessed by means of Spearman’s r’ coefficient of rank correlation.

RESULTS As shown in Table 2, subjects with the highest WHR had significantly higher baseline factor VII and insulin levels (~~0.05). No significant correlations were found

Fibrinolysis

Table 2 Comparison

19

of median (range) values calculated at baseline in obese subjects

included in the tertile with highest WHR values (for women and men separately), versus subjects of the other tertiles (see also Fig. I) I st+2nd tertiles

3rd tertiles n. I8

n. 34 Age Weight BMI Fibrinogen Factor VII:C PAI Glucose Insulin Zinsulin Cholesterol HDL Cholesterol Triglycerides Apolipoprotein A I Apolipoprotein B

31 102 36 312 I04 28 5.49 21 242 5.39 I .Ol

(14-56) (76-163) (32-54) (215492) (699165) (l-39) (4.16-6.77) (22149) (97- 1043) (3.73-7.41) (0.67-l .68) I.30 (0.55-5.59)

(Y) (Kg) (mg/dl) (S) (AU/ml) (mmol/l) (ktLJ/ml) (l.tU/ml) (mmol/l) (mmol/l) (mmol/l)

*

*

I .52

(I .27-2.28) l.l4(0.55-1.53)

(g/l) (g/l)

39 I I2 39 304 II5 29 5.38 27 246 5.37 I .02 I .47 I .4 I 1.23

(I 8-53) (81-145) (33-55) (2 17-392) (94-148) (7-40) (4.66-9.32) ( 12-60) (92-893) (4.2557.12) (0.65-I .29) (0.67-2.43) (I .03-l .75) (0.81-1.55)

*=p
between the levels of fibrinogen, factor VII or PAI, and the WHR values. Correlation coefficients were also calculated between the hematologic risk factor levels at baseline, and between these latter and the other variables. No significant correlations were found between the three hematologic risk factor levels. Fibrinogen was correlated with cholesterol (0.33, ~~0.05); factor VII with insulin (0.30, ~~0.05) and triglyceride (0.28, p
changes in BMI (p
DISCUSSION Previous studies7,‘4-‘x reported that highly obese subjects often present with high levels of factor VII, PA1 and, to some extent, fibrinogen. These hematological factors have proved to be of predictive value for cardiovascular morbidity and mortality.‘-’ Evidence from epidemiological studies shows that the

Table 3 Median and range recorded in compliant obese subjects (n. 34) at beginning and end of 3 months diet period After VLCD

Baseline Weight BMI Fibrinogen Factor VII:C PA1 Glucose Insulin Zinsulin Cholesterol HDL Cholesterol Triglycerides Apolipoprotein A I Apolipoprotein B

(Kg) (mddl) (%) (AU/ml) (mmol/l) (Nml) (NJ/ml) (mmolll) (mmol/l) (mmol/l) (gfl) (g/l)

104 37 296 108 28 5.38 22 245 5.33 0.98 I .30 I .39 I .2l

(76-145) (32-55) (2 155492) (699148) (l-40) (4.16-9.32) (2-83) (97- 1043) (3.73-7.28) (0.65-I .68) (0.562.43) (I .0332.07) (0.55-I .53)

*** *** ns *** *** *** *** *** ***

** ***

*=p
86 (60-125) 31 (2544) 297 (2 10-428) 83 (655127) 8 (O-40) 4.86 (3.83-6.10) I I (5-25) 149(5&317) 4.79 (2647.33) 0.98 (0.75-I .39) 0.99 (0.45-3.76) I .2 I (0.96-I .46) I .06 (0.47-I .70) low calorie diet

20

Prolonged Very Low Calorie Diet in Obese Subjects

incidence of cardiovascular disease in obese subjects varies according to the type of fat distribution, androidabdominal obesity being more at risk than gluteofemoral type. *v9Recent studies indicate higher fibrinogen and PA1 levels in women with abdominal obesity I9 as well as significant correlations between these factors and WHR values.‘9-20 Our study, however, which considered obese subjects of both sexes, only partly confirmed these results. Only factor VII and insulin levels were significantly higher in individuals of tertiles with the highest WHR value (those with most abdominal obesity); fibrinogen and PA1 levels proved to be no higher in cases with highest WHR levels. No significant correlation was found between the investigated hematologic risk factor levels and WHR values. As reported by other authors,2’*22 the present study confirmed at baseline the relationship between factor VII and insulin/triglyceride levels, and between PA1 and insulin levels. In line with our previous results in obese children’ and those of other authors in adult women,19 we found only a weak correlation between PA1 and triglyceride levels, a correlation reported as being stronger in other studies. ‘5,20This discrepancy may in part be ascribed to the fact that in the present as well as our previous study of obese children’ the triglyceride levels were almost normal. Furthermore, in line with the views of Landin et a1,l9 it may be assumed that the correlation between PA1 and triglycerides is mainly mediated by insulin resistance and hyperinsulinemia. Although the question of the effect of dietary restriction and associated weight reduction on hematological CVD risk factor levels is of great relevance, results to date have been scant. Reduced factor VII (but not librinogen) by a short-term (7 days) VLCD in 9 obese subjects was recently reported by Slabber et a1.23To our knowledge, no data are available on the effects of a long period of very low calorie diet in a consistent number of obese subjects. Our study shows that such a diet, maintained for 3 months, significantly affects certain hemostatic factors (factor VII and PAI) relevant to CVD in relation to extent of the obtained weight loss. Factor VII markedly decreased in all but one subject, thus confirming its close relationship with dietary and nutritional conditions.24 Our data, however, only partially agree with those of Baron et a125who, in less overweight subjects kept on a milder diet, found only transient factor VII reduction (after 1 month but no more), with no correlation with weight loss. It seems likely that many factors (such as degree of obesity, severity/duration of diet, and degree of compliance) may well explain these different results. Reduced factor VII levels have also been reported in a recent study after weight reduction from physical training in young sedentary men.26 A sharp drop in PA1 levels after diet was recorded in our study in correlation with reduction of BMI. PA1 levels decreased in all obese women, regardless of the level of baseline values. Other studies, performed in smaller, male-female, moderately obese groups of subjects, recently reported a reduction in PA1 levels after weight loss and/or physical exercise.2628

High fibrinogen levels in healthy obese subjects have been consistently reported, not only in adults but in children too.‘*‘“‘* In a previous study” we found no significant reduction in fibrinogen levels after dietary slimming, a result confirmed by both recent papers’8*23.28 and the present study where average fibrinogen levels did not change despite severe dietary restriction. This result confirms that fibrinogen levels are only fractionally regulated by dietary and nutritional factors. In conclusion, the present study in a group of highly obese subjects shows that: (a) higher factor VII and insulin levels are found in subjects with the highest WHR values, while fibrinogen and PA1 levels do not differ in subjects according to the type of fat distribution, and (b) severe and prolonged dietary restriction associated with marked weight loss consistently lowers the levels of factor VII and PAI, but not those of fibrinogen.

ACKNOWLEDGEMENTS We are grateful to Stephen Jewkes for his revision of the English.

REFERENCES I. Cattaneo M, Mannucci P M. Predictive value of coagulation tests in arterial thrombosis. Res Clin Lab 1988; 18: l-7. 2. Meade T W, Mellows S, Brozovic M, et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; ii: 533-537. 3. Hamsten A, Wiman B, De Faire U, Blomback M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 1985; 313: 1557-1563. 4. Hubert H B, Feinlierb M, McNamara P M, Castelli W P. Obesity as an independent risk factor for cardiovascular disease. A 26year follow-up of participants in the Framingham Heart Study (1983). Circulation 1983; 67: 968-974. 5. Tuomilehto J, Salonen J T, Martti B, et al. Body weight and risk of myocardial infarction and death in the adult population of eastem Finland. Br Med J 1987; 295: 623-627. 6. Welin L, Svardsudd K, Wilhelmsen L, Larsson B, Tibblin Analysis of risk factors for stroke in a cohort of men born in 1913. NEngl J Med 1987; 317: 521-526. 7. Legnani C, Maccaferri M, Tonini P, Cassio A, Cacciari E, Coccheri S. Reduced fibrinolytic response in obese children: association with high baseline activity of the fast acting plasminogen activator inhibitor (PAI). Fibrinolysis 1988; 2: 21 I-214. 8. Lapidus L, Bengtsson C, Larsson B, Pennert K, Rybo E, Sjiistrom L. Distribution of adipose tissue and risk for cardiovascular disease and death: a I2 year follow up of participants in the population study of women in Gothenburg, Sweden. Br Med J 1984; 289: 1257-1261. 9. Larsson B, Svardsudd K, Welin L, Wilhelmsen L, Bjomtorp P, Tibblin Cl. Abnormal adipose tissue distribution, obesity, and risk of cardiovascular disease and death: I3 year follow up of participants in the study of men born in 1913. Br Med J 1984; 288: 1401-1404. IO. WHO Expert Committee on Diabetes mellitus: Technical report series. Geneva, World Health Organisation 1985. I I. Cigolini M, Seidell J C, Charzewska J, et al. Fasting serum insulin in relation to fat distribution, serum lipid profile, and blood pressure in european women: The European Fat Distribution Study. Metabolism 1991; 40: 781-787. 12. Von Clauss A. Gerinnungsphysiologische Schnellmethode zur

Fibrinolysis

13. 14.

15.

16.

17.

18.

19.

20.

Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-246. Ingram GIG. The determination of plasma fibrinogen by the clotweight method. Biochem J 1952; 51: 583-585. Folsom A R, Wu K K, Davis C E, Conlan M G Sorlie P D, Szklo M. Population correlates of plasma fibrinogen and factor VII, putative cardiovascular risk factors. Atherosclerosis I991 ; 9 I : 191-205. Juhan-Vague I, Vague P, Alessi M C, et al. Relationships between plasma insulin, triglyceride, body mass index, and plasminogen activator inhibitor I. Diab Metabol 1987; 13: 33 l-336. Cacciari E, Balsam0 A, Palareti G, et al. Haemorheologic and fibrinolytic evaluation in obese children and adolescents. Eur J Pediat 1988; 147: 381-384. Parenti M, Palareti G, Babini A C, Poggi M, Torricelli P Melchionda N. Effect of slimming on metabolic and haemorheologic patterns in a group of obese subjects. Int J Obesity 1988: 12: I79- I 84. Craveri A, Tornaghi G, Paganardi L, Ranieri R, Giavardi L.Weight loss does not affect the hemorheological and fibrinolytic changes in obesity. Clin Hemorheol 1990; IO: 541-546. Landin K, Stigendal L, Eriksson E, et al. Abdominal obesity is associated with an impaired fibrinolytic activity and elevated plasminogen activator inhibitor- I. Metabolism 1990: 39: 1044-1048. Vague P, Juahn-Vague I, Chabert V, Alessi M C, Atlan C. Fat

Received: 6 April I993 Accepted after revision: I2 July 1993 Offprints orders to: Dr G. Palareti, Dept. Angiology and Blood Coagulation, University Hospital S. Orsola, Via Massarenti, 40138 Bologna, Italy. Tel: 39 5 1 6363420; Fax: 39 5 I 34 1642.

21.

22.

23.

24.

25. 26.

27.

28.

2I

distribution and plasminogen activator inhibitor activity in nondiabetic women. Metabolism 1989; 38: 913-915. Carvalho de Sousa J, Soria C, Ayrault-Jarrier M, et al. Association between coagulation factors VII and X with triglyceride rich lipoproteins. J Clin Pathol 1988; 41: 940-944. Juhan-Vague I, Alessi M C, Vague P. Increased plasma plasminogen activator inhibitor I levels. A possible link between insulin resistance and atherothrombosis. Diabetologia I99 I ; 34: 457462. Stabber M, Barnard H C, Kuyl J M, Badenhorst C J. Effect of a short-term very low calorie diet on plasma lipids, fibrinogen, and factor VII in obese subiects. Clin Biochem 1992: 25: 334-335. Meade T W. Epidemiology of atheroma, thrombosis and ischaemic heart disease. In: Bloom A L, Thomas D P, eds. Haemostasis and Thrombosis. Edinburgh: Churchill Livingstone, 1987: 697-720. Baron J A, Mann J, Stukel T. Effect of weight loss on coagulation factors VII and X. Amer J Cardiol 1989; 64: 5 19-522. Gris J C, Schved J F, Feugeas 0, et al. Impact of smoking, physical training and weight reduction on FVII, PAI-I and hemostatic markers in sedentary men. Thromb Haemost 1990; 64: 5 16-520. Sundell I B, Dahlgren S, Ranby M, Lundin E, Stenling R, Nilsson T K. Reduction of elevated plasminogen activator inhibitor levels during modest weight loss. Fibrinolysis 1989; 3: 5 l-53. Mehrabian M, Peter J B, Barnard R J, Lusis A J. Dietary regulation of tibrinolytic factors, Atherosclerosis 1990; 84: 25-32.