25
Atherosclerosis, 84 (1990) 25-32 Elsevier Scientific Publishers Ireland, Ltd.
ATHERO
04516
Dietary regulation of fibrinolytic factors Margarete Mehrabian I, James B. Peter I, R. James Barnard ’ and Aldons J. Lusis 3 ’Specialty Laboratories, Inc., Santa Monica, CA 90404 (U.S.A.), ’ Department of Kinesiology and 3 Departments
of Medicine and Microbiology,
University of California, Los Angeles, CA 90024 (U.S.A.)
(Received 18 December, 1989) (Revised, received 23 April, 1990) (Accepted 24 April, 1990)
W examined the short-term effects of a high-complex carbohydrate, low fat diet on the plasmin-dependent fibrinolytic pathway. A population of 27 adult American Caucasians exposed to the diet for 3 weeks showed highly significant reductions in the levels of plasminogen (P = O.OOOl), tissue plasminogen activator (tPA) (P = 0.0001) and plasminogen activator inhibitor (tPA1) (P = 0.0017). Fibrinogen levels also decreased, but the changes did not reach statistical significance (P = 0.07). In contrast, the levels of the Lpa(a) lipoprotein, a potential inhibitor of fibrinolysis, remained remarkably constant despite a marked decrease in the levels of apolipoprotein B, a major constituent of Lp(a). Correlations between the levels of tPA, tPA1 and plasma triglyceride were observed among the individuals both before and after the dietary challenge. Although the mechanisms responsible for the effects are unknown, the dramatic responsiveness of the thrombolytic pathway to dietary challenge is likely to be of importance in understanding the etiology of coronary artery disease and other vascular disorders.
Key words: Lipoproteins;
Fibrinolysis;
Lp(a); tPA; tPA1; Fibrinogen;
Introduction
Increased levels of fibrinogen [l-3], Lp(a) [4-61 and tissue plasminogen activator inhibitor (tPA1) [7,8] are risk factors for myocardial infarction. Levels of Lp(a) greater than 30 mg/dl are associated with a risk of atherosclerotic disease 2-5 times greater than that of normal subjects [5,6].
Correspondence to: Dr. Margarete Mehrabian, University of California, Department of Medicine, Division of Cardiology, Los Angeles, CA 90024, U.S.A. Phone: (2!3)825-1359.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Plasminogen;
Diet
How Lp(a) exerts its atherogenic effects is not known. However, recent studies have shown that ape(a) shares remarkable homology with a primary thrombolytic enzyme, plasminogen [9]. Plasminogen binding sites are widely distributed on vascular cells, promoting thrombolysis by accelerating plasminogen activation to plasmin and protecting plasmin from inhibition. There is accumulating evidence indicating that Lp(a) competes with plasminogen for binding sites on endothelial cells [lO,ll] and, moreover, inhibits plasmin generation by tPA [ll] and streptokinase 1121.Therefore Lp(a) has the potential to either regulate fibrinolytic
Ireland, Ltd.
26
activity or, at elevated concentrations, to inhibit fibrinolysis. Several studies [l-3,7,8] have provided clear evidence that impaired fibrinolysis may contribute to the development of coronary artery disease (CAD). The complex regulation of the fibrinolytic system is partly the result of a delicate balance between plasminogen activators and inhibitors. Epidemiological studies have provided evidence that elevated plasma tPA1 [7,8], fibrinogen [l-3] and Lp(a) [4-61 levels are associated with the development of cardiovascular disease. The levels of these factors are regulated by both genetic and environmental influences. There is a high incidence of CAD and stroke among populations consuming a high fat, low fiber “Western” diet as compared to other populations [13]. This appears to be due in part to the effects of diet on cholesterol and plasma lipoprotein metabolism. Elevated LDL levels appear to be a prerequisite for atherosclerosis, the major cause of CAD. Diets high in saturated fat have been shown to increase levels of total plasma cholesterol and LDL. However, Lp(a) levels do not seem to be affected by diet or cholesterol lowering drugs [4]. In the present study, we have sought to determine whether a correlation exists between plasma levels of Lp(a) and several fibrinolytic factors and whether levels of plasminogen, tPA or the cardiovascular risk factors, fibrinogen, tPA1 and Lp(a), are influenced by diet. Our results shows that a shift from traditional “Western” diet to a low fat, high complex-carbohydrate diet for three weeks results in dramatic changes in the levels of factors of the thrombolytic pathway but not Lp(a). Thus, it is quite possible that the differences in incidence of CAD and stroke between “Western” and other populations result in part from dietary effects on the thrombolytic system as well as on lipoprotein parameters. Experimental procedures
The experimental group consisted of 12 males and 15 females with a mean age of 57.9 (SD = 14.1) years. All subjects signed an “Informed Consent” form before entering into the study. The group members were participants in the Pritikin Longevity Center 26-day program. They were housed in
an inpatient facility where they were placed on a high complex-carbohydrate, high fiber, low fat and low cholesterol diet [18]. Less than 10% of the calories were obtained from fat (polyunsaturated/ saturated = 2.4), 13% from protein and remainder from carbohydrate (primarily complex). The diet contained 25 mg cholesterol per day and 35-45 g dietary fiber per 1000 kcal. Protein was derived primarily from vegetable sources with the exception of a daily portion of skimmed milk and 85 g chicken or fish per week. The subjects were encouraged to participate in light exercise (initially 0.5 h/day of treadmill walking). Body weight decreased during this period from 96.5 ( f 25.2) to 91.9 (f 23.9) kg. Blood samples were obtained from fasted subjects between 6:30 and 7:30 a.m. (to minimize the effects of diurinal variations on fibrinolytic activity) on days 0 and 21. The subjects were upright and well rested during phlebotomy. Samples for fibrinolytic studies were collected into 0.1 volume of citrate anticoagulant (Becton Dickinson, Rutherford, NJ), centrifuged at 3000 rpm for 15 min at 22” C, and then immediately aliquoted and frozen at - 80 o C. EDTA-plasma specimens were collected for cholesterol, triglyceride and lipoprotein measurements. Cholesterol and triglyceride assays were performed on the day of sample collection. Total serum cholesterol, HDL cholesterol and triglycerides were determined enzymatically as described by Warnick [14] and Bachorik and Albers [15]. LDL cholesterol was calculated as described by Friedewald et al. [16] when triglyceride levels were below 375 mg/dl. Apo A-I and B-100 levels were determined by rate nephelometry with a Beckman Array (coefficient of variation 8-10%) (Beckman, Brea, CA). Lp(a) levels were determined by a two-site immunoradiometric assay (coefficient of variation 5%) (Pharmacia, Uppsala, Sweden). tPA antigen was measured by a solid-phase double antibody immunoassay which measures free tPA as well ai tPA complexed with tPA1 (coefficient of variation 2-4%) (Diagnostica Stago, France). tPA1 activity was evaluated by a synthetic chromogenic substrate method (Diagnostica Stago, France) which measures free tPA1 as well as tPA1 complexed with tPA. Plasma was diluted with
27 Results
plasma that is specifically depleted of tPA1. The diluted plasma was then incubated with a constant amount of purified tPA, and then, in the presence of fibrin and plasminogen excess, the residual tPA was determined by the action of the plasmin it generates. The plasmin formed was measured by its activity on the synthetic substrate CBS 10.65 releasing p-nitroaniline which is then measured at 405 nm (coefficient of variation: 6-8%). Plasminogen activity was analyzed by a twostep method (Diagnostica Stago, France) in which an excess of streptokinase is added to the diluted plasma containing plasminogen. A plasminogenstreptokinase complex is formed possessing a plasmin like activity which acts on the synthetic substrate CBS 30.41, releasing p-nitroaniline which is then measured at 405 nm (coefficient of variation: 2-38). Fibrinogen levels were determined by use of an assay based on a method originally developed by Clauss [17] (Diagnostica Stago, France). All samples were performed at least in duplicate. The two-tailed Student t-test was used to analyze differences in mean values, and group distributions are expressed as means + SD. Significance was defined as a P value of less than 0.05. Correlation between variables was assessed with simple linear regression.
TABLE
Twenty seven adults participating in a 21-day medically supervised residential program were evaluated for changes in their lipoprotein and fibrinolytic factor profiles in response to strict dietary restrictions. The participants served as their own controls. Other studies have shown that there are no significant changes in lipoprotein profiles or levels of thrombolytic factors over a short period of time in individuals not undergoing the program. Two control individuals examined in parallel with individuals undergoing dietary modification showed no significant changes in the fibrinolytic parameters plasrninogen, tPA, tPAI and fibrinogen (the average changes of the controls were within the coefficients of variation of the assays). As previously observed, significant reductions occured in plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and apolipoproteins A-I and B-100, with a modest decrease in the LDL/HDL ratio (Table 1). The levels of the Lp(a) lipoprotein varied widely, ranging from 6 mg/dl to 85 mg/dl. As expected from previous studies [5], the levels of Lp(a) remained remarkably constant during the course of the program (Table 1). A small fraction of the individuals exhibited slight changes (Fig. l),
1
CONCENTRATIONS OF PLASMA LIPOPROTEIN PARAMETERS BEFORE AND AFTER A 21 DAY DIETARY PROGRAM Results
are expressed
AND THROMBOLYTIC
FACTORS
IN PARTICIPANTS
as means ( f SD).
Day 0
Day 21 171
(33.4)
Lp(a) (mg/dI) Plasminogen (% activity) tPA1 (IU/ml)
233 (39.7) 47 (16.1) 140 (37.2) 194 (122.4) 149 (34.9) 93 (22.0) 3.23 (1.2) 27 (24.0) 101 (13.4) 15.3 (14.4)
tPA (ng@) Fibrinogen (mg/dl)
11.5 395
37 106 143 112 73 2.97 27 86 10.2 8.2 365
(9.8) (27.2) (81.2) (23.9) (18.0) (1.1) (24.0) (14.5) (7.8) (3.7) (79.1)
Total cholesterol (mg/dI) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) Apo AI (mg/dI) APO B (mg/dI) LDL/HDL ratio
(4.9) (68.5)
P value
n
0.0001 O.oO@l 0.0001 0.0015 0.0001 0.0005 0.2925 0.9783 0.0001 0.0117 0.0001 0.0698
26 26 24 26 19 19 25 27 27 27 27 27
28 suggesting that some individuals may be responsive to diet in terms of Lp(a) levels, possibly for genetic reasons. No relationship was found between Lp(a) levels and plasminogen or any other lipoprotein or fibrinolytic factor (Table 2). The levels of plasminogen, tPA and tPA1 decreased significantly (P = 0.0001, P = 0.0001 and
P = 0.012, respectively) during the 3-week duration of the program (Table 1). The decrease for fibrinogen was less significant (P = 0.07). Plaminogen activity correlated with fibrinogen levels, and tPA1 activity correlated with tPA antigen levels. In addition, levels of triglyceride were significantly correlated with the levels of tPA and
250__.‘7
Day
0
Day 21
.~__
b
Day
0
Day
0
__. Day
21
Day
21
Fig. 1. Individual response in HDL cholesterol (a), apo A-I (b), triglycerides (c). tPA1 (d) and Lp(a) (e; see overleaf) levels among participants of the 21 day dietary program. Values were obtained on day 0 (pre-diet) and day 21.
29 affected. Similar trends occurred for levels of triglycerides (Fig. lc) and tPA1 (Fig. Id) but not for other parameters examined. The levels of Lp(a) were most resistant to dietary change (Fig. le). We did not observe any significant differences in mean levels of lipoprotein or fibrinolytic factors due to sex. Also, CRP levels (an acute phase reactant) were not elevated during the course of the study (data not shown). Therefore, it is unlikely that changes in the fibrinolytic factors are the result of an acute phase reaction. Discussion
The goal of this study was to determine whether a change from a typical “Western” diet, high in fats and low in fiber, to a low fat, high fiber diet would significantly affect the levels of several clotting factors known to be correlated with the risk of CAD and stroke. A “Western” diet includes about 500 mg of cholesterol per day and about 40% of the total calories derived from fat. The individuals participating in the program consumed only 25 mg of cholesterol per day and less than 10% of their calories were derived from fat. Clearly, one important risk factor affected by diet is lipoprotein metabolism. Our studies suggest strongly that clotting parameters are also likely to be importantly affected by diet, and, therefore, may be responsible in part for the differences in the incidence of CAD between Western and non-Western countries. It was not possible to determine precisely the “lifestyles” of the participants prior to their entering the program, although none of the participants
0
Day 21
Da;~ 0 Fig. 1 (continued).
tPA1, both before and after the dietary program (Table 2). For several of the parameters examined (HDL cholesterol, apo A-I, triglycerides and tPAI), the most striking changes occurred among the individuals with the highest pre-diet levels (Fig. 1). Thus, for example, individuals with initial HDL cholesterol levels above 80 mg/dl exhibited about a 30% decrease in response to the diet, whereas individuals with initial HDL cholesterol levels below 30 mg/dl showed essentially no change (Fig. la). The change in HDL cholesterol was reflected almost exactly by the change in apo A-I (Fig. lb), suggesting that the number of HDL particles rather than the cholesterol content per HDL particle was TABLE 2 CORRELATION MATRIX (r) BETWEEN FIBRINOLYTIC 21 DAY DIETARY PROGRAM
AND LIPOPROTEIN
PARAMETERS
BEFORE AND AFTER A
Plasminogen
tPA1
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Triglycerides Plasminogen tPA1 tPA
0.093 -
0.289 -
0.592 a 0.295 _ _
0.631 a 0.174
0.543 b - 0.085 0.487 b _
0.320 0.216 0.665 a
0.158 0.428 d 0.163 0.439 c
Fibrinogen
-
-
_
0.281 0.475 c 0.382 0.287 _
-0.156 0.110 -0.122 - 0.109 0.106
-0.116 - 0.096 - 0.082 - 0.195 -0.111
_
a P < 0.001; b P i 0.01; c P i 0.02; d P < 0.05.
tPA
_ _
Fibrinogen
_
Lp(a)
30 used in this study were smokers. Thus, it is possible that the results for certain individuals were influenced in part by factors such as drugs or exercise. It is noteworthy, however, that while the individuals were quite heterogeneous in terms of age, weight and “lifestyle”, similar trends were observed among most of the participants for changes both in fibrinolytic factors and lipoproteins in response to the dietary program. It is unlikely that the observed changes in the levels of fibrinolytic factors were affected by exercise. Although previous studies [22,23] have shown that exercise dramatically affects levels of fibrinolytic factors, individuals in those studies were tested immediately following exercise. As previously shown [23], exercise-induced changes in the fibrinolytic factors are very transient, with values returning to baseline levels within 90 min. In contrast, in our study individuals were tested following a minimum of an overnight rest. We cannot conclude from our results whether the observed changes in clotting factors are beneficial or harmful with respect to the development of atherosclerosis. Fibrinogen and tPA1 levels are positively correlated with CAD in epidemiologic studies, whereas tPA levels are negatively correlated with CAD. In our study, concentrations of all three factors decreased. As yet, the mechanisms mediating these changes are also totally unknown. It should be possible, using animal models, to determine whether high fat diets affect the synthesis or turnover of these and related factors. Plasminogen and fibrinogen are synthe-
sized in liver, while tPA and tPA1 are produced primarily by endothelial cells. Previous epidemiological studies have revealed a correlation between the levels of tPA1 and triglycerides [7,8], and our studies also show a strong correlation between the parameters both before (r = 0.59) and after (r = 0.63) the 3-week dietary program. The molecular basis of this relationship is unknown. Our studies also revealed weaker, but noteworthy, correlations between the levels of triglyceride and tPA, fibrinogen and plasrninogen, and tPA and tPA1. In contrast to a previous study [25], we did not find a significant correlation between cholesterol levels either with tPA1 activity (r = 0.346) or tPA (r = 0.120). The expression of clotting factors is undoubtedly regulated by feedback mechanisms to ensure proper rates of thrombosis and thrombolysis, and the correlations noted in this study may well be a reflection of such mechanisms. Our studies have also confirmed previous findings [18] that levels of total cholesterol, total triglycerides, HDL cholesterol, apolipoprotein A-I (the major protein of HDL), LDL cholesterol, and apolipoprotein B (the major protein of LDL), are all dramatically altered by the dietary program. As in the case of the clotting factors, it is not possible to draw firm conclusions about the impact of such changes on the development of atherosclerosis. Thus while LDL levels (which are positively correlated with CAD) decreased, HDL levels (which are negatively correlated with atherosclerosis) also decreased. On the other hand, the ratio of LDL cholesterol to HDL cholesterol, perhaps a better
TABLE 3 HETEROGENEITY
AMONG
INDIVIDUALS
IN THE RESPONSE TO THE DIETARY
PROGRAM
Ternary classification by initial values (mean & SEM). Highest trinal
n
Middle trinal
n
Lowest trinal
339 224
8 8
165 129
9 9
93 91
Triglycerides (mg/dl)
day 0 day 21
+45 +37
+lO *17
tPA1 (IU/ml)
day 0 day 21
33.4* 18.4f
5.5 3.4
8 8
9.6f 8.5*
0.5 1.3
9 9
HDL cholesterol (mg/dl)
day 0 day 21
66.9* 47.9*
3.7 3.2
8 8
46.2f 36.4+
1.0 1.9
9 9
Lp(a) (mg/dI)
day 0 day 21
61 60
+ 6.3 f 6.6
8 8
20 20
f 1.8 f. 3.2
9 9
n
*7 +8
9 9
5.2k0.7 4.3 f 0.8
9 9
30 29
*1.2 f1.5
7.3 f 0.8 7.9f0.8
9 9 10 10
31 indicator of CAD risk than either alone, exhibited a decrease of approximately 10%. These studies have also revealed an interesting heterogeneity of the population with respect to dietary responsiveness of levels of HDL, cholesterol, apo A-I, tPA1 and triglycerides. For each of these parameters, individuals with highest initial levels were highly responsive whereas individuals with lowest levels exhibited little or no responsiveness (Fig. 1). This is clearly seen when the individuals are grouped into the highest, middle, and lowest thirds with respect to the parameters (Table 3). Thus, the individuals in the highest trinal for triglyceride, HDL cholesterol and tPA1 levels exhibited decreases of about 34% 29%, and 45%, respectively, whereas the individuals in the lowest trinal were not significantly affected by the dietary program. In contrast, Lp(a) values did not exhibit this same trend and remained unresponsive in all three groupings. The changes of tPA1 and triglyceride levels were highly correlated among individuals; however, there was no obvious correlation between these parameters and HDL cholesterol responsiveness (data not shown). The biochemical basis of this heterogeneity, and whether the heterogeneity results from genetic or environmental influences, are unclear. If would be important to examine larger numbers of individuals exhibiting the remarkable responsiveness to dietary challenge. In marked contrast to the dramatic changes observed for the above lipoprotein parameters and clotting factors, the levels of Lp(a) were remarkably resistant to the dietary change. Previous studies have documented the relative resistance of Lp(a) levels to dietary and other environmental influences [4], and our studies demonstrate that even extreme dietary alterations have little effect on Lp(a) levels. High levels of Lp(a) are strongly correlated with risk of CAD, and recent studies suggest that Lp(a) may compete with plasminogen for binding to endothelial cell receptors, thereby possibly inhibiting plasmin production [lO,ll]. It is noteworthy that Lp(a) levels were not affected despite the fact that the dietary challenge resulted in substantial reductions in LDL cholesterol and apolipoprotein B-100 (up to 50% in certain individuals). This suggests that the processes controlling apo B-100 and LDL synthesis or catabolism
in response to dietary change do not similarly act on Lp(a), despite the presumably similar metabolic processes involved in the assembly, secretion, lipolytic processing and catabolism of the two lipoprotein particles. It has been shown that the genes for plasminogen and ape(a) are tightly linked on chromosome 6q27 [19]. A DNA polymorphism in the plasminogen gene was shown to segregate with an ape(a) isoform in a large family with high frequency of CAD. Although the correlation between this linkage and CAD remains unclear, it prompted us to examine if any correlations existed between Lp(a) levels and plasminogen levels. We did not see any relationship between Lp(a) levels and other fibrinolytic or lipoprotein parameters measured. In conclusion, our results have shown that a diet very low in fats and cholesterol can dramatically alter the levels of thrombolytic factors that have been associated with the incidence of CAD. This finding may be important in explaining the observed differences in the incidence of CAD and stroke between various populations. It may also be of use in developing therapy in the treatment and prevention of atherosclerosis. Acknowledgements
We thank Dr. Andrew Lot Le and American Bioproducts for providing a portion of reagents used in the fibrinolytic studies (tPA, tPA1, fibrinogen and plasminogen) and for helpful advice and collaboration. We sincerely appreciate the technical efforts of R.L. Bowman, Jr. in figure preparation. We also thank Dr. Steven Inkeles for coordination of patient evaluation, sample collection and valuable discussion. Supported in part by funds from the Nathan Pritikin Research Foundation, and by grants HL43429 and HL30568 from National Heart, Lung, and Blood Institute. References 1 Karmel, W.B., D’Agostino, R.B. and Belanger, A.J., Fibrinogen, cigarette smoking and risk of cardiovascular disease: Insights from the Framingham study. Am. Heart. J., 113 (1987) 1006. 2 Meade, T.W., Mellows, S., Brozovic, M. et al., Principal results of the Northwick Park heart study. Lancet, 2 (1986) 533.
32 3 Wilhelmsen, L., Svardsudd, K., Korsan-Bengtsen, K., Larsson, B., Welin, L. and Tibblin, G., Fibrinogen as a risk factor for stroke and myocardial infarction. N. Engl. J. Med. 311 (1984) 501. 4 Scanu, A.M., Lipoprotein (a) A potential bridge between the fields of atherosclerosis and thrombosis. Arch. Pathol. Lab. Med., 112 (1988) 1045. 5 Dahlen, G.H., Guyton, J.R., Arrar, M., Farmer, J.A. and Gotto, A.M., Association of level of Lp(a), plasma lipids and other lipoproteins with coronary heart disease documented by angiography. Circulation, 84 (1986) 758. 6 Rhoads, G.G., Dahlen, G., Berg, K., Morton, N.E. and Dannenberg, A.L., Lp(a) lipoprotein as a risk factor for myocardial infarction. JAMA, 256 (1986) 2540. 7 Hamsten, A., Wiman, B., de Faire, U. and Blomback, M., Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N. Engl. J. Med., 313 (1985) 1557. 8 Juhan-Vague, I., Alessi, M.C., Joly, P. et al., Plasma plasminogen activator inhibitor-l in angina pectoris. Influence of plasma insulin and acute-phase response. Arteriosclerosis, 9 (1989) 362. 9 McLean, J.W., Tomlinson, J.E. and Kuang, W.J., cDNA sequence of human apolipoprotein (a) is homologous to plasminogen. Nature, 330 (1987) 132. 10 Miles, L.A., Fless, G.M., Levin, E.G., Scanu, A.M. and Plow, E.F., A potential basis for the thrombotic risks associated with lipoprotein (a). Nature, 339 (1989) 301. 11 Hajjar, K.A., Gavish, D., Breslow, J.L. and Nachman, R.L., Lipoprotein (a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature, 339 (1989) 303. 12 Edelberg, J.M., Gonzalez-Gronow, M. and Pizza, S.V., Lipoprotein (a) inhibits streptokinase mediated activation of human plasminogen. B&hem., 28 (1989) 2370. 13 Connor, W.E. and Connor, S.L., Dietary cholesterol and fat and the prevention of coronary heart disease risks and benefits of nutritional change. In: Hallgren, B., Levin, O., Rossner, S., Vessby, B. (Eds.), Diet and Prevention of Coronary Heart Disease and Cancer, New York, Raven Press, 1986, pp. 113-148.
14 Wamick, R., Enzymatic methods for quantification of lipoprotein lipids. Methods Enzymol., 129 (1986) 101. 15 Bachorik, P.S. and Albers, J.J., Precipitation methods for quantification of lipoproteins. Methods Enzymol., 129 (1986) 78. 16 Friedewald, W.T., Levy, R.I. and Fredrickson, D.S., Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem., 18 (1972) 499. 17 Clauss, A., Gerinnungphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol., 17 (1957) 237. 18 Barnard, R.J., Peter, J.B., Hall, J.A. and Kinsella, CA., Effects of a short-term diet and exercise program on serum apoproteins. J. Appl. Nutr., 40 (1988) 5. 19 Frank, S.L., Klisak, I., Sparkes, R.S. et al., The apolipoprotein (a) gene resides on human chromosome 6q26-27, in close proximity to the homologous gene for plasminogen. Hum. Genet., 79 (1988) 352. 20 Gaubatz, J.W., Heidman, C., Gotto, A.M., Jr., Morrisett, J.D. and Dahlen, G.H., Human plasma lipoprotein (a). J. Biol. Chem., 258 (1983) 4582. 21 Fleas, G.M., Rolih, C.A. and Scanu, A.M., Heterogeneity of human plasma lipoprotein (a). Isolation and characterization of the lipoprotein subspecies and their apoproteins. J. Biol. Chem., 259 (1984) 11470. 22 Crenn, Y. and Huene, M., Etude des modifications de l’activid fibrinolytique et de l’hemostase apres un exercice physique proline. Sci. Sport, 4 (1989) 65. 23 Marsh, N. and Gaffney, P., Some observations on the release of extrinsic and intrinsic plaminogen activators during exercise in man. Haemost., 9 (1980) 238. 24 Collen, D., Semanoro, D., Tricot, J.P. and Vermylen, J., Turnover of fibrinogen, plasminogen, and prothrombin during exercise in man. J. Appl. Physiol., 42 (1977) 865. 25 Crutchley, D.J., McPhee, G.V., Terris, M.F. and CanossaTerris, M.A., Levels of three hemostatic factors in relation to serum lipids. Arteriosclerosis, 9 (1989) 934.
Atherosclerosis, 84 (1990) 25-32 Elsevier Scientific Publishers Ireland, Ltd.
ATHERO
04516
Dietary regulation of fibrinolytic factors Margarete Mehrabian I, James B. Peter I, R. James Barnard ’ and Aldons J. Lusis 3 ’Specialty Laboratories, Inc., Santa Monica, CA 90404 (U.S.A.), ’ Department of Kinesiology and 3 Departments
of Medicine and Microbiology,
University of California, Los Angeles, CA 90024 (U.S.A.)
(Received 18 December, 1989) (Revised, received 23 April, 1990) (Accepted 24 April, 1990)
W examined the short-term effects of a high-complex carbohydrate, low fat diet on the plasmin-dependent fibrinolytic pathway. A population of 27 adult American Caucasians exposed to the diet for 3 weeks showed highly significant reductions in the levels of plasminogen (P = O.OOOl), tissue plasminogen activator (tPA) (P = 0.0001) and plasminogen activator inhibitor (tPA1) (P = 0.0017). Fibrinogen levels also decreased, but the changes did not reach statistical significance (P = 0.07). In contrast, the levels of the Lpa(a) lipoprotein, a potential inhibitor of fibrinolysis, remained remarkably constant despite a marked decrease in the levels of apolipoprotein B, a major constituent of Lp(a). Correlations between the levels of tPA, tPA1 and plasma triglyceride were observed among the individuals both before and after the dietary challenge. Although the mechanisms responsible for the effects are unknown, the dramatic responsiveness of the thrombolytic pathway to dietary challenge is likely to be of importance in understanding the etiology of coronary artery disease and other vascular disorders.
Key words: Lipoproteins;
Fibrinolysis;
Lp(a); tPA; tPA1; Fibrinogen;
Introduction
Increased levels of fibrinogen [l-3], Lp(a) [4-61 and tissue plasminogen activator inhibitor (tPA1) [7,8] are risk factors for myocardial infarction. Levels of Lp(a) greater than 30 mg/dl are associated with a risk of atherosclerotic disease 2-5 times greater than that of normal subjects [5,6].
Correspondence to: Dr. Margarete Mehrabian, University of California, Department of Medicine, Division of Cardiology, Los Angeles, CA 90024, U.S.A. Phone: (2!3)825-1359.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Plasminogen;
Diet
How Lp(a) exerts its atherogenic effects is not known. However, recent studies have shown that ape(a) shares remarkable homology with a primary thrombolytic enzyme, plasminogen [9]. Plasminogen binding sites are widely distributed on vascular cells, promoting thrombolysis by accelerating plasminogen activation to plasmin and protecting plasmin from inhibition. There is accumulating evidence indicating that Lp(a) competes with plasminogen for binding sites on endothelial cells [lO,ll] and, moreover, inhibits plasmin generation by tPA [ll] and streptokinase 1121.Therefore Lp(a) has the potential to either regulate fibrinolytic
Ireland, Ltd.
26
activity or, at elevated concentrations, to inhibit fibrinolysis. Several studies [l-3,7,8] have provided clear evidence that impaired fibrinolysis may contribute to the development of coronary artery disease (CAD). The complex regulation of the fibrinolytic system is partly the result of a delicate balance between plasminogen activators and inhibitors. Epidemiological studies have provided evidence that elevated plasma tPA1 [7,8], fibrinogen [l-3] and Lp(a) [4-61 levels are associated with the development of cardiovascular disease. The levels of these factors are regulated by both genetic and environmental influences. There is a high incidence of CAD and stroke among populations consuming a high fat, low fiber “Western” diet as compared to other populations [13]. This appears to be due in part to the effects of diet on cholesterol and plasma lipoprotein metabolism. Elevated LDL levels appear to be a prerequisite for atherosclerosis, the major cause of CAD. Diets high in saturated fat have been shown to increase levels of total plasma cholesterol and LDL. However, Lp(a) levels do not seem to be affected by diet or cholesterol lowering drugs [4]. In the present study, we have sought to determine whether a correlation exists between plasma levels of Lp(a) and several fibrinolytic factors and whether levels of plasminogen, tPA or the cardiovascular risk factors, fibrinogen, tPA1 and Lp(a), are influenced by diet. Our results shows that a shift from traditional “Western” diet to a low fat, high complex-carbohydrate diet for three weeks results in dramatic changes in the levels of factors of the thrombolytic pathway but not Lp(a). Thus, it is quite possible that the differences in incidence of CAD and stroke between “Western” and other populations result in part from dietary effects on the thrombolytic system as well as on lipoprotein parameters. Experimental procedures
The experimental group consisted of 12 males and 15 females with a mean age of 57.9 (SD = 14.1) years. All subjects signed an “Informed Consent” form before entering into the study. The group members were participants in the Pritikin Longevity Center 26-day program. They were housed in
an inpatient facility where they were placed on a high complex-carbohydrate, high fiber, low fat and low cholesterol diet [18]. Less than 10% of the calories were obtained from fat (polyunsaturated/ saturated = 2.4), 13% from protein and remainder from carbohydrate (primarily complex). The diet contained 25 mg cholesterol per day and 35-45 g dietary fiber per 1000 kcal. Protein was derived primarily from vegetable sources with the exception of a daily portion of skimmed milk and 85 g chicken or fish per week. The subjects were encouraged to participate in light exercise (initially 0.5 h/day of treadmill walking). Body weight decreased during this period from 96.5 ( f 25.2) to 91.9 (f 23.9) kg. Blood samples were obtained from fasted subjects between 6:30 and 7:30 a.m. (to minimize the effects of diurinal variations on fibrinolytic activity) on days 0 and 21. The subjects were upright and well rested during phlebotomy. Samples for fibrinolytic studies were collected into 0.1 volume of citrate anticoagulant (Becton Dickinson, Rutherford, NJ), centrifuged at 3000 rpm for 15 min at 22” C, and then immediately aliquoted and frozen at - 80 o C. EDTA-plasma specimens were collected for cholesterol, triglyceride and lipoprotein measurements. Cholesterol and triglyceride assays were performed on the day of sample collection. Total serum cholesterol, HDL cholesterol and triglycerides were determined enzymatically as described by Warnick [14] and Bachorik and Albers [15]. LDL cholesterol was calculated as described by Friedewald et al. [16] when triglyceride levels were below 375 mg/dl. Apo A-I and B-100 levels were determined by rate nephelometry with a Beckman Array (coefficient of variation 8-10%) (Beckman, Brea, CA). Lp(a) levels were determined by a two-site immunoradiometric assay (coefficient of variation 5%) (Pharmacia, Uppsala, Sweden). tPA antigen was measured by a solid-phase double antibody immunoassay which measures free tPA as well ai tPA complexed with tPA1 (coefficient of variation 2-4%) (Diagnostica Stago, France). tPA1 activity was evaluated by a synthetic chromogenic substrate method (Diagnostica Stago, France) which measures free tPA1 as well as tPA1 complexed with tPA. Plasma was diluted with
27 Results
plasma that is specifically depleted of tPA1. The diluted plasma was then incubated with a constant amount of purified tPA, and then, in the presence of fibrin and plasminogen excess, the residual tPA was determined by the action of the plasmin it generates. The plasmin formed was measured by its activity on the synthetic substrate CBS 10.65 releasing p-nitroaniline which is then measured at 405 nm (coefficient of variation: 6-8%). Plasminogen activity was analyzed by a twostep method (Diagnostica Stago, France) in which an excess of streptokinase is added to the diluted plasma containing plasminogen. A plasminogenstreptokinase complex is formed possessing a plasmin like activity which acts on the synthetic substrate CBS 30.41, releasing p-nitroaniline which is then measured at 405 nm (coefficient of variation: 2-38). Fibrinogen levels were determined by use of an assay based on a method originally developed by Clauss [17] (Diagnostica Stago, France). All samples were performed at least in duplicate. The two-tailed Student t-test was used to analyze differences in mean values, and group distributions are expressed as means + SD. Significance was defined as a P value of less than 0.05. Correlation between variables was assessed with simple linear regression.
TABLE
Twenty seven adults participating in a 21-day medically supervised residential program were evaluated for changes in their lipoprotein and fibrinolytic factor profiles in response to strict dietary restrictions. The participants served as their own controls. Other studies have shown that there are no significant changes in lipoprotein profiles or levels of thrombolytic factors over a short period of time in individuals not undergoing the program. Two control individuals examined in parallel with individuals undergoing dietary modification showed no significant changes in the fibrinolytic parameters plasrninogen, tPA, tPAI and fibrinogen (the average changes of the controls were within the coefficients of variation of the assays). As previously observed, significant reductions occured in plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and apolipoproteins A-I and B-100, with a modest decrease in the LDL/HDL ratio (Table 1). The levels of the Lp(a) lipoprotein varied widely, ranging from 6 mg/dl to 85 mg/dl. As expected from previous studies [5], the levels of Lp(a) remained remarkably constant during the course of the program (Table 1). A small fraction of the individuals exhibited slight changes (Fig. l),
1
CONCENTRATIONS OF PLASMA LIPOPROTEIN PARAMETERS BEFORE AND AFTER A 21 DAY DIETARY PROGRAM Results
are expressed
AND THROMBOLYTIC
FACTORS
IN PARTICIPANTS
as means ( f SD).
Day 0
Day 21 171
(33.4)
Lp(a) (mg/dI) Plasminogen (% activity) tPA1 (IU/ml)
233 (39.7) 47 (16.1) 140 (37.2) 194 (122.4) 149 (34.9) 93 (22.0) 3.23 (1.2) 27 (24.0) 101 (13.4) 15.3 (14.4)
tPA (ng@) Fibrinogen (mg/dl)
11.5 395
37 106 143 112 73 2.97 27 86 10.2 8.2 365
(9.8) (27.2) (81.2) (23.9) (18.0) (1.1) (24.0) (14.5) (7.8) (3.7) (79.1)
Total cholesterol (mg/dI) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) Apo AI (mg/dI) APO B (mg/dI) LDL/HDL ratio
(4.9) (68.5)
P value
n
0.0001 O.oO@l 0.0001 0.0015 0.0001 0.0005 0.2925 0.9783 0.0001 0.0117 0.0001 0.0698
26 26 24 26 19 19 25 27 27 27 27 27
28 suggesting that some individuals may be responsive to diet in terms of Lp(a) levels, possibly for genetic reasons. No relationship was found between Lp(a) levels and plasminogen or any other lipoprotein or fibrinolytic factor (Table 2). The levels of plasminogen, tPA and tPA1 decreased significantly (P = 0.0001, P = 0.0001 and
P = 0.012, respectively) during the 3-week duration of the program (Table 1). The decrease for fibrinogen was less significant (P = 0.07). Plaminogen activity correlated with fibrinogen levels, and tPA1 activity correlated with tPA antigen levels. In addition, levels of triglyceride were significantly correlated with the levels of tPA and
250__.‘7
Day
0
Day 21
.~__
b
Day
0
Day
0
__. Day
21
Day
21
Fig. 1. Individual response in HDL cholesterol (a), apo A-I (b), triglycerides (c). tPA1 (d) and Lp(a) (e; see overleaf) levels among participants of the 21 day dietary program. Values were obtained on day 0 (pre-diet) and day 21.
29 affected. Similar trends occurred for levels of triglycerides (Fig. lc) and tPA1 (Fig. Id) but not for other parameters examined. The levels of Lp(a) were most resistant to dietary change (Fig. le). We did not observe any significant differences in mean levels of lipoprotein or fibrinolytic factors due to sex. Also, CRP levels (an acute phase reactant) were not elevated during the course of the study (data not shown). Therefore, it is unlikely that changes in the fibrinolytic factors are the result of an acute phase reaction. Discussion
The goal of this study was to determine whether a change from a typical “Western” diet, high in fats and low in fiber, to a low fat, high fiber diet would significantly affect the levels of several clotting factors known to be correlated with the risk of CAD and stroke. A “Western” diet includes about 500 mg of cholesterol per day and about 40% of the total calories derived from fat. The individuals participating in the program consumed only 25 mg of cholesterol per day and less than 10% of their calories were derived from fat. Clearly, one important risk factor affected by diet is lipoprotein metabolism. Our studies suggest strongly that clotting parameters are also likely to be importantly affected by diet, and, therefore, may be responsible in part for the differences in the incidence of CAD between Western and non-Western countries. It was not possible to determine precisely the “lifestyles” of the participants prior to their entering the program, although none of the participants
0
Day 21
Da;~ 0 Fig. 1 (continued).
tPA1, both before and after the dietary program (Table 2). For several of the parameters examined (HDL cholesterol, apo A-I, triglycerides and tPAI), the most striking changes occurred among the individuals with the highest pre-diet levels (Fig. 1). Thus, for example, individuals with initial HDL cholesterol levels above 80 mg/dl exhibited about a 30% decrease in response to the diet, whereas individuals with initial HDL cholesterol levels below 30 mg/dl showed essentially no change (Fig. la). The change in HDL cholesterol was reflected almost exactly by the change in apo A-I (Fig. lb), suggesting that the number of HDL particles rather than the cholesterol content per HDL particle was TABLE 2 CORRELATION MATRIX (r) BETWEEN FIBRINOLYTIC 21 DAY DIETARY PROGRAM
AND LIPOPROTEIN
PARAMETERS
BEFORE AND AFTER A
Plasminogen
tPA1
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Triglycerides Plasminogen tPA1 tPA
0.093 -
0.289 -
0.592 a 0.295 _ _
0.631 a 0.174
0.543 b - 0.085 0.487 b _
0.320 0.216 0.665 a
0.158 0.428 d 0.163 0.439 c
Fibrinogen
-
-
_
0.281 0.475 c 0.382 0.287 _
-0.156 0.110 -0.122 - 0.109 0.106
-0.116 - 0.096 - 0.082 - 0.195 -0.111
_
a P < 0.001; b P i 0.01; c P i 0.02; d P < 0.05.
tPA
_ _
Fibrinogen
_
Lp(a)
30 used in this study were smokers. Thus, it is possible that the results for certain individuals were influenced in part by factors such as drugs or exercise. It is noteworthy, however, that while the individuals were quite heterogeneous in terms of age, weight and “lifestyle”, similar trends were observed among most of the participants for changes both in fibrinolytic factors and lipoproteins in response to the dietary program. It is unlikely that the observed changes in the levels of fibrinolytic factors were affected by exercise. Although previous studies [22,23] have shown that exercise dramatically affects levels of fibrinolytic factors, individuals in those studies were tested immediately following exercise. As previously shown [23], exercise-induced changes in the fibrinolytic factors are very transient, with values returning to baseline levels within 90 min. In contrast, in our study individuals were tested following a minimum of an overnight rest. We cannot conclude from our results whether the observed changes in clotting factors are beneficial or harmful with respect to the development of atherosclerosis. Fibrinogen and tPA1 levels are positively correlated with CAD in epidemiologic studies, whereas tPA levels are negatively correlated with CAD. In our study, concentrations of all three factors decreased. As yet, the mechanisms mediating these changes are also totally unknown. It should be possible, using animal models, to determine whether high fat diets affect the synthesis or turnover of these and related factors. Plasminogen and fibrinogen are synthe-
sized in liver, while tPA and tPA1 are produced primarily by endothelial cells. Previous epidemiological studies have revealed a correlation between the levels of tPA1 and triglycerides [7,8], and our studies also show a strong correlation between the parameters both before (r = 0.59) and after (r = 0.63) the 3-week dietary program. The molecular basis of this relationship is unknown. Our studies also revealed weaker, but noteworthy, correlations between the levels of triglyceride and tPA, fibrinogen and plasrninogen, and tPA and tPA1. In contrast to a previous study [25], we did not find a significant correlation between cholesterol levels either with tPA1 activity (r = 0.346) or tPA (r = 0.120). The expression of clotting factors is undoubtedly regulated by feedback mechanisms to ensure proper rates of thrombosis and thrombolysis, and the correlations noted in this study may well be a reflection of such mechanisms. Our studies have also confirmed previous findings [18] that levels of total cholesterol, total triglycerides, HDL cholesterol, apolipoprotein A-I (the major protein of HDL), LDL cholesterol, and apolipoprotein B (the major protein of LDL), are all dramatically altered by the dietary program. As in the case of the clotting factors, it is not possible to draw firm conclusions about the impact of such changes on the development of atherosclerosis. Thus while LDL levels (which are positively correlated with CAD) decreased, HDL levels (which are negatively correlated with atherosclerosis) also decreased. On the other hand, the ratio of LDL cholesterol to HDL cholesterol, perhaps a better
TABLE 3 HETEROGENEITY
AMONG
INDIVIDUALS
IN THE RESPONSE TO THE DIETARY
PROGRAM
Ternary classification by initial values (mean & SEM). Highest trinal
n
Middle trinal
n
Lowest trinal
339 224
8 8
165 129
9 9
93 91
Triglycerides (mg/dl)
day 0 day 21
+45 +37
+lO *17
tPA1 (IU/ml)
day 0 day 21
33.4* 18.4f
5.5 3.4
8 8
9.6f 8.5*
0.5 1.3
9 9
HDL cholesterol (mg/dl)
day 0 day 21
66.9* 47.9*
3.7 3.2
8 8
46.2f 36.4+
1.0 1.9
9 9
Lp(a) (mg/dI)
day 0 day 21
61 60
+ 6.3 f 6.6
8 8
20 20
f 1.8 f. 3.2
9 9
n
*7 +8
9 9
5.2k0.7 4.3 f 0.8
9 9
30 29
*1.2 f1.5
7.3 f 0.8 7.9f0.8
9 9 10 10
31 indicator of CAD risk than either alone, exhibited a decrease of approximately 10%. These studies have also revealed an interesting heterogeneity of the population with respect to dietary responsiveness of levels of HDL, cholesterol, apo A-I, tPA1 and triglycerides. For each of these parameters, individuals with highest initial levels were highly responsive whereas individuals with lowest levels exhibited little or no responsiveness (Fig. 1). This is clearly seen when the individuals are grouped into the highest, middle, and lowest thirds with respect to the parameters (Table 3). Thus, the individuals in the highest trinal for triglyceride, HDL cholesterol and tPA1 levels exhibited decreases of about 34% 29%, and 45%, respectively, whereas the individuals in the lowest trinal were not significantly affected by the dietary program. In contrast, Lp(a) values did not exhibit this same trend and remained unresponsive in all three groupings. The changes of tPA1 and triglyceride levels were highly correlated among individuals; however, there was no obvious correlation between these parameters and HDL cholesterol responsiveness (data not shown). The biochemical basis of this heterogeneity, and whether the heterogeneity results from genetic or environmental influences, are unclear. If would be important to examine larger numbers of individuals exhibiting the remarkable responsiveness to dietary challenge. In marked contrast to the dramatic changes observed for the above lipoprotein parameters and clotting factors, the levels of Lp(a) were remarkably resistant to the dietary change. Previous studies have documented the relative resistance of Lp(a) levels to dietary and other environmental influences [4], and our studies demonstrate that even extreme dietary alterations have little effect on Lp(a) levels. High levels of Lp(a) are strongly correlated with risk of CAD, and recent studies suggest that Lp(a) may compete with plasminogen for binding to endothelial cell receptors, thereby possibly inhibiting plasmin production [lO,ll]. It is noteworthy that Lp(a) levels were not affected despite the fact that the dietary challenge resulted in substantial reductions in LDL cholesterol and apolipoprotein B-100 (up to 50% in certain individuals). This suggests that the processes controlling apo B-100 and LDL synthesis or catabolism
in response to dietary change do not similarly act on Lp(a), despite the presumably similar metabolic processes involved in the assembly, secretion, lipolytic processing and catabolism of the two lipoprotein particles. It has been shown that the genes for plasminogen and ape(a) are tightly linked on chromosome 6q27 [19]. A DNA polymorphism in the plasminogen gene was shown to segregate with an ape(a) isoform in a large family with high frequency of CAD. Although the correlation between this linkage and CAD remains unclear, it prompted us to examine if any correlations existed between Lp(a) levels and plasminogen levels. We did not see any relationship between Lp(a) levels and other fibrinolytic or lipoprotein parameters measured. In conclusion, our results have shown that a diet very low in fats and cholesterol can dramatically alter the levels of thrombolytic factors that have been associated with the incidence of CAD. This finding may be important in explaining the observed differences in the incidence of CAD and stroke between various populations. It may also be of use in developing therapy in the treatment and prevention of atherosclerosis. Acknowledgements
We thank Dr. Andrew Lot Le and American Bioproducts for providing a portion of reagents used in the fibrinolytic studies (tPA, tPA1, fibrinogen and plasminogen) and for helpful advice and collaboration. We sincerely appreciate the technical efforts of R.L. Bowman, Jr. in figure preparation. We also thank Dr. Steven Inkeles for coordination of patient evaluation, sample collection and valuable discussion. Supported in part by funds from the Nathan Pritikin Research Foundation, and by grants HL43429 and HL30568 from National Heart, Lung, and Blood Institute. References 1 Karmel, W.B., D’Agostino, R.B. and Belanger, A.J., Fibrinogen, cigarette smoking and risk of cardiovascular disease: Insights from the Framingham study. Am. Heart. J., 113 (1987) 1006. 2 Meade, T.W., Mellows, S., Brozovic, M. et al., Principal results of the Northwick Park heart study. Lancet, 2 (1986) 533.
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