Gynecol. Invest. 6: 329-336 (1975)
Hormonal Steroids: Effects on the Vascular System' J. Solash , R. P erez , J.S. K eates, N. R amasamy, H. Stein , D. T royansky , E. A teyeh , jr., J.R. J ones , S. S rinivasan and P.N. Sawyer Electrochemical and Biophysical Laboratories of the Vascular Surgical Services, Departments of Surgery and Surgical Research, and Obstetrics and Gynecology, Downstate Medical Center, State University of New York, Brooklyn, N.Y.
Key Words. Contraceptive steroids • Clinical studies • Animal studies • Electro phoretic mobility • Blood vessels • Blood cells Abstract. An increase in the incidence of thromboembolic disorders has been associated with oral contraceptive use, though the causative mechanisms remain un clear. Our studies indicate that the contraceptive steroids, irrespective of the inter mediary metabolic processes involved, cause changes in the surface charge charac teristics of the blood vessel wall and blood cells in the following cases: (i) in experi ments using dogs, the hormonal steroids result in a greater reduction in the pore sur face charge of veins than in arteries; (ii) in rats, the current induced mesenteric occlusion times are significantly lowered following administration of combined con traceptive steroids; (iii) in humans, the electrophoretic mobilities of erythrocytes and platelets from women taking Ovral and Demulen are lower than in controls, and (iv) there is no significant alteration of plasma coagulation times of women who are on injectable progestin therapy. Demulen and Ovral appear to result in a slight decrease in activated partial thromboplastin times compared to controls.
Introduction
It is now known that the use of contraceptive hormonal steroids is asso ciated with thromboembolic disorders [5, 8, 18]. It has been found that women using oral contraceptives show alterations in the concentrations of a number of plasma-clotting factors [17]. In addition, changes in platelet function [2, 12] and other vascular components [6, 19] have been de scribed.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:30:04 AM
1 This work was supported in part by a grant (HL 15123-02) from the National Heart and Lung Institute, National Institutes of Health, Bethesda, Md.
330
SOLASH cl III.
In a preliminary communication [16] we suggested that changes in the surface charge characteristics of vascular elements induced by oral contra ceptives could account for, at least in part, some of the thromboembolic side effects. We now wish to report the results of long-term studies of patients and experimental animals who were administered contraceptive steroids. Materials and Methods Animat Studies Rats. The effects of mestranol, estradiol, norcthindrone, dimethysterone and Provera on current induced thrombosis times of rat mesenteric vessels were determined. Female Wistar rats (90-120 g) were used for this study. The contraceptive hormonal steroids were given intraperitoneally daily for 10 days (estrogens 1.4 ug/day/100 g/rat; progestins 14 ug/day/100 g/rat). On the 10th day the rats were anesthetized, the mesoappendix was exposed, and the time taken for complete occlusion of the vessels under the influence of a small applied electric current of 10 //A was determined. The experimental details have been elaborated elsewhere [1].
Clinical Studies 34 women taking various oral or injectable steroid contraceptives were chosen for this study. Specifically excluded were patients with a previous history of thrombo embolic disease, gynecologic disorders or known metabolic defects (e.g., hypertension, diabetes). Four groups of women were examined: (i) 10 control subjects, age-matched, who were not on any therapy; (ii) 13 subjects were on Depo-Provera (medroxy progesterone acetate, injectable progestin; Upjohn); (iii) 9 subjects were using Ovral (combination of ethinyl estradiol 50 fig and norgestrel 500 itg; Wyeth) and (iv) 11 subjects were on Demulen (combination of ethinyl estradiol 50 «g and ethynodiol diacetate 1.0 mg; Searle). 20 ml of blood were drawn from these subjects for blood coagulation studies and determination of electrophoretic mobilities of erythrocytes and platelets. The study was carried out as a double-blind trial. The Fibrometer test system (BBL, Md.) was used for carrying out plasma co-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:30:04 AM
Dogs. The effect of mestranol (M), norethindrone (N), and a combination of both (M + N) on the intimal surface charge of arteries and veins was determined in this study. 16 adult female dogs (6-8 kg) were divided into four groups. Group i served as the control. (1.5 mg/kg) was given orally to group ii. Groups iii and iv, respectively, received N (15 mg/kg) and a combination of M and N (1.5 mg/kg of M + 15 mg/kg of N). The therapy was continued for 4 weeks. At the end of therapy, segments of the aorta and vena cava were excised for elcctroosmotic flow measurements. The electroosmotic study consists of determining the flow rate across a porous membrane, i.e.. blood vessel wall, under the influence of an electric field applied across the mem brane. The details of this experimental procedure have been described in our earlier communications [15, 16).
Hormonal Steroids: Effects on the Vascular System
331
agulation tests. Thrombin clot times (TRT), activated partial thromboplastin times (APTT), and recalcification times (RCT) were determined. TRT were determined in the Fibrometcr system by mixing 0.1 ml of the patient’s plasma with 0.1 ml of normal saline and incubating for 2 min at 37 °C. Finally, 0.1 ml (1 NIH unit) thrombin (Upjohn) was added and the coagulation time recorded. All measurements were triplicated, and the average clotting times of three measurements (single sample) were used. F.ach day the thrombin was standardized by using Fibrotro! (normal coagulation control plasma, lyophilized; BBL). The APTT was determined using Fibrolet, lyophilized activated platelet factor reagent (BBL), which is a chloroform extract of brain activated by Cclite. 0.1 ml of Fibrolet was mixed with 0.1 ml of the patient's plasma for 1-2 min and gently shaken. 0.1 ml of 0.025 M CaCL was then added and the APTT was determined in the Fibrotest system. Three determinations were made per sample. Standard plasma (Fibrotrol) controls were also determined. RCT was measured (manually) by mixing 0.2 ml of the patient’s plasma (or Fibrotrol standard) with 0.2 ml of 0.025 M CaCI2 and noting the time taken for the first appearance of fibrin strands. At least three determinations were made per sample. Due to inherent variations in enzymatic activity of reagents used in the TRT. APTT and RCT determinations, the (Fibrotrol) control values were different for different days. Therefore, the results are reported as ratios, i.e., TRT, APTT or RCT of patient's plasma to standard plasma (Fibrotrol). The electrophoretic mobilities (BM) of erythrocytes and platelets from human subjects were determined using Seaman's microelectrophoresis apparatus [15, 16]. Cells were suspended in calcium-free Tyrode solution, and the mobility of the cells at 37 °C' under the influence of an electric field was determined. Cell movement across an eye-piece grid was timed using a stop watch. At least 10 cells were ob served (5 in each direction) for each run. The detailed procedure has been elaborated in earlier publications [15, 16].
Results
The current-induced occlusion time of mesenteric vessels was deter mined in rats fed various contraceptive steroids for 10 days (table 1). The progestin, dimethisterone (p< 0.001), significantly lowered the occlusion times while another progestin, Provera, did not significantly alter the occlu sion time (p>0.1). Of the two estrogenic steroids, 17/?-ethynyI estradiol (when compared to the control) caused a threefold reduction in the mesenteric vessel occlusion time (p0.05 0.8610.22 0.05 1.2110.10 >0.10
1 Ratio of experimental value to standard plasma (Fibrotrol) value.
Table IV. Electrophoretic mobility of blood cells from women taking contraceptive steroids Name of the contraceptive drug
Number Electrophoretic mobility, u sec-1 V 1cm of erythrocytes platelets patients m eaniSD p m eaniSD P
Control Depo-Provera Demulen Ovral
9 13 10 9
1.4210.07 1.3610.09
Hormonal Steroids: Effects on the Vascular System' J. Solash , R. P erez , J.S. K eates, N. R amasamy, H. Stein , D. T royansky , E. A teyeh , jr., J.R. J ones , S. S rinivasan and P.N. Sawyer Electrochemical and Biophysical Laboratories of the Vascular Surgical Services, Departments of Surgery and Surgical Research, and Obstetrics and Gynecology, Downstate Medical Center, State University of New York, Brooklyn, N.Y.
Key Words. Contraceptive steroids • Clinical studies • Animal studies • Electro phoretic mobility • Blood vessels • Blood cells Abstract. An increase in the incidence of thromboembolic disorders has been associated with oral contraceptive use, though the causative mechanisms remain un clear. Our studies indicate that the contraceptive steroids, irrespective of the inter mediary metabolic processes involved, cause changes in the surface charge charac teristics of the blood vessel wall and blood cells in the following cases: (i) in experi ments using dogs, the hormonal steroids result in a greater reduction in the pore sur face charge of veins than in arteries; (ii) in rats, the current induced mesenteric occlusion times are significantly lowered following administration of combined con traceptive steroids; (iii) in humans, the electrophoretic mobilities of erythrocytes and platelets from women taking Ovral and Demulen are lower than in controls, and (iv) there is no significant alteration of plasma coagulation times of women who are on injectable progestin therapy. Demulen and Ovral appear to result in a slight decrease in activated partial thromboplastin times compared to controls.
Introduction
It is now known that the use of contraceptive hormonal steroids is asso ciated with thromboembolic disorders [5, 8, 18]. It has been found that women using oral contraceptives show alterations in the concentrations of a number of plasma-clotting factors [17]. In addition, changes in platelet function [2, 12] and other vascular components [6, 19] have been de scribed.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:30:04 AM
1 This work was supported in part by a grant (HL 15123-02) from the National Heart and Lung Institute, National Institutes of Health, Bethesda, Md.
330
SOLASH cl III.
In a preliminary communication [16] we suggested that changes in the surface charge characteristics of vascular elements induced by oral contra ceptives could account for, at least in part, some of the thromboembolic side effects. We now wish to report the results of long-term studies of patients and experimental animals who were administered contraceptive steroids. Materials and Methods Animat Studies Rats. The effects of mestranol, estradiol, norcthindrone, dimethysterone and Provera on current induced thrombosis times of rat mesenteric vessels were determined. Female Wistar rats (90-120 g) were used for this study. The contraceptive hormonal steroids were given intraperitoneally daily for 10 days (estrogens 1.4 ug/day/100 g/rat; progestins 14 ug/day/100 g/rat). On the 10th day the rats were anesthetized, the mesoappendix was exposed, and the time taken for complete occlusion of the vessels under the influence of a small applied electric current of 10 //A was determined. The experimental details have been elaborated elsewhere [1].
Clinical Studies 34 women taking various oral or injectable steroid contraceptives were chosen for this study. Specifically excluded were patients with a previous history of thrombo embolic disease, gynecologic disorders or known metabolic defects (e.g., hypertension, diabetes). Four groups of women were examined: (i) 10 control subjects, age-matched, who were not on any therapy; (ii) 13 subjects were on Depo-Provera (medroxy progesterone acetate, injectable progestin; Upjohn); (iii) 9 subjects were using Ovral (combination of ethinyl estradiol 50 fig and norgestrel 500 itg; Wyeth) and (iv) 11 subjects were on Demulen (combination of ethinyl estradiol 50 «g and ethynodiol diacetate 1.0 mg; Searle). 20 ml of blood were drawn from these subjects for blood coagulation studies and determination of electrophoretic mobilities of erythrocytes and platelets. The study was carried out as a double-blind trial. The Fibrometer test system (BBL, Md.) was used for carrying out plasma co-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:30:04 AM
Dogs. The effect of mestranol (M), norethindrone (N), and a combination of both (M + N) on the intimal surface charge of arteries and veins was determined in this study. 16 adult female dogs (6-8 kg) were divided into four groups. Group i served as the control. (1.5 mg/kg) was given orally to group ii. Groups iii and iv, respectively, received N (15 mg/kg) and a combination of M and N (1.5 mg/kg of M + 15 mg/kg of N). The therapy was continued for 4 weeks. At the end of therapy, segments of the aorta and vena cava were excised for elcctroosmotic flow measurements. The electroosmotic study consists of determining the flow rate across a porous membrane, i.e.. blood vessel wall, under the influence of an electric field applied across the mem brane. The details of this experimental procedure have been described in our earlier communications [15, 16).
Hormonal Steroids: Effects on the Vascular System
331
agulation tests. Thrombin clot times (TRT), activated partial thromboplastin times (APTT), and recalcification times (RCT) were determined. TRT were determined in the Fibrometcr system by mixing 0.1 ml of the patient’s plasma with 0.1 ml of normal saline and incubating for 2 min at 37 °C. Finally, 0.1 ml (1 NIH unit) thrombin (Upjohn) was added and the coagulation time recorded. All measurements were triplicated, and the average clotting times of three measurements (single sample) were used. F.ach day the thrombin was standardized by using Fibrotro! (normal coagulation control plasma, lyophilized; BBL). The APTT was determined using Fibrolet, lyophilized activated platelet factor reagent (BBL), which is a chloroform extract of brain activated by Cclite. 0.1 ml of Fibrolet was mixed with 0.1 ml of the patient's plasma for 1-2 min and gently shaken. 0.1 ml of 0.025 M CaCL was then added and the APTT was determined in the Fibrotest system. Three determinations were made per sample. Standard plasma (Fibrotrol) controls were also determined. RCT was measured (manually) by mixing 0.2 ml of the patient’s plasma (or Fibrotrol standard) with 0.2 ml of 0.025 M CaCI2 and noting the time taken for the first appearance of fibrin strands. At least three determinations were made per sample. Due to inherent variations in enzymatic activity of reagents used in the TRT. APTT and RCT determinations, the (Fibrotrol) control values were different for different days. Therefore, the results are reported as ratios, i.e., TRT, APTT or RCT of patient's plasma to standard plasma (Fibrotrol). The electrophoretic mobilities (BM) of erythrocytes and platelets from human subjects were determined using Seaman's microelectrophoresis apparatus [15, 16]. Cells were suspended in calcium-free Tyrode solution, and the mobility of the cells at 37 °C' under the influence of an electric field was determined. Cell movement across an eye-piece grid was timed using a stop watch. At least 10 cells were ob served (5 in each direction) for each run. The detailed procedure has been elaborated in earlier publications [15, 16].
Results
The current-induced occlusion time of mesenteric vessels was deter mined in rats fed various contraceptive steroids for 10 days (table 1). The progestin, dimethisterone (p< 0.001), significantly lowered the occlusion times while another progestin, Provera, did not significantly alter the occlu sion time (p>0.1). Of the two estrogenic steroids, 17/?-ethynyI estradiol (when compared to the control) caused a threefold reduction in the mesenteric vessel occlusion time (p0.05 0.8610.22 0.05 1.2110.10 >0.10
1 Ratio of experimental value to standard plasma (Fibrotrol) value.
Table IV. Electrophoretic mobility of blood cells from women taking contraceptive steroids Name of the contraceptive drug
Number Electrophoretic mobility, u sec-1 V 1cm of erythrocytes platelets patients m eaniSD p m eaniSD P
Control Depo-Provera Demulen Ovral
9 13 10 9
1.4210.07 1.3610.09
