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Single dose of nandrolone does not prevent postoperative fibrinolytic shutdown
Fibrinolysis(1995)9, 350-355
© 1995PearsonProfessionalLtd
Single Dose of Nandrolone Does Not Prevent Postoperative Fibrinolytic Shutdown
M. Szczepaflski, T. Duchiflski, P. Kretowicz
SUMMARY Nandrolone 50 mg, 100 mg, or placebo was intramuscularly injected prior to surgery into patients
admitted for gynecological operations. Plasminogen and fibrinogen concentration, plasminogen activator inhibitor (PAI) activity and fibrinolytic activity in euglobulins were determined until the seventh postoperative day. Injection of nandrolone resulted in a delay in the postoperative rise of fibrinogen, but an earlier increase of plasminogen after surgery, as compared with the control group. 50 mg of this anabolic steroid given before surgery prevented the postoperative increase of PAI activity observed in the placebo group, while a 100 mg dose caused a temporary decline of this activity until the third postoperative day. The fibrinolytic activity in euglobulins was significantly decreased on the first postoperative day in the patients of all groups and no differences were found among the groups up to the end of determinations. It is concluded that the injection of nandrolone prior to surgery temporarily inhibits the postoperative rise in PAI activity but does not prevent fibrinolytic shutdown occurring after surgery.
stanozolol 1°-14 and nandrolone. 15-t7 According to Psuja et a1,15'16 a single dose of nandrolone phenylpropionate injected prior to surgery prevented the postoperative decrease of fibrinolytic activity. The aim of the present investigations was to estimate the influence of a single dose of nandrolone, injected preoperatively, on the postoperative fibrinolytic activity in euglobulins and on the activity of PAI; the levels of plasminogen and fibrinogen were also assessed.
The decline of flbrinolytic activity observed after abdominal operations, 1 gynecological and urological surgery,2 and after total hip replacement 3 has been connected with the postoperative increase in plasminogen activator inhibitor (PAl-1), rather than with the reduced level of tissue-type plasminogen activator (t-PA). 3-8 According to D'Angelo et al4 and Kluft et al, 8 the level of t-PA antigen did not decline in patients after surgery but the sharp rise of PAI- 1 on the first postoperative day was reflected by the low activity of t-PA and was responsible for the postoperative fibrinolytic shutdown. There are diverse opinions regarding the associations between venous thromboembolism and impaired fibrinolytic activity, but Prins and Hirsh 9 claimed in their critical collective review that such an association existed in patients with postoperative deep vein thrombosis. Therefore, efforts aimed at the prevention of postoperative fibrinolytic shutdown might result in the diminished frequency of this complication. Clinical studies aimed at the prevention of this shutdown and reduction of the frequency of postoperative venous thrombosis were performed using two anabolic steroids,
PATIENTS AND METHODS Thirty patients admitted to the Department of Obstetrics and Gynecology for surgical treatment were randomized into three groups of 10 patients. They were injected i.m. 24h before surgery with 2ml of isotonic sodium chloride (placebo) in group A, 50mg of nandrolone phenylpropionate (Nerobolil, Richter) in group B, and 100mg of this anabolic steroid in group C. The Table presents their age and operation performed. Exclusion criteria were: malignant tumours, diabetes, arterial hypertension, and thromboembolic disease within the last 5 years. Informed consent was obtained from all patients entering this study, which was approved by the local ethical committee. The preoperative treatment with nandrolone or placebo was blind to all concerned until the end of the trial.
M. Szczepahski, T. Duchihski, P. Kretowicz,
Laboratory of Hemostasis, Department of Biochemistry and Department of Obstetrics and Gynecology, Medical Center of Postgraduate Education, Warsaw, Poland. 350
Fibrinolysis Table Clinical features of patients
Groups of patients
Patients Age (mean_+ SD) Surgery: total abdominal hysterectomy (t.a.h.) t.a.h, plus bilateral salpingo-oophorectomy t.a.h, plus unilateral salpingo-oophorectomy myomectomy salpingo-oophorectomy oophorectomy ovarian cystectomy salpingectomy splenectomy
Placebo (A)
Nandrolone (B) 50mg (C) 100rag
I0 38.6+7.8
l0 41.5_+13.2
10 44.0_+8.7
2
0
1
3
6
5
1
1
2
0 2 l 0 1 0
0 0 0 2 0 1
1 0 0 1 0 0
The patient who underwent a splenectomy was preoperatively diagnosed as having a benign tumour of the left ovary, but an unusually mobile, floating spleen was found during surgery and a splenectomy was performed. All patients had blood samples taken on the day prior to surgery, and on the first, third, and seventh days after surgery. Blood samples were collected between 07:00 and 08:00 from a cubital vein with minimal venous occlusion and anticoagulated with 0.13 M trisodium citrate (9:1, v:v), centrifuged at 2500 g for 15 rain at 4°C, and separated plasma samples were kept at -20°C until determinations. In order to avoid the possible influence of blood platelets, the plasma specimens for PAI activity were obtained from the uppermost layer of centrifuged samples. Fibrinogen Concentration in plasma was determined by a colorimetric method. Is Plasminogen concentration in plasma was measured by an amidolytic method, using the commercial kit (Berichrom Plasminogen).
g/t
A
351
PAl activity was estimated with a synthetic chromogenic substrate, using the commercial kit (Berichrom PAI). Fibrinolytic activity in euglobulins was assayed basically according to Kowalski et a119 with some modifications. 1 ml of plasma was diluted with 7.5 ml of distilled water, acidified with 1% acetic acid to pH 5.8, and left for 30 rain at 4°C for complete precipitation. The euglobulin sediment was separated by centrifugation at 2500g for 15 rain at 4°C and dissolved in 0.25 ml of 0.22M borate buffer, pH 7.6. The solubilized euglobulins were placed as 30 ~tl drops on the 1 mm thick fibrin plates and incubated for 22h at 37°C. The fibrin plates were prepared from 0.2% human fibrinogen (Behring) dissolved in 25 mM sodium barbital buffer of pH 7.8, containing 57 mM sodium chloride and 0.58 mM calcium chloride. 2° The fibrinolytic activity in euglobulins was expressed in mm 2, as a product of two perpendicular diameters of corresponding circular zones of fibrin plate lysis. Student's t-test for paired data was used for comparisons between the results obtained on consecutive days of determinations within groups. The differences between the results were regarded as significant on the probability level of P < 0.02.
RESULTS The fibrinogen concentration in group A patients rose immediately after surgery and was significantly increased already on the first postoperative day; this increase was most pronounced on the third postoperative day. The administration of nandrolone resulted in a delay in the rise of fibrinogen and in both groups, B as well as C, its concentration increased not earlier than between the first and third day after surgery (Fig. 1). The differences in fibrinogen level disappeared then among the groups of patients, starting from the third postoperative day.
B
C
5-
.
3'
-~b ~ ~ Fig. 1 Fibrinogen concentration in patients of group A (placebo), B (nandrolone, 50 mg), and C (nandrolone, 100 mg). Ordinate: concentration of fibrinogen in g/1. Abscissa: days before and after surgery. Values are means+SD. I~*l : difference significant on the probability level of P<0.02.
352
Single Dose of Nandrolone Does Not Prevent Postoperative Fibrinolytic Shutdown
%
A
160-
B
C
140t20 100. Off
80
o
•
=
•
,
i
•
,i
,~i
,,
il
~
,
il~
,,
Q
,,
~
,
Fig. 2 Plasminogen concentration. Ordinate: percentage of plasminogen concentration; the concentration of plasminogen in reference normal plasma (Behring) was assigned as 100%. Other details as in Figure 1.
The plasminogen concentration fell immediately after the operation in all groups of patients. This concentration rose again in group A between the third and seventh postoperative day but this rise was observed already between the first and third, and continued until the seventh day in patients of B and C groups (Fig. 2). The activity of PAI increased in the patients of group A on the first postoperative day and the second increase of this activity was observed in them between the third and seventh day. The early postoperative rise of PAI activity was not seen in patients injected preoperatively with 50 mg of nandrolone. The significant decline of this activity was noted between the first and third postoperative day in patients injected prior to surgery with 100 mg of this anabolic hormone. The increase of PAI activity between the third and seventh postoperative day was found in the patients of all groups, placebo as well as nandrolone-treated (Fig. 3). The fibrinolytic activity in euglobulins followed the same pattern in patients of all groups: the decline on the first postoperative day and absence of any other significant changes until the end of determination (Fig. 4).
w',,,L
A
Increased oozing at the beginning of operation was observed in five patients of group C but the postoperative course was not complicated by a wound hematoma in any of them; the only case with such a hematoma and prolonged wound healing occurred in a group A. Side-effects were observed in one patient 10 min after the i.m. injection of 100rag of nandrolone. They consisted of a hot flush and generalized spotted rash; both of these symptoms subsided within one hour.
DISCUSSION The fibrinogen concentration in plasma of patients in the control group rose immediately after the operation and was greatest on the third postoperative day. This pattern of fibrinogen response to surgical stress has been reported by others. 4's'16'21 The fibrinogen molecules are synthesized in and released from hepatocytes and it is now believed that fibrinogen response to surgery is mediated by hepatocyte-stimulating factor identical to interleukin-6. 22,23
B
8765 4 3 2 1
0
!
Fig. 3 PAl activity. Ordinate: PAl activity in urokinase-inhibiting units/ml. Other details as in Figure 1.
, ,9,,
~
,
Fibrinolysis 353 ,S~ , rt'l rf}
C
B
A
4OO 300
200 iO0
I
•
i
i
~
I
i
J
a
-~b~ Fig. 4 Fibrinolytic activity in euglobulins assayed on human fibrin plates. Ordinate: zones of lysis on fibrin plates, estimated in mm 2. Other details as in Figure I.
The preoperative injection of 50mg or 100mg of nandrolone in our patients resulted in the delayed postoperative increase of fibrinogen concentration. Contrary to the subjects of the control group, the nandrolone-treated patients did not reveal a significant rise of fibrinogen on the first postoperative day but the concentration of this protein in their plasma reached the values of control group patients after the next 2 days. These findings are different from the data of Blamey et a112 and of Preston et al, ~3 who did not observe any difference in postoperative fibrinogen concentration between the controls and stanozololtreated patients. Psuja et a116 injected surgical patients with 50 mg of nandrolone before operation and also observed significant increase of fibrinogen level as soon as the first postoperative day. We are inclined to explain the delay in the postoperative rise of fibrinogen in our nandrolone-treated patients by the interference between this anabolic steroid and interleukin-6, mediating the fibrinogen response to surgery. The plasminogen concentration in the plasma of our patients in all three groups was significantly diminished on the first postoperative day. Berridge et al]° observed a decrease of plasminogen two days after vascular surgery, Mellbring et al 21 noted this phenomenon one to three hours after abdominal surgery, and Paramo et a124 encountered the decline of plasminogen level even during cardiopulmonary bypass surgery. This decline in plasminogen concentration in plasma can be considered as resulting from an intraoperative activation of the fibrinolytic system and the consumption of this zymogen. We have observed the secondary increase of plasminogen level between the third and seventh postoperative day and a similar rise was observed by some authors 1121 ' but not by others. ]°'13"16 The rise of plasminogen level in subjects receiving stanozolol was noted by some authors: peroral administration of this drug, 10 mg 24h, resulted in an elevated plasminogen level after three weeks 25
or one month 26 of this treatment; Small e t a127 injected healthy adults i.m. with 50rag of stanozolol and an increased concentration of plasminogen was found two days later. In our investigation, patients injected with 50 mg or 100mg of nandrolone prior to surgery showed an accelerated postoperative rise of plasminogen level compared with control group patients, and this is in agreement with the observations of Berridge et al 1° and of Blamey et al ]1 but not with those of Psuja et al. 16 It seems evident, therefore, that preoperative administration of a single dose of anabolic steroid does not prevent the intraoperative consumption of the fibrinolytic zymogen, plasminogen, but stimulates its postoperative compensatory rise. We have observed a significant increase in PAI activity on the first postoperative day in the patients of the control group and a similar response of PAI to surgery was reported by others. 2 - 6 8' 2'4 The rise of PAI was accompanied by a decline in the fibrinolytic activity in the euglobulin fraction. This activity was regarded in the past as expressing the plasminogen activator activity 12'27-29 but it later became evident that PAI is also partially precipitated together with plasminogen activator in the euglobulin fraction. 3°'31 It is now believed that the fibrinolytic activity in euglobulins is strongly influenced by both t-PA and PAI, 32'33 and, according to Kluft, 34 a net effect of t-PA and its inhibitor is recorded in the euglobulin fraction. The significant decline in the fibrinolytic activity of this fraction in the patients of our control group, corresponding to the postoperative fibrinolytic shutdown, could eventually be explained by the concomitant rise of PAI activity. However, a similar shutdown observed in nandrolone-treated patients was not accompanied by an increase of PAI activity. This increase, observed immediately after the operation in placebo-receiving patients, was abolished by the preoperative injection of 50 mg of nandrolone; moreover, the 100 mg dose of this drug resulted in a significant
354
Single Dose of Nandrolone Does Not Prevent Postoperative Fibrinolytic Shutdown
d e c l i n e in PAI a c t i v i t y b e t w e e n the first and third p o s t o p e r a t i v e day. T h e a c t i v i t y o f PAI in s u r g i c a l patients, p r e t r e a t e d w i t h a n a b o l i c steroids, has not b e e n d e t e r m i n e d by o t h e r s so far, and the d a t a on f i b r i n o l y t i c a c t i v i t y in e u g l o b u l i n s in t h e s e p a t i e n t s are d i s c r e p a n t . B l a m e y et a111'12 n o t e d a s i g n i f i c a n t l y i n c r e a s e d a c t i v i t y in e u g l o b u l i n s on the first p o s t o p e r a t i v e day in p a t i e n t s i n j e c t e d p r i o r to s u r g e r y w i t h 50 m g o f s t a n o z o l o l , as c o m p a r e d w i t h u n t r e a t e d p a t i e n t s ; t h e y e x p l a i n e d this f i n d i n g by the rise in p l a s m i n o g e n a c t i v a t o r activity. S i m i l a r o b s e r v a t i o n s w e r e m a d e by P r e s t o n et al ~3 in s u r g i c a l p a t i e n t s p r e o p e r a t i v e l y t r e a t e d w i t h 75 m g o f s t a n o z o l o l . B e r r i d g e et al ~° w e r e n o t a b l e to s u p p r e s s the e a r l y f i b r i n o l y t i c s h u t d o w n in p a t i e n t s u n d e r g o i n g f e m o r o p o p l i t e a l b y p a s s by the p r e o p e r a t i v e i.m. i n j e c tion o f 5 0 m g o f s t a n o z o l o l , f o l l o w e d by a p e r o r a l d o s e o f 10 m g o f this d r u g p e r 24 hours. O n the o t h e r hand, P s u j a et a115'16 p r e v e n t e d the p o s t o p e r a t i v e d e c l i n e in f i b r i n o l y t i c a c t i v i t y by a s i n g l e i.m. i n j e c tion o f n a n d r o l o n e p r i o r to surgery. W e h a v e o b s e r v e d an e a r l y p o s t o p e r a t i v e i n h i b i t i o n o f P A I a c t i v i t y by this a n a b o l i c s t e r o i d but this w a s not r e f l e c t e d b y any c h a n g e s in c o n c o m i t a n t f i b r i n o l y t i c s h u t d o w n . W e are i n c l i n e d to s p e c u l a t e , t h e r e f o r e , that the f i b r i n o l y t i c s h u t d o w n o c c u r r i n g a f t e r s u r g e r y is i n f l u e n c e d by the d e c r e a s e o f t - P A a c t i v i t y r a t h e r than by the i n c r e a s e o f PAI activity.
REFERENCES I. Psuja P, Sowier J, Gizlo J, Zawilska K. Actual and reserve fibrinolytic activity in the postoperative period - possibility of exhaustion of the reserve of vascular plasminogen activator (In Polish). Acta Haemat Pol 1980; 11: I-7. 2. Aranda A, Paramo J A, Rocha E. Fibrinolytic activity in plasma after gynecological and urological surgery. Haemostasis 1988; 18: 129-134. 3. Paramo J A, Alfaro M J, Rocha E. Postoperative changes in the plasmatic levels of tissue-type plasminogen activator and its fast-acting inhibitor - relationship to deep vein thrombosis and influence of prophylaxis. Thromb Haemostas 1985; 54: 713-716. 4. D'Angelo A, Kluft C, Verheijen J H, Rijken D C, Mozzi E, Manucci P M. Fibrinolytic shut-down after surgery: impairment of the balance between tissue-type plasminogen activator and its specific inhibitor. Europ J Clin Invest 1985; 15: 308-312. 5. Juhan-Vague I, Aillaud M F, De Cock Met al. The fast acting inhibitor of tissue-type plasminogen activator is an acute phase reactant protein. In: Davidson J F et al, eds. Progress in fibrinolysis VII. Edinburgh: Churchill Livingstone, 1985: 146-149. 6. Kassis J, Hirsh J, Podor T J. Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis. Blood 1992; 80: 1758-1764. 7. Kluft C, Verheijen J H, Cooper P et al. Post-operative changes in the activity on blood of extrinsic (tissue-type) plasminogen activator and its fast-acting inhibitor (abstract 41 ). Haemostasis 1984; 14-26. 8. Kluft C, Verheijen J H, Jie A F H et al. The postoperative fibrinolytic shutdown: a rapidly reverting acute phase pattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma. Scand J Clin Lab Invest 1985; 45: 605-610.
9. Prins M H, Hirsh J A. A critical review of the evidence supporting a relationship between impaired fibrinolytic activity and venous thromboembolism. Arch Intern Med 1991 ; 151 : 1721-1731. 10. Berridge D C, Frier M, Westby J C, Hopkinson B R, Makin G S. Double-blind randomized trial of perioperative fibrinolytic enhancement for femoropopliteal bypass. Brit J Surg 1991 ; 78: 101-104. I I. Blarney S L, McArdle B M, Burns Pet al. Prevention of fibrinolytic shutdown after major surgery by intramuscular stanozolol. Thromb Res 1983; 31:451-459. 12. Blarney S L, McArdle B M, Burns P, Carter D C, Lowe G D O, Forbes C D. A double-blind trial of intramuscular stanozolol in the prevention of postoperative deep vein thrombosis following elective abdominal surgery. Thromb Haemostas 1984; 51: 71-74. 13. Preston F E, Kluft C, Simms M, Malia R G, Graeves M. Comparison of effects of stanozolol and subcutaneous heparin on coagulation and fibrinolysis in surgical patients (abstract 51). Haemostasis 1984; 14:31. 14. Sue-Ling H M, Davies J A, Prentice C R M, Verheijen J H, Kluft C. Effects of oral stanozolol used in prevention of postoperative deep vein thrombosis on fibrinolytic activity. Thromb Haemostas 1985; 54: 141-142. 15. Psuja P, Tokarz A, Szymczak O, Zozulifiska M, Sowier J, Zawilska K. The use of anabolic steroid nandrolone phenylpropionate in the prevention of deep vein thrombosis after abdominal surgery. In: Maurer P C et al, eds. What is new in angiology? Proceedings of the 14th World Congress International Union of Angiology, July 6-11, 1986:211-212. 16. Psuja P, Tokarz A, Szymczak P, Zozuliflska M, Sowier J, Zawilska K. Use of nandrolone in the prevention of postoperative deep vein thrombosis (In Polish). Pol Tyg Lek 1986; 41: 1589-1591. 17. Zawilska K, Tokarz A, Misiak A et al. Nebulized heparin and anabolic steroid in the prevention of postoperative deep vein thrombosis following elective abdominal surgery. Folia Haematol 1990; 117: 699-707. 18. Swaim W R, Feders M B. Fibrinogen assay. Clin Chemistry 1967; 13: 1026-1028. 19. Kowalski E, Kope6 M, Niewiarowski S. An evaluation of the euglobulin method for the determination of fibrinolysis. J Clin Path 1959; 12: 215-218. 20. Brakman P. Fibrinolysis. A standardized fibrin plate method and a fibrinolytic assay of plasminogen. Amsterdam: Scheltema and Holkema N.V., 1967. 21. Mellbring G, Dahlgren S, Reiz S, Wiman B. Fibrinolytic activity in plasma and deep vein thrombosis after major abdominal surgery. Thromb Res 1983; 32: 575-584. 22. Heinrich P C, Castell J V, Andus T. Interleukin-6 and the acute phase response. Biochem J 1990; 265: 621-636. 23. DiMinno G, Mancini M. Drugs affecting plasma fibrinogen levels. Cardiovasc Drugs Ther 1992; 6: 25-27. 24. Paramo J A, Rifon J, Llorens R, Casares J, Paloma M J, Rocha E. Intra- and postoperative fibrinolysis in patients undergoing cardiopulmonary bypass surgery. Haemostasis 1991; 21: 58-64. 25. Kluft C, Preston F E, Malia R Get al. Stanozolol-induced changes in fibrinolysis and coagulation in healthy adults. Thromb Haemostas 1984; 51 : 157-164. 26. Davidson J F, Lockhead M, McDonald G A, McNicol G P. Fibrinolytic enhancement by stanozolol: a double blind trial. Br J Haematol 1972; 22: 543-559. 27. Small M, McArdle B M, Lowe G D O, Forbes C D, Prentice C R M. The effect of intramuscular stanozolol on fibrinolysis and blood lipids. Thromb Res 1982; 28: 27-36. 28. Verheijen J H, Chang G T G, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51 : 392-395. 29. Preston F E, Burakowski B K, Porter N R, Malia R G. The fibrinolytic response to stanozolol in normal subjects. Thromb Res 1981 ; 22:543-551. 30. Kluft C, Jie A F H. Underestimation of tissue-type plasminogen activator activity in plasma when euglobulin fractions are used for assessment (abstract 147). Fibrinolysis suppl. 1986.
Fibrinolysis 355 31. DeMaat MPM, Kluft C, de Boer K, Knot E A R, Jie A F H. Acid treatment of plasma for the inactivation of plasminogen activator inhibitor-1 (PAl-1). Thromb Res 1988; 52: 425-430. 32. Urano T, Sahakibara K, Rydzewski A, Urano S, Takada Y, Takada A. Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and
Received: 15 November 1994 Accepted after revision: I 1 August 1995 Corresponding author: Maciej Szczepafiski, Laboratory of Hemostasis, Postgraduate Teaching Hospital, ul.Czerniakowska 23 I, 00-416 Warsaw, Poland. Tel: 34.12.28, Fax: 34.04.70.
plasminogen activator inhibitor I. Thromb Haemostas 1990; 63: 82-86. 33. Urano T, Sumiyoshi K, Pietraszek M H, Takada Y, Takada A. PAI-I plays an important role in the expression of t-PA activity in the euglobulin clot lysis by controlling the concentration of free t-PA. Thromb Haemostas 1991 ; 66: 474478. 34. Kluft C. Personal communication.
© 1995PearsonProfessionalLtd
Single Dose of Nandrolone Does Not Prevent Postoperative Fibrinolytic Shutdown
M. Szczepaflski, T. Duchiflski, P. Kretowicz
SUMMARY Nandrolone 50 mg, 100 mg, or placebo was intramuscularly injected prior to surgery into patients
admitted for gynecological operations. Plasminogen and fibrinogen concentration, plasminogen activator inhibitor (PAI) activity and fibrinolytic activity in euglobulins were determined until the seventh postoperative day. Injection of nandrolone resulted in a delay in the postoperative rise of fibrinogen, but an earlier increase of plasminogen after surgery, as compared with the control group. 50 mg of this anabolic steroid given before surgery prevented the postoperative increase of PAI activity observed in the placebo group, while a 100 mg dose caused a temporary decline of this activity until the third postoperative day. The fibrinolytic activity in euglobulins was significantly decreased on the first postoperative day in the patients of all groups and no differences were found among the groups up to the end of determinations. It is concluded that the injection of nandrolone prior to surgery temporarily inhibits the postoperative rise in PAI activity but does not prevent fibrinolytic shutdown occurring after surgery.
stanozolol 1°-14 and nandrolone. 15-t7 According to Psuja et a1,15'16 a single dose of nandrolone phenylpropionate injected prior to surgery prevented the postoperative decrease of fibrinolytic activity. The aim of the present investigations was to estimate the influence of a single dose of nandrolone, injected preoperatively, on the postoperative fibrinolytic activity in euglobulins and on the activity of PAI; the levels of plasminogen and fibrinogen were also assessed.
The decline of flbrinolytic activity observed after abdominal operations, 1 gynecological and urological surgery,2 and after total hip replacement 3 has been connected with the postoperative increase in plasminogen activator inhibitor (PAl-1), rather than with the reduced level of tissue-type plasminogen activator (t-PA). 3-8 According to D'Angelo et al4 and Kluft et al, 8 the level of t-PA antigen did not decline in patients after surgery but the sharp rise of PAI- 1 on the first postoperative day was reflected by the low activity of t-PA and was responsible for the postoperative fibrinolytic shutdown. There are diverse opinions regarding the associations between venous thromboembolism and impaired fibrinolytic activity, but Prins and Hirsh 9 claimed in their critical collective review that such an association existed in patients with postoperative deep vein thrombosis. Therefore, efforts aimed at the prevention of postoperative fibrinolytic shutdown might result in the diminished frequency of this complication. Clinical studies aimed at the prevention of this shutdown and reduction of the frequency of postoperative venous thrombosis were performed using two anabolic steroids,
PATIENTS AND METHODS Thirty patients admitted to the Department of Obstetrics and Gynecology for surgical treatment were randomized into three groups of 10 patients. They were injected i.m. 24h before surgery with 2ml of isotonic sodium chloride (placebo) in group A, 50mg of nandrolone phenylpropionate (Nerobolil, Richter) in group B, and 100mg of this anabolic steroid in group C. The Table presents their age and operation performed. Exclusion criteria were: malignant tumours, diabetes, arterial hypertension, and thromboembolic disease within the last 5 years. Informed consent was obtained from all patients entering this study, which was approved by the local ethical committee. The preoperative treatment with nandrolone or placebo was blind to all concerned until the end of the trial.
M. Szczepahski, T. Duchihski, P. Kretowicz,
Laboratory of Hemostasis, Department of Biochemistry and Department of Obstetrics and Gynecology, Medical Center of Postgraduate Education, Warsaw, Poland. 350
Fibrinolysis Table Clinical features of patients
Groups of patients
Patients Age (mean_+ SD) Surgery: total abdominal hysterectomy (t.a.h.) t.a.h, plus bilateral salpingo-oophorectomy t.a.h, plus unilateral salpingo-oophorectomy myomectomy salpingo-oophorectomy oophorectomy ovarian cystectomy salpingectomy splenectomy
Placebo (A)
Nandrolone (B) 50mg (C) 100rag
I0 38.6+7.8
l0 41.5_+13.2
10 44.0_+8.7
2
0
1
3
6
5
1
1
2
0 2 l 0 1 0
0 0 0 2 0 1
1 0 0 1 0 0
The patient who underwent a splenectomy was preoperatively diagnosed as having a benign tumour of the left ovary, but an unusually mobile, floating spleen was found during surgery and a splenectomy was performed. All patients had blood samples taken on the day prior to surgery, and on the first, third, and seventh days after surgery. Blood samples were collected between 07:00 and 08:00 from a cubital vein with minimal venous occlusion and anticoagulated with 0.13 M trisodium citrate (9:1, v:v), centrifuged at 2500 g for 15 rain at 4°C, and separated plasma samples were kept at -20°C until determinations. In order to avoid the possible influence of blood platelets, the plasma specimens for PAI activity were obtained from the uppermost layer of centrifuged samples. Fibrinogen Concentration in plasma was determined by a colorimetric method. Is Plasminogen concentration in plasma was measured by an amidolytic method, using the commercial kit (Berichrom Plasminogen).
g/t
A
351
PAl activity was estimated with a synthetic chromogenic substrate, using the commercial kit (Berichrom PAI). Fibrinolytic activity in euglobulins was assayed basically according to Kowalski et a119 with some modifications. 1 ml of plasma was diluted with 7.5 ml of distilled water, acidified with 1% acetic acid to pH 5.8, and left for 30 rain at 4°C for complete precipitation. The euglobulin sediment was separated by centrifugation at 2500g for 15 rain at 4°C and dissolved in 0.25 ml of 0.22M borate buffer, pH 7.6. The solubilized euglobulins were placed as 30 ~tl drops on the 1 mm thick fibrin plates and incubated for 22h at 37°C. The fibrin plates were prepared from 0.2% human fibrinogen (Behring) dissolved in 25 mM sodium barbital buffer of pH 7.8, containing 57 mM sodium chloride and 0.58 mM calcium chloride. 2° The fibrinolytic activity in euglobulins was expressed in mm 2, as a product of two perpendicular diameters of corresponding circular zones of fibrin plate lysis. Student's t-test for paired data was used for comparisons between the results obtained on consecutive days of determinations within groups. The differences between the results were regarded as significant on the probability level of P < 0.02.
RESULTS The fibrinogen concentration in group A patients rose immediately after surgery and was significantly increased already on the first postoperative day; this increase was most pronounced on the third postoperative day. The administration of nandrolone resulted in a delay in the rise of fibrinogen and in both groups, B as well as C, its concentration increased not earlier than between the first and third day after surgery (Fig. 1). The differences in fibrinogen level disappeared then among the groups of patients, starting from the third postoperative day.
B
C
5-
.
3'
-~b ~ ~ Fig. 1 Fibrinogen concentration in patients of group A (placebo), B (nandrolone, 50 mg), and C (nandrolone, 100 mg). Ordinate: concentration of fibrinogen in g/1. Abscissa: days before and after surgery. Values are means+SD. I~*l : difference significant on the probability level of P<0.02.
352
Single Dose of Nandrolone Does Not Prevent Postoperative Fibrinolytic Shutdown
%
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Fig. 2 Plasminogen concentration. Ordinate: percentage of plasminogen concentration; the concentration of plasminogen in reference normal plasma (Behring) was assigned as 100%. Other details as in Figure 1.
The plasminogen concentration fell immediately after the operation in all groups of patients. This concentration rose again in group A between the third and seventh postoperative day but this rise was observed already between the first and third, and continued until the seventh day in patients of B and C groups (Fig. 2). The activity of PAI increased in the patients of group A on the first postoperative day and the second increase of this activity was observed in them between the third and seventh day. The early postoperative rise of PAI activity was not seen in patients injected preoperatively with 50 mg of nandrolone. The significant decline of this activity was noted between the first and third postoperative day in patients injected prior to surgery with 100 mg of this anabolic hormone. The increase of PAI activity between the third and seventh postoperative day was found in the patients of all groups, placebo as well as nandrolone-treated (Fig. 3). The fibrinolytic activity in euglobulins followed the same pattern in patients of all groups: the decline on the first postoperative day and absence of any other significant changes until the end of determination (Fig. 4).
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A
Increased oozing at the beginning of operation was observed in five patients of group C but the postoperative course was not complicated by a wound hematoma in any of them; the only case with such a hematoma and prolonged wound healing occurred in a group A. Side-effects were observed in one patient 10 min after the i.m. injection of 100rag of nandrolone. They consisted of a hot flush and generalized spotted rash; both of these symptoms subsided within one hour.
DISCUSSION The fibrinogen concentration in plasma of patients in the control group rose immediately after the operation and was greatest on the third postoperative day. This pattern of fibrinogen response to surgical stress has been reported by others. 4's'16'21 The fibrinogen molecules are synthesized in and released from hepatocytes and it is now believed that fibrinogen response to surgery is mediated by hepatocyte-stimulating factor identical to interleukin-6. 22,23
B
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Fig. 3 PAl activity. Ordinate: PAl activity in urokinase-inhibiting units/ml. Other details as in Figure 1.
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The preoperative injection of 50mg or 100mg of nandrolone in our patients resulted in the delayed postoperative increase of fibrinogen concentration. Contrary to the subjects of the control group, the nandrolone-treated patients did not reveal a significant rise of fibrinogen on the first postoperative day but the concentration of this protein in their plasma reached the values of control group patients after the next 2 days. These findings are different from the data of Blamey et a112 and of Preston et al, ~3 who did not observe any difference in postoperative fibrinogen concentration between the controls and stanozololtreated patients. Psuja et a116 injected surgical patients with 50 mg of nandrolone before operation and also observed significant increase of fibrinogen level as soon as the first postoperative day. We are inclined to explain the delay in the postoperative rise of fibrinogen in our nandrolone-treated patients by the interference between this anabolic steroid and interleukin-6, mediating the fibrinogen response to surgery. The plasminogen concentration in the plasma of our patients in all three groups was significantly diminished on the first postoperative day. Berridge et al]° observed a decrease of plasminogen two days after vascular surgery, Mellbring et al 21 noted this phenomenon one to three hours after abdominal surgery, and Paramo et a124 encountered the decline of plasminogen level even during cardiopulmonary bypass surgery. This decline in plasminogen concentration in plasma can be considered as resulting from an intraoperative activation of the fibrinolytic system and the consumption of this zymogen. We have observed the secondary increase of plasminogen level between the third and seventh postoperative day and a similar rise was observed by some authors 1121 ' but not by others. ]°'13"16 The rise of plasminogen level in subjects receiving stanozolol was noted by some authors: peroral administration of this drug, 10 mg 24h, resulted in an elevated plasminogen level after three weeks 25
or one month 26 of this treatment; Small e t a127 injected healthy adults i.m. with 50rag of stanozolol and an increased concentration of plasminogen was found two days later. In our investigation, patients injected with 50 mg or 100mg of nandrolone prior to surgery showed an accelerated postoperative rise of plasminogen level compared with control group patients, and this is in agreement with the observations of Berridge et al 1° and of Blamey et al ]1 but not with those of Psuja et al. 16 It seems evident, therefore, that preoperative administration of a single dose of anabolic steroid does not prevent the intraoperative consumption of the fibrinolytic zymogen, plasminogen, but stimulates its postoperative compensatory rise. We have observed a significant increase in PAI activity on the first postoperative day in the patients of the control group and a similar response of PAI to surgery was reported by others. 2 - 6 8' 2'4 The rise of PAI was accompanied by a decline in the fibrinolytic activity in the euglobulin fraction. This activity was regarded in the past as expressing the plasminogen activator activity 12'27-29 but it later became evident that PAI is also partially precipitated together with plasminogen activator in the euglobulin fraction. 3°'31 It is now believed that the fibrinolytic activity in euglobulins is strongly influenced by both t-PA and PAI, 32'33 and, according to Kluft, 34 a net effect of t-PA and its inhibitor is recorded in the euglobulin fraction. The significant decline in the fibrinolytic activity of this fraction in the patients of our control group, corresponding to the postoperative fibrinolytic shutdown, could eventually be explained by the concomitant rise of PAI activity. However, a similar shutdown observed in nandrolone-treated patients was not accompanied by an increase of PAI activity. This increase, observed immediately after the operation in placebo-receiving patients, was abolished by the preoperative injection of 50 mg of nandrolone; moreover, the 100 mg dose of this drug resulted in a significant
354
Single Dose of Nandrolone Does Not Prevent Postoperative Fibrinolytic Shutdown
d e c l i n e in PAI a c t i v i t y b e t w e e n the first and third p o s t o p e r a t i v e day. T h e a c t i v i t y o f PAI in s u r g i c a l patients, p r e t r e a t e d w i t h a n a b o l i c steroids, has not b e e n d e t e r m i n e d by o t h e r s so far, and the d a t a on f i b r i n o l y t i c a c t i v i t y in e u g l o b u l i n s in t h e s e p a t i e n t s are d i s c r e p a n t . B l a m e y et a111'12 n o t e d a s i g n i f i c a n t l y i n c r e a s e d a c t i v i t y in e u g l o b u l i n s on the first p o s t o p e r a t i v e day in p a t i e n t s i n j e c t e d p r i o r to s u r g e r y w i t h 50 m g o f s t a n o z o l o l , as c o m p a r e d w i t h u n t r e a t e d p a t i e n t s ; t h e y e x p l a i n e d this f i n d i n g by the rise in p l a s m i n o g e n a c t i v a t o r activity. S i m i l a r o b s e r v a t i o n s w e r e m a d e by P r e s t o n et al ~3 in s u r g i c a l p a t i e n t s p r e o p e r a t i v e l y t r e a t e d w i t h 75 m g o f s t a n o z o l o l . B e r r i d g e et al ~° w e r e n o t a b l e to s u p p r e s s the e a r l y f i b r i n o l y t i c s h u t d o w n in p a t i e n t s u n d e r g o i n g f e m o r o p o p l i t e a l b y p a s s by the p r e o p e r a t i v e i.m. i n j e c tion o f 5 0 m g o f s t a n o z o l o l , f o l l o w e d by a p e r o r a l d o s e o f 10 m g o f this d r u g p e r 24 hours. O n the o t h e r hand, P s u j a et a115'16 p r e v e n t e d the p o s t o p e r a t i v e d e c l i n e in f i b r i n o l y t i c a c t i v i t y by a s i n g l e i.m. i n j e c tion o f n a n d r o l o n e p r i o r to surgery. W e h a v e o b s e r v e d an e a r l y p o s t o p e r a t i v e i n h i b i t i o n o f P A I a c t i v i t y by this a n a b o l i c s t e r o i d but this w a s not r e f l e c t e d b y any c h a n g e s in c o n c o m i t a n t f i b r i n o l y t i c s h u t d o w n . W e are i n c l i n e d to s p e c u l a t e , t h e r e f o r e , that the f i b r i n o l y t i c s h u t d o w n o c c u r r i n g a f t e r s u r g e r y is i n f l u e n c e d by the d e c r e a s e o f t - P A a c t i v i t y r a t h e r than by the i n c r e a s e o f PAI activity.
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Fibrinolysis 355 31. DeMaat MPM, Kluft C, de Boer K, Knot E A R, Jie A F H. Acid treatment of plasma for the inactivation of plasminogen activator inhibitor-1 (PAl-1). Thromb Res 1988; 52: 425-430. 32. Urano T, Sahakibara K, Rydzewski A, Urano S, Takada Y, Takada A. Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and
Received: 15 November 1994 Accepted after revision: I 1 August 1995 Corresponding author: Maciej Szczepafiski, Laboratory of Hemostasis, Postgraduate Teaching Hospital, ul.Czerniakowska 23 I, 00-416 Warsaw, Poland. Tel: 34.12.28, Fax: 34.04.70.
plasminogen activator inhibitor I. Thromb Haemostas 1990; 63: 82-86. 33. Urano T, Sumiyoshi K, Pietraszek M H, Takada Y, Takada A. PAI-I plays an important role in the expression of t-PA activity in the euglobulin clot lysis by controlling the concentration of free t-PA. Thromb Haemostas 1991 ; 66: 474478. 34. Kluft C. Personal communication.