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A comparative study of the influence of smoking on skin flap survival in pregnant rats
British Journal of Plastic Surgery (2005) 58, 812–816
A comparative study of the influence of smoking on skin flap survival in pregnant rats Zekeriya Tosuna,*, Uldis Biteb a
Division of Plastic Surgery, Meram Medical Faculty, Selcuk University, Konya, Turkey Division of Plastic Surgery, Mayo Clinic, Rochester, MN, USA
b
Received 15 July 2004; accepted 21 March 2005
KEYWORDS Flap viability; Pregnancy; Smoking
Summary In this study, the effects of smoking on skin flap survival were evaluated. For purposes of comparison 12 female rats were divided into four groups. Group 1 comprised nonsmoker, nonpregnant rats; group 2 nonsmoker, pregnant rats; group 3 smoker, nonpregnant rats and group 4 smoker pregnant. On each subject a standard 4!10 cm McFarlane dorsal skin flap was raised. On the seventh day postoperatively, the flaps were traced on a transparent sheet marking off the necrotic areas. The area of skin flap necrosis was assessed by ‘netcad’ digital imaging analysis program. The area of skin flap viability in the pregnant groups was greater than in the nonpregnant groups. The nonsmoker pregnant group’s flap viability was the highest and the smoker nonpregnant group’s viability was the lowest. This result supports the conclusion that pregnancy enhances flap viability and produces some defensive factors against some of the adverse effects of smoking. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
It has been demonstrated that smoking can cause a decrease in skin flap viability in pregnant and nonpregnant patients.1–3 While this knowledge is shared by many investigators there is no study of flap viability in pregnancy in the plastic surgery literature. Although it is well known that nicotine has a deleterious effect on skin flap survival, the
effects of pregnancy to reduce or negate these effects is not yet known. The purpose of this study was to observe and compare the effects of smoking and pregnancy on pregnant and nonpregnant skin flap viability in rats.
Materials and methods * Corresponding author. Address: Selcuk Universitesi Meram TIP Fakultesi, Plastik ve Rekonstruktif Cerrahi Klinigi, 42080 Konya, Turkey. Tel.: C90 332 223 60 73; fax: C90 332 223 61 81. E-mail addresses: [email protected] (Z. Tosun), ztosun@ selcuk.edu.tr (Z. Tosun).
After the approval of the animal care committee 48 Wistar female rats weighing from 240 to 270 g were used and divided into four equal groups. The rats were housed in individual cages and were exposed
S0007-1226/$ - see front matter q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2005.03.004
A comparative study of the influence of smoking on skin flap survival in pregnant rats
813
814 to 12 h light/dark cycles and offered a rat diet and water ad libitum. The rats were between 16 and 20 weeks of age. Pregnant rats were included in the study 1 week after coitus, in order to obtain haematological, hormonal and metabolic standardisation. All the rats were anaesthetised with intraperitoneal 10 mg/kg ketamin and 5 mg/kg xylazine. Their backs were shaved and cleaned with povidone iodine. A 4!10 cm caudal based standard McFarlane dorsal skin flap was raised.4 The flaps were replaced in their beds and sutured with 4/0 silk interrupted sutures. Nonsmoker groups: group 1 comprised 12 nonsmoker, nonpregnant rats; group 2, 12 nonsmoker, pregnant rats.
Smoker groups: group 3 comprised 12 smoker, nonpregnant rats; group 4, 12 smoker, pregnant rats. We exposed groups 3 and 4 to the acute smoking model in a Maddox smoking chamber, 5 days before and 7 days after surgery.5 We used 30 ml puffs per cigarette with 8 cigarette exposures per 8 h day. The area of skin flap necrosis was assessed at the seventh day postoperatively. Blood was taken for assessment of haemoglobin levels 1 h after cigarette exposure and then the animals were euthanised with an overdose of ketamine. Each flap was traced on a transparent sheet at the seventh day postoperatively, marking off the necrotic areas. These film membranes were scanned and the area of skin flap necrosis was assessed by the ‘netcad’ digital imaging analysis program (Fig. 1(A)–(D)).6 No prophylactic antibiotics were used. However, rats that did not survive the entire 7 days were eliminated from this study. New rats replaced the ones lost to the study. In group 1 (nonsmoker nonpregnant) one rat was lost; one was lost from group 2 (nonsmoker pregnant), two were lost from group 3 (smoker nonpregnant). No rats were lost from group 4 (smoker pregnant). Flaps were evaluated on the seventh day postoperatively. In order to assess the effects of both the pregnancy and smoking on flap viability univariate analysis of variance two-way ANOVA test was used. Haemoglobin values were used as covariant for standardisation.
Z. Tosun, U. Bite
Results Nonsmoker group: both (12 nonpregnant and 12 pregnant) groups were not exposed to smoke. The rats in groups 1 and 2 had smaller necrosed areas so flap viability was greater. In group 2 (nonsmoker, pregnant) flap viability was the highest. Smoker groups: skin flap survival decreased in both smoker groups (12 nonpregnant and 12 pregnant rats) compared with the nonsmoker groups. The nonpregnant smoker group’s viability was the lowest having the largest necrosed areas. It appears that pregnancy and smoking either individually or alone affects flap viability (smoking F: 20.94 P: 0.000) (pregnancy F: 10.18 P: 0.003). After assessment of haemoglobin values there were no differences among the groups except between the groups 1 and 4 (F: 3.103 P: 0.036). There was no correlation between the haemoglobin values and necrosed areas (r: 0.156 P: 0.290). Mean necrosis level and standard deviation, haemoglobin (Hb) values and standard deviation of all the groups are seen in Table 1.
Discussion There is insufficient information in the plastic surgery literature about flap viability and the effects of smoking on pregnancy. Although elective surgery can be delayed in pregnancy, emergency surgery is an obligation. In this situation survival of the flap cannot be estimated due to lack of studies on this topic. What are the of effects of smoking on blood vessels? Zimmerman and Mc-Geachie7 have shown that nicotine exposed endothelium had greater cytoplasmic vacuolation, mitocondrial swelling and, subendothelial oedema than control endothelium and is also more permeable. Nicotine has no detrimental effects on the intimal layer of arteries but might lead to fibrosis and calcification in the media.8 In our study we raised acute random skin flaps that contained no major arteries. We, therefore, did not study changes in large vessel morphology caused by nicotine administration. However, Forrest et al.9 has shown that nicotine can significantly decrease capillary blood flow, distal perfusion and flap survival in rats.
Figure 1 (A) A sample for postoperative seventh day result of nonsmoker nonpregnant group. (B) A sample for postoperative seventh day result of smoker nonpregnant rats. (C) A sample for postoperative seventh day result of smoker pregnant rats. (D) A sample for postoperative seventh day result of nonsmoker pregnant rats.
A comparative study of the influence of smoking on skin flap survival in pregnant rats
815
Table 1 Mean necrosis level and standard deviation, haemoglobin (Hb) values and standard deviation of all the groups (meanGSD) Groups
Necrosis
Hb
1. Nonsmoker nonpregnant 2. Nonsmoker pregnant 3. Nonpregnant smoker 4. Pregnant smoker Total
7.63G1.98 6.00G1.71 10.59G2.33a 8.06G1.48 8.07G2.48
16.10G2.18 14.27G1.95 15.36G1.87 13.84G2.03b 14.89G2.15
a b
Statistically significant result among the all the other groups (P!0.05). Statistically significant result between group 1 (P!0.05). According to Tukey HSD test after ANOVA.
It is well known that nicotine causes cutaneous vasoconstriction.10 Sarin et al.11 showed a reduction in human digital blood flow after smoking one cigarette. Forrest et al. showed that nicotine can significantly decrease capillary blood flow, distal perfusion and acute random pattern skin flap survival in the rat.9 Smoking is known to produce cutaneous vasoconstriction. This has been demonstrated by skin temperature measurements, the plethysmograph and observation of capillary beds. Furthermore, during the smoking a decrease in digital blood flow has also been observed.1 Smoking has been known to cause intravascular thrombosis and acutely increase platelet aggregation.12 Dintenfass reported that in smokers, levels of fibrinogen, haemoglobin and blood viscosity are increased.13 Nicotine also leads to direct endothelial cell damage. Nicotine affects prostaglandin and thromboxane synthesis and/or release.10 It was thought that loss of rats in our study was due to individual factors. However, differences between the groups in the number of rats lost does not affect the result. Interestingly, we lost no rats in group 4 (smoker pregnant). Some pregnancy factors that we do not know about may have had a critical role. The mechanism of increased flap survival in nonsmoker pregnant rats has not been explained clearly. However, there are some studies on peripheral blood flow in pregnancy. A review of plasma volume measurements showed an increase of 42%, or approximately 1100 cm3, compared to nonpregnant controls in a human study.14 We also know that a decrease in viscosity of a fluid in a vessel improves the flow.15 Gati et al.16 showed that anaemia or haemodilution in animals leads to increased flap survival compared to normal and polycythemic controls. Nevertheless anaemia and haemodilution are not enough to explain enhanced flap survival in pregnancy. Our haemoglobin values were normal and there were no anaemic and polycythemic rats.
In this study, a statistically significant increase in random skin flap survival was demonstrated both in the nonsmoking and smoking groups and similarly between pregnant and nonpregnant groups. Some unknown factors in pregnancy have positive effects on flap survival. These factors may also have a defensive role against the effects of smoking. Further experimental studies are necessary for the explanation of these factors.
References 1. Lawrence WT, Murphy RC, Robson MC, Hegger JP. The detrimental effect of cigarette smoking on flap survival; an experimental study in the rat. Br J Plast Surg 1984;37:216–9. 2. Kaufman T, Eichenlaub EH, Levin M, Hurwitz DJ, Klain M. Tobacco smoking: impairment of experimental flap survival. Ann Plast Surg 1984;13:468–72. 3. Bailet JW, Hoffman LF, Trachy RE, Weymuller EA. The effect of nifedipine on skin flap survival in rats. Laryngoscope 1994; 104:253–8. 4. McFarlane RM, DeYoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177. 5. Maddox WL, Creasia DA, Dalbey WE, Guerin WE, Stokely JR, Kendrick J. A tobacco smoke inhalation exposure device for rodents. Arch Environ Health 1978;33:64–71. 6. Dursun S, Ozdemir C, Karabork H, Kocak S. Noise pollution map of Konya city centre and effect of factors on noise pollution, international conference on the environmental problems of the Mediterranean region, Cyprus; 2002. 7. Zimmerman M, McGeachie J. The effect of nicotine on aortic endothelium: a quantitative ultrastructural study. Atherosclerosis 1987;63:33–41. 8. Forrest CR, Pang CY, Lindsay WK. Pathogenesis of ischemic necrosis in random-pattern skin flaps induced by long-term low-dose nicotine treatment in the rat. Plast Reconstr Surg 1991;87:518–28. 9. Forrest CR, Pang CY, Lindsay WK. Dose and time effects of nicotine treatment on the capillary blood flow and viability of random pattern skin flaps in the rat. Br J Plast Surg 1987; 40:295–9. 10. Reus WF, Robson MC, Zachary L, Heggers JP. Acute effects of tobacco smoking on blood flow in the cutaneous microcirculation. Br J Plast Surg 1984;37:213–5. 11. Sarin CL, Austin JC, Nickel WO. Effects of smoking on digital blood-flow velocity. JAMA 1974;229:1327–8.
816 12. Davis JW, Davis RF. Acute effect of tobacco cigarette smoking on the platelet aggregate ratio. Am J Med Sci 1979;278:139. 13. Dintenfass L. Elevation of blood viscosity, aggregation of red cells, haematocrit values, and fibrinogen levels in cigarette smokers. Med J Aust 1975;17:617–20. 14. Cabaniss CD, Cabaniss ML. In: Kitay DZ, editor. Physiologic hematology of pregnancy in hematologic problems in pregnancy. New Jersey: Medical Economic Books; 1987. p. 3–14.
Z. Tosun, U. Bite 15. Guyton AC. Overview of the circulation; medical physics of pressure, flow and resistance. 9th ed. Textbook of medical physiology. Philadelphia, PA: Saunders; 1996 p. 161–9. 16. Gati I, Nagy I, Szekey JA, Than E, Bognar J, Panka J. Determination of steroid elimination and blood coagulation during normal and toxemic pregnancy. Zentralbl Gynakol 1969;24:777–82.
A comparative study of the influence of smoking on skin flap survival in pregnant rats Zekeriya Tosuna,*, Uldis Biteb a
Division of Plastic Surgery, Meram Medical Faculty, Selcuk University, Konya, Turkey Division of Plastic Surgery, Mayo Clinic, Rochester, MN, USA
b
Received 15 July 2004; accepted 21 March 2005
KEYWORDS Flap viability; Pregnancy; Smoking
Summary In this study, the effects of smoking on skin flap survival were evaluated. For purposes of comparison 12 female rats were divided into four groups. Group 1 comprised nonsmoker, nonpregnant rats; group 2 nonsmoker, pregnant rats; group 3 smoker, nonpregnant rats and group 4 smoker pregnant. On each subject a standard 4!10 cm McFarlane dorsal skin flap was raised. On the seventh day postoperatively, the flaps were traced on a transparent sheet marking off the necrotic areas. The area of skin flap necrosis was assessed by ‘netcad’ digital imaging analysis program. The area of skin flap viability in the pregnant groups was greater than in the nonpregnant groups. The nonsmoker pregnant group’s flap viability was the highest and the smoker nonpregnant group’s viability was the lowest. This result supports the conclusion that pregnancy enhances flap viability and produces some defensive factors against some of the adverse effects of smoking. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
It has been demonstrated that smoking can cause a decrease in skin flap viability in pregnant and nonpregnant patients.1–3 While this knowledge is shared by many investigators there is no study of flap viability in pregnancy in the plastic surgery literature. Although it is well known that nicotine has a deleterious effect on skin flap survival, the
effects of pregnancy to reduce or negate these effects is not yet known. The purpose of this study was to observe and compare the effects of smoking and pregnancy on pregnant and nonpregnant skin flap viability in rats.
Materials and methods * Corresponding author. Address: Selcuk Universitesi Meram TIP Fakultesi, Plastik ve Rekonstruktif Cerrahi Klinigi, 42080 Konya, Turkey. Tel.: C90 332 223 60 73; fax: C90 332 223 61 81. E-mail addresses: [email protected] (Z. Tosun), ztosun@ selcuk.edu.tr (Z. Tosun).
After the approval of the animal care committee 48 Wistar female rats weighing from 240 to 270 g were used and divided into four equal groups. The rats were housed in individual cages and were exposed
S0007-1226/$ - see front matter q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2005.03.004
A comparative study of the influence of smoking on skin flap survival in pregnant rats
813
814 to 12 h light/dark cycles and offered a rat diet and water ad libitum. The rats were between 16 and 20 weeks of age. Pregnant rats were included in the study 1 week after coitus, in order to obtain haematological, hormonal and metabolic standardisation. All the rats were anaesthetised with intraperitoneal 10 mg/kg ketamin and 5 mg/kg xylazine. Their backs were shaved and cleaned with povidone iodine. A 4!10 cm caudal based standard McFarlane dorsal skin flap was raised.4 The flaps were replaced in their beds and sutured with 4/0 silk interrupted sutures. Nonsmoker groups: group 1 comprised 12 nonsmoker, nonpregnant rats; group 2, 12 nonsmoker, pregnant rats.
Smoker groups: group 3 comprised 12 smoker, nonpregnant rats; group 4, 12 smoker, pregnant rats. We exposed groups 3 and 4 to the acute smoking model in a Maddox smoking chamber, 5 days before and 7 days after surgery.5 We used 30 ml puffs per cigarette with 8 cigarette exposures per 8 h day. The area of skin flap necrosis was assessed at the seventh day postoperatively. Blood was taken for assessment of haemoglobin levels 1 h after cigarette exposure and then the animals were euthanised with an overdose of ketamine. Each flap was traced on a transparent sheet at the seventh day postoperatively, marking off the necrotic areas. These film membranes were scanned and the area of skin flap necrosis was assessed by the ‘netcad’ digital imaging analysis program (Fig. 1(A)–(D)).6 No prophylactic antibiotics were used. However, rats that did not survive the entire 7 days were eliminated from this study. New rats replaced the ones lost to the study. In group 1 (nonsmoker nonpregnant) one rat was lost; one was lost from group 2 (nonsmoker pregnant), two were lost from group 3 (smoker nonpregnant). No rats were lost from group 4 (smoker pregnant). Flaps were evaluated on the seventh day postoperatively. In order to assess the effects of both the pregnancy and smoking on flap viability univariate analysis of variance two-way ANOVA test was used. Haemoglobin values were used as covariant for standardisation.
Z. Tosun, U. Bite
Results Nonsmoker group: both (12 nonpregnant and 12 pregnant) groups were not exposed to smoke. The rats in groups 1 and 2 had smaller necrosed areas so flap viability was greater. In group 2 (nonsmoker, pregnant) flap viability was the highest. Smoker groups: skin flap survival decreased in both smoker groups (12 nonpregnant and 12 pregnant rats) compared with the nonsmoker groups. The nonpregnant smoker group’s viability was the lowest having the largest necrosed areas. It appears that pregnancy and smoking either individually or alone affects flap viability (smoking F: 20.94 P: 0.000) (pregnancy F: 10.18 P: 0.003). After assessment of haemoglobin values there were no differences among the groups except between the groups 1 and 4 (F: 3.103 P: 0.036). There was no correlation between the haemoglobin values and necrosed areas (r: 0.156 P: 0.290). Mean necrosis level and standard deviation, haemoglobin (Hb) values and standard deviation of all the groups are seen in Table 1.
Discussion There is insufficient information in the plastic surgery literature about flap viability and the effects of smoking on pregnancy. Although elective surgery can be delayed in pregnancy, emergency surgery is an obligation. In this situation survival of the flap cannot be estimated due to lack of studies on this topic. What are the of effects of smoking on blood vessels? Zimmerman and Mc-Geachie7 have shown that nicotine exposed endothelium had greater cytoplasmic vacuolation, mitocondrial swelling and, subendothelial oedema than control endothelium and is also more permeable. Nicotine has no detrimental effects on the intimal layer of arteries but might lead to fibrosis and calcification in the media.8 In our study we raised acute random skin flaps that contained no major arteries. We, therefore, did not study changes in large vessel morphology caused by nicotine administration. However, Forrest et al.9 has shown that nicotine can significantly decrease capillary blood flow, distal perfusion and flap survival in rats.
Figure 1 (A) A sample for postoperative seventh day result of nonsmoker nonpregnant group. (B) A sample for postoperative seventh day result of smoker nonpregnant rats. (C) A sample for postoperative seventh day result of smoker pregnant rats. (D) A sample for postoperative seventh day result of nonsmoker pregnant rats.
A comparative study of the influence of smoking on skin flap survival in pregnant rats
815
Table 1 Mean necrosis level and standard deviation, haemoglobin (Hb) values and standard deviation of all the groups (meanGSD) Groups
Necrosis
Hb
1. Nonsmoker nonpregnant 2. Nonsmoker pregnant 3. Nonpregnant smoker 4. Pregnant smoker Total
7.63G1.98 6.00G1.71 10.59G2.33a 8.06G1.48 8.07G2.48
16.10G2.18 14.27G1.95 15.36G1.87 13.84G2.03b 14.89G2.15
a b
Statistically significant result among the all the other groups (P!0.05). Statistically significant result between group 1 (P!0.05). According to Tukey HSD test after ANOVA.
It is well known that nicotine causes cutaneous vasoconstriction.10 Sarin et al.11 showed a reduction in human digital blood flow after smoking one cigarette. Forrest et al. showed that nicotine can significantly decrease capillary blood flow, distal perfusion and acute random pattern skin flap survival in the rat.9 Smoking is known to produce cutaneous vasoconstriction. This has been demonstrated by skin temperature measurements, the plethysmograph and observation of capillary beds. Furthermore, during the smoking a decrease in digital blood flow has also been observed.1 Smoking has been known to cause intravascular thrombosis and acutely increase platelet aggregation.12 Dintenfass reported that in smokers, levels of fibrinogen, haemoglobin and blood viscosity are increased.13 Nicotine also leads to direct endothelial cell damage. Nicotine affects prostaglandin and thromboxane synthesis and/or release.10 It was thought that loss of rats in our study was due to individual factors. However, differences between the groups in the number of rats lost does not affect the result. Interestingly, we lost no rats in group 4 (smoker pregnant). Some pregnancy factors that we do not know about may have had a critical role. The mechanism of increased flap survival in nonsmoker pregnant rats has not been explained clearly. However, there are some studies on peripheral blood flow in pregnancy. A review of plasma volume measurements showed an increase of 42%, or approximately 1100 cm3, compared to nonpregnant controls in a human study.14 We also know that a decrease in viscosity of a fluid in a vessel improves the flow.15 Gati et al.16 showed that anaemia or haemodilution in animals leads to increased flap survival compared to normal and polycythemic controls. Nevertheless anaemia and haemodilution are not enough to explain enhanced flap survival in pregnancy. Our haemoglobin values were normal and there were no anaemic and polycythemic rats.
In this study, a statistically significant increase in random skin flap survival was demonstrated both in the nonsmoking and smoking groups and similarly between pregnant and nonpregnant groups. Some unknown factors in pregnancy have positive effects on flap survival. These factors may also have a defensive role against the effects of smoking. Further experimental studies are necessary for the explanation of these factors.
References 1. Lawrence WT, Murphy RC, Robson MC, Hegger JP. The detrimental effect of cigarette smoking on flap survival; an experimental study in the rat. Br J Plast Surg 1984;37:216–9. 2. Kaufman T, Eichenlaub EH, Levin M, Hurwitz DJ, Klain M. Tobacco smoking: impairment of experimental flap survival. Ann Plast Surg 1984;13:468–72. 3. Bailet JW, Hoffman LF, Trachy RE, Weymuller EA. The effect of nifedipine on skin flap survival in rats. Laryngoscope 1994; 104:253–8. 4. McFarlane RM, DeYoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177. 5. Maddox WL, Creasia DA, Dalbey WE, Guerin WE, Stokely JR, Kendrick J. A tobacco smoke inhalation exposure device for rodents. Arch Environ Health 1978;33:64–71. 6. Dursun S, Ozdemir C, Karabork H, Kocak S. Noise pollution map of Konya city centre and effect of factors on noise pollution, international conference on the environmental problems of the Mediterranean region, Cyprus; 2002. 7. Zimmerman M, McGeachie J. The effect of nicotine on aortic endothelium: a quantitative ultrastructural study. Atherosclerosis 1987;63:33–41. 8. Forrest CR, Pang CY, Lindsay WK. Pathogenesis of ischemic necrosis in random-pattern skin flaps induced by long-term low-dose nicotine treatment in the rat. Plast Reconstr Surg 1991;87:518–28. 9. Forrest CR, Pang CY, Lindsay WK. Dose and time effects of nicotine treatment on the capillary blood flow and viability of random pattern skin flaps in the rat. Br J Plast Surg 1987; 40:295–9. 10. Reus WF, Robson MC, Zachary L, Heggers JP. Acute effects of tobacco smoking on blood flow in the cutaneous microcirculation. Br J Plast Surg 1984;37:213–5. 11. Sarin CL, Austin JC, Nickel WO. Effects of smoking on digital blood-flow velocity. JAMA 1974;229:1327–8.
816 12. Davis JW, Davis RF. Acute effect of tobacco cigarette smoking on the platelet aggregate ratio. Am J Med Sci 1979;278:139. 13. Dintenfass L. Elevation of blood viscosity, aggregation of red cells, haematocrit values, and fibrinogen levels in cigarette smokers. Med J Aust 1975;17:617–20. 14. Cabaniss CD, Cabaniss ML. In: Kitay DZ, editor. Physiologic hematology of pregnancy in hematologic problems in pregnancy. New Jersey: Medical Economic Books; 1987. p. 3–14.
Z. Tosun, U. Bite 15. Guyton AC. Overview of the circulation; medical physics of pressure, flow and resistance. 9th ed. Textbook of medical physiology. Philadelphia, PA: Saunders; 1996 p. 161–9. 16. Gati I, Nagy I, Szekey JA, Than E, Bognar J, Panka J. Determination of steroid elimination and blood coagulation during normal and toxemic pregnancy. Zentralbl Gynakol 1969;24:777–82.