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The effect of acute maternal hemorrhage on the release of catecholamines in the pregnant ewe and the fetus
The effect of acute maternal hemorrhage on the release of catecholamines in the pregnant ewe and the fetus RAUL
ARTAL,
M.D.
THEODORE
H.
ROBERT PETER
LAM, W.
GLATZ,
M.D.
B.Sc.
NATHANIELSZ,
CALVIN
J.
HOBEL,
Los Angeles,
Calijornin
M.D.,
PH.D.
M.D.
Five chronically instrumented pregnant ewes and their fetuses were studied to assess the effect of acute maternal hemorrhage. The hemorrhage was produced in two stages; during the first stage 15% of the total maternal blood volume was removed, and then in a second stage an addltiinal 15% was removed. Biophysical and biochemical variables were monitored. Plasma dopamine, norepinephrine, and epinephrine were determined by a sensitive radioenzymatic assay. A significant rise in maternal and fetal catecholamines was observed following an estimated maternal blood volume depletion of 30%. During a 15minute recovery period, the maternal catecholamine concentrations returned to normal, while the fetal concentrations remained elevated. These changes were accompanied by significant changes in maternal and fetal homeoatasis. The signiftcance of these findings is discussed. (AM. J. OBSTET. GYNECOL. 135:818, 1979.)
HEMORRHAGE IS KNOWN to affect the sympathetic nervous system and the release of catecholamines, which results in significant hemodynamic and metabolic changes. ‘* * The effect of maternal hemorrhage on the circulating fetal catecholamines has not been studied, in part because of numerous technical difficulties in the measurement of catecholamines in small amounts of biologic fluids. These measurements are now possible following the development of sensitive radioenzymatic assays.3. ’ The present study was conducted to assess the effect of acute maternal hemorrhage on maternal and fetal homeostasis. To investigate this condition we utilized the chronically instrumented sheep preparation and a modified exFrom the Departments of Obstetrics and Gynecology and Pediatrics, Harbor-Univwsity of Cal(forniu, Los Angeles Medical Center. Presented at the Twenty-sixth Annual Society for Gynecologic Investigation, California, March 21-24, 1979.
Meeting of the San Drkgo,
Reprint requests: Dr. Raul Artal, Department of Obstetrics and Gynecology, Los Angeles CountylUniverstiy of Southern Calzyorma Medical Center, 1240 N. Mission Road, Los Angeles, California 90033.
018
perimental study design acute hemorrhage.’
developed
in other studies of
Material and methods Care of animals and experimental protocol. Five date-mated pregnant Rambouillet-Columbia ewes and their fetuses were used in this study. The animals were chronically instrumented utilizing previously described techniques.6 Catheters were implanted in the maternal femoral artery and vein and in the fetal carotid artery and internal jugular vein. The animals were allowed to recover for at least 72 hours following surgery before any experimental procedure was performed. Maternal and fetal heart rates were monitored continuously. Fetal and maternal arterial blood gases and base excess were measured on a Radiometer ABL2 blood gas analyzer. PO, and Pco, are expressed throughout as millimeters of mercury and base excess as milliequivalent per liter. All the studies were performed on animals that were stable at the time of experiment and had a fetal arterial pH greater than 7.33 and a Pop greater’ than 18 mm Hg. The gestational age at the time of experiment was 128 to 134 days. 0002-9378/79/220818+05$00.50/0~
1979
The
C. V.
Masby Co.
Volume
Number
1% 6
The experimental procedure protocol was as follows: Simultaneous maternal and fetal arterial blood samples were obtained for hormone determinations at the beginning of the study; biophysical variables were monitored during a control period of 15 minutes. At the end of the control period the first phlebotOmy was performed during which 15% of the total estimated maternal blood volume (12.5 ml of blood/kg of body weight) was removed over approximately 5 minutes. Fifteen minutes later a second phlebotomy was performed to remove an additional 15% of the total estimated maternal blood volume. Additional simultaneous maternal and fetal blood samples were obtained for hormone determinations. At ‘this point, the blood that had been removed was returned to the ewe. The experiment was concluded by a 15-minute recovery period. after which the last simultaneous maternal and fetal blood samples were drawn. pH, PO,, and Pcop were measured in all samples and were within the range previously reported.6 Special precautions were taken to avoid any degradation of catecholamines during sampling. The blood samples for catecholamine determination were collected in tubes containing a stabilizing solution of 90 mg/ml of ethyleneguanosinetetraacetic acid and 60 mg/ml of glutathione. The plasma was separated immediately at 4” C in a refrigerated centrifuge. Samples were stored at -80” C until measured. All estimations were performed within 12 weeks. Dopamine, norepinephrine, and epinephrine were determined by a radioenzymatic assay in duplicates of 50 ~1 of plasma. The within-assay coefficients of variation were 4.8% for dopamine, 4.3% for norepinephrine, and 3.5% for epinephrine; the betweenassay coefficients of variation were 12.OYc for dopamine, 9.7% for norepinephrine, and 8.4% for epinephrine. The radioenzymatic assay included the modification described by Peuler and Johnson3 of adding O.lM benzyloxyamine to the catechol-O-methyltransferase (COMT) preparation to prevent any increase in dopamine levels by the decarboxylase-catalyzed conversion of 3,4-dihydroxyphenylalanine (DOPA) to dopamine. To analyze statistical differences between tbe various stages of the experiment the data were tested by the Rankit test and found to be nongaussian.’ Therefore, each data set was compared by the nonparametric statistic Mann-Whitney U test.
Results Blood respiratory gases and pH. No significant changes were found between the control samples and the first phlebotomy samples. However, significant
Effect
of hemorrhage
on catecholamine
release
819
MATERNALVALUES
&M+2SEM
OCONTROL
-2nd
PHLEBOTOMY
Fig. 1. l-he effect ot‘the second phlebotomy and f’etal respiratory gases and pH. ,o,ooo,---
.-.-.-
._-~
*p
on the maternal
-
-
?
ELE m 95% C.L. - p
CONTROL 1st PHll
Fig. 2. The maternal (El and f’etal (L) dopamine concentrations at the different stages of the experiment. The horizontal lines at the bottom of the figure link two bars to indicate a statistical difference of at least 0.0 1. The bars represent mean and 95%
confidence
limits
(C.L.).
changes in these measurements were obsrl-vcd at the end of the second phlebotomy. These changes are illustrated in Fig. 1. Initial maternal pH was 7.40 t 0.03 (mean t SEM) and fetal pH was 7.33 t 0.01. These values fell following the second phlebotomv to 7.28 t 0.04 and 7.1 1 L 0.05, respectively (p < 0.05 in each instance). The second phlebotomy did not significantly change the maternal Pco, (40 +- 3 to 33 -C 8) but significantly altered the fetal PCO, (48.3 -+ 0.8 to 61.8 + 3.9). Similarly, PO? did not significantly change in the ewe (95 rt 2 to X9 -C 3) with the second phlebolomy but
820
November Am. J. Obstet.
Artal et al.
CONTROL 1st PHLEB -------------------------------------------------------
2nd PHLEB
I
15, 1979 Gynecol.
21,000 .F T ,
RE OVER
== ==
Fig. 3. The maternal (E) and fetal (f.) norepinephrine concentrations at the different stages of the experiment. The continuous horizontal lines at the bottom of the figure link two bars to indicate a statistical difference of at least 0.01. The double dashed lines indicate a statistical difference .of 0.05. The bars represent mean and 95% confidence limits (CL.).
Fig. 4. The maternal (El and fetal (L) epinephrine concentrations at the different stages of the experiment. The continuous horizontal lines at the bottom of the figure link two bars to indicate a statistical difference of at least 0.01. The double dashed lines indicate a statistical difference of 0.05. The bars represent mean and 95% confidence limits (CL.).
was significantly reduced in the fetus (20.8 2 0.9 to 16.5 rt 1.9) (p < 0.05). The second phlebotomy significantly altered the base excess in both ewe (-0.5 t 0.2 to -6 2 2)and fetus (-0.25 ? 0.01 to -7.68 * 1.1) (p < 0.05 in each instance). Catecholamines. Dopumi~. Fig. 2 illustrates the changes in circulating dopamine in both the ewe and the fetus. The mean baseline concentrations were 35 (4 to 300) pg/ml (mean and 95% confidence limits) in the ewe and 52 (33 to 80) pg/ml in the fetus. Following the first phlebotomy, the plasma dopamine concentration rose to 60 (30 to 1,000) pg/ml in the ewe and 49 (11 to 220) pgiml in the fetus. These changes were not significant. Following the second phlebotomy, the plasma dopamine concentration rose significantly in both fetus and ewe. The concentrations rose to 290 (60 to 1,400) pgiml in the ewe (p < 0.01) and to 320 (100 to 1,000) pg/ml in the fetus (p < 0.01). During the recovery period the dopamine concentration remained significantly elevated (p < 0.01) from the control period at 160 (64 to 390) pg/ml in the ewe and 450 (90 to 2,000) pgiml in the fetus Norepiwphrine. The changes in norepinephrine are illustrated in Fig. 3. The norepinephrine concentra-
tions during the control period were 3 10 (120 to 800) pgiml in the ewes and 460 (210 to 1,000) pg/ml in the fetus. No significant changes in the norepinephrine levt:ls were observed following the first phlebotomy and the values were 560 (330 to 940) pg/ml in the ewe and 470 (2 10 to 1,050) pg/ml in the fetus. The second phlebotomy significantly increased the norepinephrine fell to concentrations significantly increased the norepinephrine levels to 3,900 (350 to 40,000) pg/ml (p < 0.01) in the ewe and to 10,000 (2,500 to 42,000) pg/ml (p < 0.05) in the fetus. During the recovery period norepinephrine fell to concentrations significantly different (p < 0.01) from those following the second phlebotomy to 430 (160 to 1,100) pgiml in the ewe and remained significantly elevated (p < 0.05 at 10,000 (2,500 to 35,000) pg/ml in the fetus. Epinephrirre. The changes in epinephrine concentrations are illustrated in Fig. 4. The baseline values determined were 190 (40 to 900) pg/ml of epinephrine in the ewe and 58 (9 to 350) pg/ml of epinephrine in the fetus. In the different stages of the experiment the levels of epinephrine followed a trend similar to that observed with the other catecholamines. No significant statistical differences in epinephrine levels were observed following the first phlebotomy. The mean val-
Volume Number
135 6
ues wet-c: 500 (IO0 to -~.OOO) pgitnl of‘ epinephrine in the ewe and XI (.? to ‘4.X)) pgiml of epinephrine in the fetus. Significant elevations in epinephrint levels were observed at the end of’ the second maternal phlebotomy. The mean epinephrine Irvels obtained were 5.000 (%O to 100.000) pgitnl in the cw and i,.iOO (I ,700 to 3Y.000) pgiml in the fetus. At the end of the recover:, period, the epinephrine levels fell to 430 (1 .il) to 1.L’OO) pgiml in the ewe and to 240 (70 to 840) pgiml in the frtus. Cardiovascular changes. During the first phlebotomy, the mean mater-nal Itcart rate increased without a change itt the blood pwssttre. \vhile in the fetus no changes were observed. tIo\vc\,er, one fetus responded to the first phlehotom~ with brad~carclia, hypotension. and a Pof of. It! tnm Hg. During the second phlebotctm\ the mean matcrn;tI blood pressure and heart rate decreased, while all ~‘CYIISCS cle\rloped bradycardia. hypertension. and tvidenittg of’ the puIse prww-c. Within 5 minutes of this brad!cardia most of. the fetuses had a gradual increase in heart rate.
Comment Experimental hemorrhage in the pregnant ewe can provide ;I useful animal model f’or the study of the complex maternal-fetal interactions that occur at the time of stress. Such a model could help us to understand the role the adrenergic nervows system plays in the adaptation of the fetus f’rom a normal to a stressful condition. Our stud) indicates that significant hemodynamic changes will ha\,e to occttr in the mother hefore they are rcHected in the fetus; a maternal blood volume depletion of‘ 30% was needed to induce significant changes in fetal homeostasis. The hemodynamic changes observed in the ewe are common to acute blood volume depletion. as demonstrated in other anitnal studie\.‘, “. ’ In the fetus the typical haroreceptor responses of bradycardia and hypertension were f’ollowcd by a recovery period during which an increase in heart rate ~i;ts observed. The alterations in the acid-base balance in the ewe were those of part11 compensated subacute metabolic. acidosis. while those
Effect of hemorrhage on catecholamine
release
821
in the fettis were characteristic. of’ ;I state (II ;I( ttte ttlc%tholic. acidosis. The high lwels of I’c:o~ trb~~~ctl at the’ end of the second phlebotomy it! rhc, f’cttt\ :11-c likei\ ;t reflection of’ decreased placental pr’t I’ttsii ~1). The humoral changes obwrvetl C~~thy, c.~~tf of tlrtt second phlebotomv resulted in ;I si,qttiftc;tnt ~xAc;tw 01’ catecholamines in both the ewe and tltc fertrs;. Prcviotta studie? I’ have demottstrated the c.;tpabilit\ of the frt;tI sheep to releasr c~ttec~holatninc~ in wspmw~ to tl if’f’c,rent stimuli, but in none ot’ the sttt(lic.\ h;t\ lhts rc=~pottw of the f’etus been so significant. l.o\~crittg 111~.ntatertl;tl blood volume by :rO’;C rrsultctl in ~111 tighttolcl inc ~‘c.tsc’ in dopamitie, a twel~~rtold inc re;iw it1 rtot~c~l~iitcf~hrirr~. and a 2%f’old increase in rpinc~pltrt~tc* in tl~c. C’IVC’.~I‘ltt~ increments observed in the f’cstus MC’IX‘ zi\ti&l for. dopamine. :! I-fold fat- tiorel)itic~f~~tt.i~~t~, aiitl I .YO-fi)lti Cot, epinephrine. The acute rcleasc oi c’atc’ctrolamittc~s itt both the ewes and their frruscs i\ Grnil,tt- IO the, IVsponse produced hy hemorrhage in attc\tlt~:ti/eci hvpotensivr dogs.“’ Of interest is that during nlic two\~~n period the catecholamine Ir~~ls w~tt~-nt~l IO not trial iti the e~‘e while the fetal c-atcc-hckm~inc l~vt.1~ t-crnainc~d significantly elevated. ‘This last finding co~~ld bc intcrpreted as being another reflection of tl<~crt~,tsutl plac~tttal perfusion or the inahilit\ of the tutus to tttc.taboli/c the catecholamines and to recovct. f’~om ~hc. altwc~d metabolic state in a hrief period. On tht’ r)thc’r hand. the possible continued release of cater hol;tntinrs cluring this altered metabolic state m;rv bc- \,t~al liar Irtai survival and the elevations in norrl,ittrpltt.tii~, xtd epinephrine during the f’etal reco\ ct.\. lwt-ic)(i ;tr(’ consistenf with this cardiovasctilar rrspott\(’ (It) 111~~wcottd phlebotomy). The significant raise in epinq)hrittc I’(*flew the ability of. the f’etal adrmats ;11tc1th(, organ of‘ Zuckerkandl (fetal extramedullar! (hrotnatin tisstw) to respond to acute stress. ‘The ef’r‘ect ot prolc~ngecl cxposure of the fetus to elevated level \ 01‘ c,irc tilitlitlg C;lt(‘cholamines is unkntwn. The authors would like to expresc fhcir qatitude to Dr. C;arland Johnson of The Ltp-john (:I).. K;tlama~oo, Michigan, for his helpf’ttl advice.
REFERENCES
1. Greever, C. J., and Watts, D. T.: Epinephrine levels in peripheral blood during irreversible hemorrhagic shock in dogs, Circ. Res. 7:192, 1959. 2. Miller. R. A., and Benfey, B. G.: The fluorimetric estimation of adrenaline and not-adrenaline during hemorrhagic hypotension, Br. J. Anaesth. 30:158, 1958. 3. Peuler. D. J., and Johnson, G. A.: Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine, Life Sci. 21:625, 1977.
4. Da Prada. M., and Zurcher, G.: Simultaneous radioertzymatic determination of plasma and tissw adren;Gttr, noradrenaline and dopamine within the t‘cmtomolc range, Life Sci. 19:1161, 1976. 5. Weitzman, R. E., Glatz, T. H., and Fisher. D. A.: ‘The effect of hemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs. Endocrinology 103:2154. 1978. 6. Nathanielsz. P. W.. .4bel, M. H.. Bass. F. (G.. Kranv. E. j..
822
Artal
November
et al.
Am.
Thomas, A. L., and Higgins, G. C.: Pituitary stalk-section and some of its effects on endocrine function in the fetal lamb, Q. J. Exp. Physibl. 63:211, 1978. 7. Kletzky, 0. A., Nakamura, R. M., Thorneycroft, I. H., and Mishell, D. R.: Log normal distribution of gonadotropins and ovarian steroid values in the normal menstrual cycle, AM. J. OBSTET. GYNECOL. 121:688, 1975. 8. Jones, C. T., and Robinson, R. 0.: Plasma catecholamines in the fetal and adult sheep, J. Physiol. 248:15, 1975.
15, 1979
J. Obstet. Gynecol.
9. Comline, R. S., and Silver, M.: The release of adrenaline and noradrenalin from the adrenal glands of the foetel sheep, J. Physiol. 156:424, 1961. 10. Darby, T. D., and Watts, D. T.: Acidosis and blood epinephrine levels in hemorrhagic hypotension, Am. J. Physiol. 206:1281, 1964.
lniormation for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership of the manuscript (&Ii of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under cotisideration by another journal, and has not been previously published. I sign for and acce,pt responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.
ARTAL,
M.D.
THEODORE
H.
ROBERT PETER
LAM, W.
GLATZ,
M.D.
B.Sc.
NATHANIELSZ,
CALVIN
J.
HOBEL,
Los Angeles,
Calijornin
M.D.,
PH.D.
M.D.
Five chronically instrumented pregnant ewes and their fetuses were studied to assess the effect of acute maternal hemorrhage. The hemorrhage was produced in two stages; during the first stage 15% of the total maternal blood volume was removed, and then in a second stage an addltiinal 15% was removed. Biophysical and biochemical variables were monitored. Plasma dopamine, norepinephrine, and epinephrine were determined by a sensitive radioenzymatic assay. A significant rise in maternal and fetal catecholamines was observed following an estimated maternal blood volume depletion of 30%. During a 15minute recovery period, the maternal catecholamine concentrations returned to normal, while the fetal concentrations remained elevated. These changes were accompanied by significant changes in maternal and fetal homeoatasis. The signiftcance of these findings is discussed. (AM. J. OBSTET. GYNECOL. 135:818, 1979.)
HEMORRHAGE IS KNOWN to affect the sympathetic nervous system and the release of catecholamines, which results in significant hemodynamic and metabolic changes. ‘* * The effect of maternal hemorrhage on the circulating fetal catecholamines has not been studied, in part because of numerous technical difficulties in the measurement of catecholamines in small amounts of biologic fluids. These measurements are now possible following the development of sensitive radioenzymatic assays.3. ’ The present study was conducted to assess the effect of acute maternal hemorrhage on maternal and fetal homeostasis. To investigate this condition we utilized the chronically instrumented sheep preparation and a modified exFrom the Departments of Obstetrics and Gynecology and Pediatrics, Harbor-Univwsity of Cal(forniu, Los Angeles Medical Center. Presented at the Twenty-sixth Annual Society for Gynecologic Investigation, California, March 21-24, 1979.
Meeting of the San Drkgo,
Reprint requests: Dr. Raul Artal, Department of Obstetrics and Gynecology, Los Angeles CountylUniverstiy of Southern Calzyorma Medical Center, 1240 N. Mission Road, Los Angeles, California 90033.
018
perimental study design acute hemorrhage.’
developed
in other studies of
Material and methods Care of animals and experimental protocol. Five date-mated pregnant Rambouillet-Columbia ewes and their fetuses were used in this study. The animals were chronically instrumented utilizing previously described techniques.6 Catheters were implanted in the maternal femoral artery and vein and in the fetal carotid artery and internal jugular vein. The animals were allowed to recover for at least 72 hours following surgery before any experimental procedure was performed. Maternal and fetal heart rates were monitored continuously. Fetal and maternal arterial blood gases and base excess were measured on a Radiometer ABL2 blood gas analyzer. PO, and Pco, are expressed throughout as millimeters of mercury and base excess as milliequivalent per liter. All the studies were performed on animals that were stable at the time of experiment and had a fetal arterial pH greater than 7.33 and a Pop greater’ than 18 mm Hg. The gestational age at the time of experiment was 128 to 134 days. 0002-9378/79/220818+05$00.50/0~
1979
The
C. V.
Masby Co.
Volume
Number
1% 6
The experimental procedure protocol was as follows: Simultaneous maternal and fetal arterial blood samples were obtained for hormone determinations at the beginning of the study; biophysical variables were monitored during a control period of 15 minutes. At the end of the control period the first phlebotOmy was performed during which 15% of the total estimated maternal blood volume (12.5 ml of blood/kg of body weight) was removed over approximately 5 minutes. Fifteen minutes later a second phlebotomy was performed to remove an additional 15% of the total estimated maternal blood volume. Additional simultaneous maternal and fetal blood samples were obtained for hormone determinations. At ‘this point, the blood that had been removed was returned to the ewe. The experiment was concluded by a 15-minute recovery period. after which the last simultaneous maternal and fetal blood samples were drawn. pH, PO,, and Pcop were measured in all samples and were within the range previously reported.6 Special precautions were taken to avoid any degradation of catecholamines during sampling. The blood samples for catecholamine determination were collected in tubes containing a stabilizing solution of 90 mg/ml of ethyleneguanosinetetraacetic acid and 60 mg/ml of glutathione. The plasma was separated immediately at 4” C in a refrigerated centrifuge. Samples were stored at -80” C until measured. All estimations were performed within 12 weeks. Dopamine, norepinephrine, and epinephrine were determined by a radioenzymatic assay in duplicates of 50 ~1 of plasma. The within-assay coefficients of variation were 4.8% for dopamine, 4.3% for norepinephrine, and 3.5% for epinephrine; the betweenassay coefficients of variation were 12.OYc for dopamine, 9.7% for norepinephrine, and 8.4% for epinephrine. The radioenzymatic assay included the modification described by Peuler and Johnson3 of adding O.lM benzyloxyamine to the catechol-O-methyltransferase (COMT) preparation to prevent any increase in dopamine levels by the decarboxylase-catalyzed conversion of 3,4-dihydroxyphenylalanine (DOPA) to dopamine. To analyze statistical differences between tbe various stages of the experiment the data were tested by the Rankit test and found to be nongaussian.’ Therefore, each data set was compared by the nonparametric statistic Mann-Whitney U test.
Results Blood respiratory gases and pH. No significant changes were found between the control samples and the first phlebotomy samples. However, significant
Effect
of hemorrhage
on catecholamine
release
819
MATERNALVALUES
&M+2SEM
OCONTROL
-2nd
PHLEBOTOMY
Fig. 1. l-he effect ot‘the second phlebotomy and f’etal respiratory gases and pH. ,o,ooo,---
.-.-.-
._-~
*p
on the maternal
-
-
?
ELE m 95% C.L. - p
CONTROL 1st PHll
Fig. 2. The maternal (El and f’etal (L) dopamine concentrations at the different stages of the experiment. The horizontal lines at the bottom of the figure link two bars to indicate a statistical difference of at least 0.0 1. The bars represent mean and 95%
confidence
limits
(C.L.).
changes in these measurements were obsrl-vcd at the end of the second phlebotomy. These changes are illustrated in Fig. 1. Initial maternal pH was 7.40 t 0.03 (mean t SEM) and fetal pH was 7.33 t 0.01. These values fell following the second phlebotomv to 7.28 t 0.04 and 7.1 1 L 0.05, respectively (p < 0.05 in each instance). The second phlebotomy did not significantly change the maternal Pco, (40 +- 3 to 33 -C 8) but significantly altered the fetal PCO, (48.3 -+ 0.8 to 61.8 + 3.9). Similarly, PO? did not significantly change in the ewe (95 rt 2 to X9 -C 3) with the second phlebolomy but
820
November Am. J. Obstet.
Artal et al.
CONTROL 1st PHLEB -------------------------------------------------------
2nd PHLEB
I
15, 1979 Gynecol.
21,000 .F T ,
RE OVER
== ==
Fig. 3. The maternal (E) and fetal (f.) norepinephrine concentrations at the different stages of the experiment. The continuous horizontal lines at the bottom of the figure link two bars to indicate a statistical difference of at least 0.01. The double dashed lines indicate a statistical difference .of 0.05. The bars represent mean and 95% confidence limits (CL.).
Fig. 4. The maternal (El and fetal (L) epinephrine concentrations at the different stages of the experiment. The continuous horizontal lines at the bottom of the figure link two bars to indicate a statistical difference of at least 0.01. The double dashed lines indicate a statistical difference of 0.05. The bars represent mean and 95% confidence limits (CL.).
was significantly reduced in the fetus (20.8 2 0.9 to 16.5 rt 1.9) (p < 0.05). The second phlebotomy significantly altered the base excess in both ewe (-0.5 t 0.2 to -6 2 2)and fetus (-0.25 ? 0.01 to -7.68 * 1.1) (p < 0.05 in each instance). Catecholamines. Dopumi~. Fig. 2 illustrates the changes in circulating dopamine in both the ewe and the fetus. The mean baseline concentrations were 35 (4 to 300) pg/ml (mean and 95% confidence limits) in the ewe and 52 (33 to 80) pg/ml in the fetus. Following the first phlebotomy, the plasma dopamine concentration rose to 60 (30 to 1,000) pg/ml in the ewe and 49 (11 to 220) pgiml in the fetus. These changes were not significant. Following the second phlebotomy, the plasma dopamine concentration rose significantly in both fetus and ewe. The concentrations rose to 290 (60 to 1,400) pgiml in the ewe (p < 0.01) and to 320 (100 to 1,000) pg/ml in the fetus (p < 0.01). During the recovery period the dopamine concentration remained significantly elevated (p < 0.01) from the control period at 160 (64 to 390) pg/ml in the ewe and 450 (90 to 2,000) pgiml in the fetus Norepiwphrine. The changes in norepinephrine are illustrated in Fig. 3. The norepinephrine concentra-
tions during the control period were 3 10 (120 to 800) pgiml in the ewes and 460 (210 to 1,000) pg/ml in the fetus. No significant changes in the norepinephrine levt:ls were observed following the first phlebotomy and the values were 560 (330 to 940) pg/ml in the ewe and 470 (2 10 to 1,050) pg/ml in the fetus. The second phlebotomy significantly increased the norepinephrine fell to concentrations significantly increased the norepinephrine levels to 3,900 (350 to 40,000) pg/ml (p < 0.01) in the ewe and to 10,000 (2,500 to 42,000) pg/ml (p < 0.05) in the fetus. During the recovery period norepinephrine fell to concentrations significantly different (p < 0.01) from those following the second phlebotomy to 430 (160 to 1,100) pgiml in the ewe and remained significantly elevated (p < 0.05 at 10,000 (2,500 to 35,000) pg/ml in the fetus. Epinephrirre. The changes in epinephrine concentrations are illustrated in Fig. 4. The baseline values determined were 190 (40 to 900) pg/ml of epinephrine in the ewe and 58 (9 to 350) pg/ml of epinephrine in the fetus. In the different stages of the experiment the levels of epinephrine followed a trend similar to that observed with the other catecholamines. No significant statistical differences in epinephrine levels were observed following the first phlebotomy. The mean val-
Volume Number
135 6
ues wet-c: 500 (IO0 to -~.OOO) pgitnl of‘ epinephrine in the ewe and XI (.? to ‘4.X)) pgiml of epinephrine in the fetus. Significant elevations in epinephrint levels were observed at the end of’ the second maternal phlebotomy. The mean epinephrine Irvels obtained were 5.000 (%O to 100.000) pgitnl in the cw and i,.iOO (I ,700 to 3Y.000) pgiml in the fetus. At the end of the recover:, period, the epinephrine levels fell to 430 (1 .il) to 1.L’OO) pgiml in the ewe and to 240 (70 to 840) pgiml in the frtus. Cardiovascular changes. During the first phlebotomy, the mean mater-nal Itcart rate increased without a change itt the blood pwssttre. \vhile in the fetus no changes were observed. tIo\vc\,er, one fetus responded to the first phlehotom~ with brad~carclia, hypotension. and a Pof of. It! tnm Hg. During the second phlebotctm\ the mean matcrn;tI blood pressure and heart rate decreased, while all ~‘CYIISCS cle\rloped bradycardia. hypertension. and tvidenittg of’ the puIse prww-c. Within 5 minutes of this brad!cardia most of. the fetuses had a gradual increase in heart rate.
Comment Experimental hemorrhage in the pregnant ewe can provide ;I useful animal model f’or the study of the complex maternal-fetal interactions that occur at the time of stress. Such a model could help us to understand the role the adrenergic nervows system plays in the adaptation of the fetus f’rom a normal to a stressful condition. Our stud) indicates that significant hemodynamic changes will ha\,e to occttr in the mother hefore they are rcHected in the fetus; a maternal blood volume depletion of‘ 30% was needed to induce significant changes in fetal homeostasis. The hemodynamic changes observed in the ewe are common to acute blood volume depletion. as demonstrated in other anitnal studie\.‘, “. ’ In the fetus the typical haroreceptor responses of bradycardia and hypertension were f’ollowcd by a recovery period during which an increase in heart rate ~i;ts observed. The alterations in the acid-base balance in the ewe were those of part11 compensated subacute metabolic. acidosis. while those
Effect of hemorrhage on catecholamine
release
821
in the fettis were characteristic. of’ ;I state (II ;I( ttte ttlc%tholic. acidosis. The high lwels of I’c:o~ trb~~~ctl at the’ end of the second phlebotomy it! rhc, f’cttt\ :11-c likei\ ;t reflection of’ decreased placental pr’t I’ttsii ~1). The humoral changes obwrvetl C~~thy, c.~~tf of tlrtt second phlebotomv resulted in ;I si,qttiftc;tnt ~xAc;tw 01’ catecholamines in both the ewe and tltc fertrs;. Prcviotta studie? I’ have demottstrated the c.;tpabilit\ of the frt;tI sheep to releasr c~ttec~holatninc~ in wspmw~ to tl if’f’c,rent stimuli, but in none ot’ the sttt(lic.\ h;t\ lhts rc=~pottw of the f’etus been so significant. l.o\~crittg 111~.ntatertl;tl blood volume by :rO’;C rrsultctl in ~111 tighttolcl inc ~‘c.tsc’ in dopamitie, a twel~~rtold inc re;iw it1 rtot~c~l~iitcf~hrirr~. and a 2%f’old increase in rpinc~pltrt~tc* in tl~c. C’IVC’.~I‘ltt~ increments observed in the f’cstus MC’IX‘ zi\ti&l for. dopamine. :! I-fold fat- tiorel)itic~f~~tt.i~~t~, aiitl I .YO-fi)lti Cot, epinephrine. The acute rcleasc oi c’atc’ctrolamittc~s itt both the ewes and their frruscs i\ Grnil,tt- IO the, IVsponse produced hy hemorrhage in attc\tlt~:ti/eci hvpotensivr dogs.“’ Of interest is that during nlic two\~~n period the catecholamine Ir~~ls w~tt~-nt~l IO not trial iti the e~‘e while the fetal c-atcc-hckm~inc l~vt.1~ t-crnainc~d significantly elevated. ‘This last finding co~~ld bc intcrpreted as being another reflection of tl<~crt~,tsutl plac~tttal perfusion or the inahilit\ of the tutus to tttc.taboli/c the catecholamines and to recovct. f’~om ~hc. altwc~d metabolic state in a hrief period. On tht’ r)thc’r hand. the possible continued release of cater hol;tntinrs cluring this altered metabolic state m;rv bc- \,t~al liar Irtai survival and the elevations in norrl,ittrpltt.tii~, xtd epinephrine during the f’etal reco\ ct.\. lwt-ic)(i ;tr(’ consistenf with this cardiovasctilar rrspott\(’ (It) 111~~wcottd phlebotomy). The significant raise in epinq)hrittc I’(*flew the ability of. the f’etal adrmats ;11tc1th(, organ of‘ Zuckerkandl (fetal extramedullar! (hrotnatin tisstw) to respond to acute stress. ‘The ef’r‘ect ot prolc~ngecl cxposure of the fetus to elevated level \ 01‘ c,irc tilitlitlg C;lt(‘cholamines is unkntwn. The authors would like to expresc fhcir qatitude to Dr. C;arland Johnson of The Ltp-john (:I).. K;tlama~oo, Michigan, for his helpf’ttl advice.
REFERENCES
1. Greever, C. J., and Watts, D. T.: Epinephrine levels in peripheral blood during irreversible hemorrhagic shock in dogs, Circ. Res. 7:192, 1959. 2. Miller. R. A., and Benfey, B. G.: The fluorimetric estimation of adrenaline and not-adrenaline during hemorrhagic hypotension, Br. J. Anaesth. 30:158, 1958. 3. Peuler. D. J., and Johnson, G. A.: Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine, Life Sci. 21:625, 1977.
4. Da Prada. M., and Zurcher, G.: Simultaneous radioertzymatic determination of plasma and tissw adren;Gttr, noradrenaline and dopamine within the t‘cmtomolc range, Life Sci. 19:1161, 1976. 5. Weitzman, R. E., Glatz, T. H., and Fisher. D. A.: ‘The effect of hemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs. Endocrinology 103:2154. 1978. 6. Nathanielsz. P. W.. .4bel, M. H.. Bass. F. (G.. Kranv. E. j..
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Artal
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Thomas, A. L., and Higgins, G. C.: Pituitary stalk-section and some of its effects on endocrine function in the fetal lamb, Q. J. Exp. Physibl. 63:211, 1978. 7. Kletzky, 0. A., Nakamura, R. M., Thorneycroft, I. H., and Mishell, D. R.: Log normal distribution of gonadotropins and ovarian steroid values in the normal menstrual cycle, AM. J. OBSTET. GYNECOL. 121:688, 1975. 8. Jones, C. T., and Robinson, R. 0.: Plasma catecholamines in the fetal and adult sheep, J. Physiol. 248:15, 1975.
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9. Comline, R. S., and Silver, M.: The release of adrenaline and noradrenalin from the adrenal glands of the foetel sheep, J. Physiol. 156:424, 1961. 10. Darby, T. D., and Watts, D. T.: Acidosis and blood epinephrine levels in hemorrhagic hypotension, Am. J. Physiol. 206:1281, 1964.
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