J. Physiol. (1975), 248, pp. 15-33 With 7 text-figures Printed in Great Britain

15

PLASMA CATECHOLAMINES IN FOETAL AND ADULT SHEEP

BY C. T. JONES AND R. 0. ROBINSON* From the Nu ffeld Institute for Medical Research, University of Oxford, Oxford OX3 9DS (Received 12 March 1974) SUMMARY

1. Foetal and maternal plasma catecholamine concentrations were measured during and after hypoxia (mean maternal P.,02 44 mmHg) in chronically catheterized sheep, 118-141 days pregnant. 2. In most foetuses the initial plasma catecholamines were < 007 ng/ml. During hypoxia plasma adrenaline and noradrenaline always rose; there was a rise in arterial pressure and a fall in heart rate. 3. The initial catecholamine concentration in the ewes was < 0052-3 ng/ml. During hypoxia there was no consistent change; the maternal plasma concentrations were less than the foetal. 4. Infusion of adrenaline at 03 ,tg kg-1 min- to the ewe resulted in plasma catecholamine concentrations higher than those observed during hypoxia. There was a rise in heart rate but no consistent change in arterial pressure. 5. Infusion of adrenaline 04 jug kg-1 min-' into the foetal jugular vein caused a rise in plasma concentration similar to that seen during hypoxia. There was a rise in heart rate but no significant change in arterial pressure. 6. The half-life of adrenaline and of noradrenaline in the maternal and foetal circulation was 0-25-1 min. There was no evidence of transfer of labelled catecholamine across the placenta. INTRODUCTION

The concentrations of catecholamines in foetal plasma have not previously been reported. Injection or infusion of catecholamines have large effects on the foetal circulation (Dawes, Mott & Rennick, 1956; Born, Dawes & Mott, 1956; Assali, Holm & Sehgal, 1962a, b; Smith, Morris & Assali, 1964; Cassin, Dawes & Ross, 1964; Friedman, Poole, Jacobowitz, Seagren & Braunwald, 1968; Adamson, Muller-Heubach & *

Present address: Department of Pediatrics, John Radcliffe Hospital, Headley

Way, Oxford.

1. T. JONES AND R. 0. ROBINSON 16 Meyers, 1971; Joelsson, Barton, Daniel, James & Adamson, 1972) but it is not known whether the doses used result in plasma concentrations that occur in vivo. Although Comline & Silver (1961) and Comline, Silver & Silver (1965) have shown that hypoxia is a powerful stimulus for adrenal catecholamine secretion in the foetus, without information on the rate of clearance it is not possible to estimate the plasma concentrations reached at the secretion rate reported. Plasma catecholamines have been measured in chronically catheterized pregnant sheep before and during hypoxia. Adrenaline concentrations similar to those observed during hypoxia were obtained by intravenous infusion and the cardiovascular effects compared. The adrenaline concentrations reached during infusion and the rate of disappearance of labelled catecholamine from the circulation have been used to determine their rate of clearance. METHODS of 118-141 foetal days gestation from seventeen ewes of various sheep Seventeen breeds were used; eleven for experiments involving hypoxaemia and six in control experiments. Foetal and maternal carotid and jugular catheters were implanted and continuous records of foetal arterial pressure, heart rate and tracheal pressure made (Dawes, Fox, Leduc, Liggins & Richards, 1971). The sheep were allowed at least 3 days to recover from operation. In order to produce maternal and foetal hypoxia eleven of the ewes were given 9 % 02 and 3 % CO2 in N2 at 40 I./min to breathe for 1 hr; the gas was delivered into the nasal end of a polyethylene bag placed over the head with the outlet round the neck. Carotid blood samples (11 ml. from the mother and 7 ml. from the foetus) were taken 60 and 10 min before, 10, 25, 45 and 60 min during and 10 and 60 min after the period of hypoxia. Six sheep were used for control experiments in which air was passed through the bag. Blood was collected into 10 ml. plastic syringes containing 15-20 u. heparin (Boots Pure Drug Co., Nottingham); it was transferred at once to polystyrene tubes (Henleys Medical Supplies, London) and placed on ice. Plasma was removed after centrifugation at 1500 g and 20 C for 20 min and extracted as described below. There was no significant loss of catecholamine before and during centrifugation. Blood samples (1 ml.) were collected into glass syringes for immediate determination of haematocrit and of P., 02 Pa C02 and pH at 38° C using a Model 27 Acid-Base Analyser (Radiometer, Copenhagen). Blood gas and pH measurements were corrected to the rectal temperature of the sheep. Adrenaline infusion. L-adrenaline (Macarthys Ltd, Romford, Essex) diluted with 0 9 % NaCl (w/v) and acidified to pH 3-5 with 1 M-HCl was infused through the foetal jugular catheter for 2 hr at 1-3 ,tg/min (6.6 ml./hr) with a constant infusion pump. The sterile infusion syringe and catheter were wrapped in aluminium foil to exclude light. In other sheep L-adrenaline was infused into the maternal jugular at 12-7 /,zg/min (9.49 ml./hr). Maternal (11 ml.) and foetal (7 ml.) carotid blood samples were collected 60 and 10 min before, 30, 60 and 120 min during and 120 min after the infusion. Metabolic clearance rate of adrenaline and noradrenaline. Three foetal sheep were injected via their jugular catheter with 10 ,c [3H]DL-adrenaline (12 c/m-inole) or [3H]L-noradrenaline (7.8 and 8-2 c/m-mole) in 1 ml. 0 9 % NaCl over approximately

FOETAL CATECHOLAMINES

17

5 sec. Maternal (10 ml.) and foetal (5 ml.) carotid arterial blood samples were taken over 14 hr at the times shown in Fig. 6; plasma was separated and catecholamines were collected as described above. The next day the ewes were injected via their jugular catheter with 100 ,uc [-3H]L-noradrenaline in 2 ml. 0-9 % NaCl over 5 sec. Maternal (10 ml.) and foetal (5 ml.) carotid samples were collected as before. Extraction of adrenaline and noradrenaline. Plasma samples were applied immediately to a column (4 x 25 mm) containing I g of Dowex 50W-X8 (H+ form). The column was then washed with 10 ml. of glass-distilled water, 10 ml. 0-2 M sodium phosphate buffer (pH 6-5) containing 2 % (w/v) ethyleneglycol tetra-acetic acid (EGTA), followed by 10 ml. glass-distilled'water. Adrenaline and noradrenaline were eluted from the column with 5 ml. 1-5 M-HCl. Addition of 10 ng dopamine to the column produced no detectable fluorescence in the adrenaline and noradrenaline fraction. Elution of adrenaline and noradrenaline from the columns only occurred above 0-4 M-HCl and was not enhanced by increasing the concentration above 1-5 M-HCl. The recovery of adrenaline off the columns was 44 ± 14 % (S.D.) and of noradrenaline was 52 + 15 %. When 10 ng of the O-methylated metabolites metanephrine and normetanephrine were added to the column they were not eluted in the adrenaline and noradrenaline fraction. The pH of the catecholamine eluate was adjusted to pH 5-5 with 20 %, 2 % and 0-2 % (w/v) KOH, using indicator paper (if K2CO3 was used a rise in pH and consequent loss of catecholamine occurred during the evaporation); 0-5 ml. of McIlvaine buffer, pH 5-5 (McIlvaine, 1921) was then added. This was evaporated to dryness in a Buchler Evapomix (Baird and Tatlock, Chadwell Heath, Essex) at 30-35° C and 0-5 mmHg. The residue was resuspended in 0-7 ml. 0-2 M sodium phosphate, pH 6-5. Minimum loss of catecholamines occurred with an evaporation time of 1 hr, at pH 5-5 and 350 C. Recovery of 1 or 10 ng adrenaline and noradrenaline in plasma or buffer after column separation and evaporation was 44-1 + 15 % (s.D.) and 36-6 ± 16 % (s.D.) respectively. It was not affected by prior acidification of plasma and thus any protein bound catecholamine (Mirkin, Brown & Ulstrom, 1966) is probably measured together with free catecholamine. After catecholamine elution, the columns were regenerated by washing with 20 ml. 2 M-HCl, 10 ml. glass-distilled water, 10 ml. 0-2 M sodium phosphate (pH 6-5) containing 2 % EGTA, and 10 ml. glass-distilled water. The same batch of resin was used throughout. Before use it was primed by applying 500 ng each of adrenaline and noradrenaline to the columns followed by the normal washing procedure. Assay of catecholamines. Adrenaline and noradrenaline were assayed by the tri-hydroxyindole method (Lund, 1949; Euler & Floding, 1955) as described by Haggendal (1962) with modifications as outlined below. After resuspension of the evaporated eluate in 0-7 ml., 0-5 ml. was added to 0-2 ml. 0-2 M sodium phosphate (pH 6-5) and 0-02 ml. 0-0025 % (w/v) CuC12. Catecholamine in this solution was oxidized for 3 min by 0-05 ml. 0-25 % (w/v) K3Fe(CN6). The oxidation was stopped with 0-3 ml. of a solution prepared by mixing 50 mg f8-mercaptoethanol with 5 ml. 0-2 % (w/v) K2SO3 and 5 ml. 5 M-NaOH immediately before use. Acidification (approx. pH 4-0) with 0-2 ml. 10 M acetic acid 3 min later resulted in fluorescent compounds stable to light for 10-15 min. Fluorescence was determined in an Aminco Bowman spectrofluorimeter. The adrenaline excitation Ama. was 445 nm and the emission Ama. was 515 nm; for noradrenaline they were 412 and 475 nm respectively. Adrenaline and noradrenaline concentrations were calculated from simultaneous equations using the total fluorescence at 475 nm during excitation at 412 nm and at 515 nm during excitation at 445 nm. The constants used in the equations were calculated on the day of assay from adrenaline

~~C. T. JONES AND B. 0. ROBINSON

18 18

and noradrenaline standards and showed little variation. The ratio of adrenaline : noradrenaline fluorescence was

Activation 412 445

Emission 475 515

Ratio 1: 1-98+0O43 (S.D.) 1: 0-27+O-08 (S.D.)

It was possible to measure adrenaline when it was not < 25 % of the total catecholamine and noradrenaline when it was not < 10 % of the total. The nonoxidized tissue blank gave a fluorescence which was about 25 % greater than that given by glass-distilled water. The standard deviation of the blank was equivalent to 0-05 ng at 475 nm and 0-06 ng at 515 nm. Thus the limit of detection of the assay was 0-1Ing noradrenaline and 0- 12 ng adrenaline (i.e. 2 S.D. of the blank). The quantity then can be measured in a plasma sample (as given by: limit of detection/plasma volume x recovery) was approximately 0-05 ng/ml. maternal, and 0-07 ng/ml. foetal. With the exception of the measurement of fluorescence all procedures involving the handling of the plasma samples and the assay mixture were conducted in dim light. 'Unless quoted as total catecholamine the results refer to adrenaline or noradrenaline concentrations. They are expressed as means + 5.E. Chemical. These were obtained from: L-adrenaline, L-noradrenaline, dopamine and Na2SO3 (Koch Light Laboratories, Colnbrook, Bucks.), /J-mercaptoethanol, metanephrine, normetanephrine, EGTA, Dowex 50W-X8 resin (Sigma London Chemical Co., Kingston-upon-Thames), HCl, NaGH and KOH (Aristar grade, B.D.H. Ltd, Poole, Dorset), K2S03 (ExP grade, Heico Laboratories Ltd, Delaware Water Gap, Pennsylvania, U.S.A.), all other chemicals used were Analar grade and obtained from BDH Ltd, Poole, Dorset. RESULTS

Six sheep, 118-138 days pregnant, which had not been the subject of previous experiments and having normal blood gas values were used for control observations. Exposure to air passed at 40 1./mmn through a polyethylene bag placed over the ewe's head produced no significant arterial change in maternal or foetal carotid arterial pH, Pa, o pressure or heart rate. In only one of the six ewes were carotid plasma catecholamines detectable before or during the period she was breathing air from the bag (adrenaline 0-8; noradrenaline 1-4 ng/ml.). Only in this

there evidence of a sustained increase in the foetal carotid plasma catecholamine concentration (maximum values, adrenaline 1-6 ng/ ml. and noradrenaline 1-7 ng/ml.). There was no change associated with the number of blood samples taken. The infusion of acidified (pH 3.5) saline into one adult sheep (9-49 ml./hr) and one foetus (6-6 ml./hr.; 1.31 days gestation) caused no cardiovascular changes and was not associated with detectable catecholamine concentrations. Hypoxia. Hypoxia lasting for 1 hr was induced by giving the ewes 9% 02 with 3 % 002 in N2 to breathe. After recovery from operation ewe was

1 FOETAL CATECHOLAMINES 19 3-4 days previously, seven sheep 118-i138 days pregnant and their foetuses had normal blood gas and pH values, arterial pressures and heart rates. The mean plasma glucose and lactate concentrations for the foetal sheep were 0-95 + O-O8 and 1'59 ± 0-08 /tmole/ml. respectively and for the ewes were 3-57 ± 0-26 and 0-67 + 0-03 /imole/ ml. respectively. Foetal breathing was present in normal amounts (a good indicator of foetal health, K. Boddy, G. S. Dawes and J. Robinson, unpublished) in

9%0~2 to ewe,

10{(a) Maternal Pa, 02 50 E

EJ 24[

j(b) Foetal P2, 0

7,-(c) 0

Foetal pH

*2

1

3

Time (hr)

Fig. 1. The changes in maternal carotid P,0 and foetal carotid P,0 and pH during administration of 9 % 02 to seven ewes.

the three from which tracheal pressure records were made. The mean packed cell volume of the seven foetuses was 31 +2 % (S.E.). Fig. 1 shows that during hypoxia the mean maternal P., 02in these seven sheep fell to 44 mmllg and the foetal fell from 26 to 18 mmHg. There were no significant changes in Pa2,CO22(maternal 30 + 4*7 mmHg; foetal 44 + 0*7 mm Hg). During hypoxia the foetal carotid pH fell from 7-37 + 0*03 to 7-24 ± 0*04; it rose to 7*27 + 0*05 during the subsequent hour when the ewe was once again breathing air. In six of the seven ewes catecholamines were not detected (< 0-05

C. T. JONES AND R. 0. ROBINSON

20

9% 02 to ewe

2

-

(a)

Foet us

0/' C

0

so

(b

E °

4' .Cd Lco I C_ U MC I

Ew Ew

(b)Ew

'J na I I

0

niCem.

rim rM

3

2 Time (hr)

Fig. 2. The changes in maternal and foetal carotid plasma catecholamine concentration during and after administration of 9 % 02 to ewe 76 (135 days gestation). 0' foetal adrenaline; *, noradrenaline; R, maternal adrenaline; *, noradrenaline. Before hypoxia the maternal and foetal catecholamine concentrations were below the limit of detection. The foetal pH before hypoxia was 7-34. 3 0

E 2

.oC

0

C

0

*

0

0

(44 4'

U

C 0

I

0

U

0 0

0 110

I

I 120 Gestational

~~~I age

130 (days)

I 140

Fig. 3. The maximum foetal plasma adrenaline and noradrenaline concentrations observed during the administration of 9% 02 to the seven normal sheep. 0O adrenaline; *, noradrenaline.

FOETAL CATECHOLAMINES 21 ng/ml.) in carotid plasma before hypoxia; in three of these there was a rise to 0 3-141 ng/ml. during hypoxia. Before hypoxia catecholamines were detected (> 0 07 ng/ml.) in only one of fourteen carotid samples from the seven foetuses. During hypoxia there was a rise in plasma adrenaline (range 0-3-2-8 ng/ml.) and noradrenaline (range 0>4-2-7 ng/ml.) within 10-25 min. in all foetuses (Fig. 2). Noradrenaline was usually present in higher concentrations than adrenaline. The catecholamine concentration fell after hypoxia in all but one foetus which subsequently died (133 days gestation, see below). The plasma adrenaline and noradrenaline concentration observed during hypoxia were similar over the age range studied (Fig. 3). 200 r

C

150 UE

0

'100 F

60

9%

F (b)

°, to ewe

Foetal arterial pressure

55 I E E

50 F 45 0

1

3 2 Time (hr) Fig. 4. The changes in foetal heart rate and arterial pressure during administration of 9 % 02 to eleven ewes. The heart rate changes in foetuses with maximum catecholamine concentrations of < 3 ng/ml. (0) and > 8 ng/ml. (@).

During hypoxia the foetal arterial pressure rose (12-25 %) while the foetal heart rate fell within a few minutes and remained low throughout. For at least 1 hr after hypoxia there was an increase in the foetal heart rate above the control value (Fig. 4).

C. T. JONES AND B. 0. ROBINSON An additional four sheep (134-140 days pregnant, three being in the same range of gestation as the previous seven sheep) have been considered separately. Two of these were allowed only 1 day to recover from the operation (one of which was in early labour at 136 days gestation) compared with the usual of at least 3-4 days. Of the other two sheep one had a post-operative maternal infection while the other had a foetus with an initial plasma pH of only 7 20 (the plasma lactate in this foetus was 2*3 ,amole/ml.). The mean plasma pH for the foetuses in this group 22

9% 02 to ewe 12

(a) Foetus

'10

-C

al~~~

,1/

0

U

E U

2

(b) Ewe

-

_

0 0

~

u*

1

ru r-.

2

r

3

Time (hr)

Fig. 5. The changes in maternal and foetal carotid plasma catecholamine concentrations during and after the administration of 9 % 02 to ewe 86 (140 days gestation). 0, Foetal adrenaline; 0, and noradrenaline; Lii, maternal adrenaline; *, and noradrenaline. The foetal pH before hypoxia was < 7-20.

was 7-34 + 0-05. A post mortem was carried out on these animals 24-48 hr after the experiment; none of the foetuses were infected and they were alive at delivery except for the one in early labour which died at approximately 10 min after the end of hypoxia (see below). The foetuses had normal resting Pa, o, (range 20-27 mmHg), Pa, cO2, heart rate and arterial pressure values but their mean packed cell volume (38.5 + 0-6 %) was significantly higher than that in the previous group. The mean plasma glucose and lactate concentration of foetuses in this group were 1-05 + 0-09 and 1-67 + 0-17 jtmole/ml. respectively and of the ewes were 3-49 + 0-1

D

FOETAL CATECHOLAMINES 23 and 0-6 + 0-04 #tmole/ml. respectively. These values are not significantly different from those reported for the normal group of animals. The three foetuses from which tracheal pressure recordings were taken made little or no breathing movements. Catecholamines were detected in all four ewes before hypoxia (0.32-1 ng/ml.) and in most samples thereafter. There was no consistent change with hypoxia. Before hypoxia two of the foetuses in this group had elevated catecholamine concentrations (1-6-44 ng/ml.). During

45j 20 -9 04 to

._

-0 W

Ch

C L_

0 U

0 to C

E (U

0 U

0 U (U

E (U

0

IF

0

10-

oL 7-1

0

ba

S

I

7.1

7-3 7.4 pH Fig. 6. The relationship between the minimum carotid pH and the maximum plasma total catecholamine concentration observed in the foetuses during hypoxia. Correlation coefficient 0-87 (P < 0-001). The figure includes data from foetuses above 120 days gestation; those with catecholamine concentrations > 10 ng/ml. are in the abnormal group of animals. 7-2

hypoxia catecholamine concentrations rose to > 8 ng/ml. (adrenaline 2-3-29-1 ng/ml.; noradrenaline 8*3-16-5 ng/ml.); in most samples adrenaline was predominant (Fig. 5). The foetal plasma pH showed a large fall to a mean value of 7-14 + 0-04 and remained at 7-16 + 0-04 after hypoxia. The foetal arterial pressure response was variable, showing little change in one, rising by about 14 % in two and by about 65 % in the

C. T. JONES AND R. 0. ROBINSON fourth. The fall in heart rate seen at the onset of hypoxia lasted for 10-15 min only (Fig. 4). The mean heart rate seen in these four foetuses between 15-40 min of hypoxia was significantly higher (P < 0.01) than that observed in the seven normal foetuses. During hypoxia there was a reciprocal correlation between the maximum total catecholamine concentration and the minimum pH observed in the foetuses (Fig. 6). There was no significant correlation with Pa °2Thus apart from the different catecholamine responses the major difference between the group of normal (seven) foetuses and the abnormal (four) foetuses was that the latter had a much lower pH during and after hypoxia (P < 0 02). A rise (three- to fivefold) in foetal plasma lactate was observed during hypoxia in the two groups; the responses were not significantly different. Of the eleven foetuses described above, two (one from the normal group of seven and one from the abnormal group of four) died within 3 hr after the end of hypoxia despite having before hypoxia Pao0 (2023 mmHg) and pH (7.33 and 7.40) values within the normal range and undetectable catecholamines. During hypoxia the maternal Pao02 fell to lower levels than usual (37 and 39 mmHg). This was associated with severe foetal acidaemia which continued together with high catecholamine concentrations (adrenaline 6-9 and 35-5 ng/ml.; noradrenaline 1-7 and 24*9 ng/ml.) until death. The gestational age of the foetuses were respectively 133 and 136 days. 24

Adrenaline infusions Foetal. Adrenaline was infused for 2 hr at 1 27 /tg/min (0.41-0.44 ,ug/ kg. min) into the jugular vein of four foetal sheep (121-141 days gestation) that were normal by the criterion previously outlined. Steady-state conditions were observed within 30 min when foetal carotid adrenaline concentrations were raised from < 0 07 to 1 0-3 4 ng/ml. (i.e. in the same range as that observed during hypoxia). In one of the four foetuses adrenaline was present in detectable amounts 2 hr after the infusion. No adrenaline was detected in maternal blood during foetal infusion. Foetal carotid PaIo2 fell by 2-5 mmHg and pH by 0*01-0*06 (mean 0.05) from an initial mean value of 7-34. There was no significant rise in foetal arterial pressure (from 51 + 3 to 57 + 4 mmHg); the heart rate rose from 167 + 11 to 190 + 17 beats/min. The heart rate returned to the initial values within 10 min after the end of the infusion. Maternal. Infusion of adrenaline 12 8 ,tg/min (approx. 0 26 ,tg/kg. min) into the jugular vein of three ewes 121-143 days pregnant (considered normal by the criterion previously described) caused a rise in maternal carotid adrenaline concentration from < 0.05 ng/ml. to 0-6-2-3 ng/ml.

FOETAL CATECHOLAMINES

25

TABLE 1. Half-life of adrenaline in the maternal and foetal circulation calculated from infusion data

Foetal infusion 3 816 Sheep no.... 121 Gestational age (days) 141 2 3 Steady-state adrenaline (ng/ml.) Half-life (min)* 0-26 0-27 Maternal infusion 45 824 Sheep no.... Days pregnant 134 143 7 0-8 Steady-state adrenaline (nglml.) 1-2 0-13 Half-life (min)*

839 129 1 0-23

69 128 2 0-26

848 121 2 0-33

* The half-life was calculated from the plasma volume, infusion rate and steadystate adrenaline concentration assuming that the clearance obeys first-order kinetics and that endogenous secretion makes a small contribution only. Plasma

volumes were estimated from the data of Broughton Pipkin and Christopherson & Webster (1972).

&

Kirkpatrick (1973)

10-3(L

0

\X

>

L

to-6\

10-'

4

8 'Time (hr)

12

Fig. 7. The disappearance of [3HIL-noradrenaline from the maternal(a and foetal ( O) circulation following a single injection to each. It is expressed as the fraction of the total injected radioactivity remaining/ml. plasma at Various times after the injection.

26 C. T. JONES AND R. 0. ROBINSON within 30 min and to 0-9-5-6 ng/ml. by 2 hr. In one ewe there was a rise in plasma noradrenaline to 1.5 ng/ml. No catecholamines were detected 2 hr after the end of the infusion. There was no change in maternal Pa,02 or PaC02; the carotid pH fell by 0*05-0*08 (mean 0.06) from the initial values of 7-33-7-52 (mean 7.45). The heart rate rose from 124 + 7 to 162+4 beats/min, but there was no consistent change in arterial pressure. The change in carotid P- 02 in the three foetuses was variable, but in all three arterial pH fell by 0*07-0-13 (mean 0.09) from an initial mean value of 7-37. There was no consistent change in foetal heart rate or arterial pressure. Adrenaline (3 7 ng/ml.) and noradrenaline (1 ng/ml.) appeared in detectable amounts in one foetus (121 days gestation) in which there was a substantial although temporary fall in P.,o2 (from 20 to 12 mmHg) and pH (7.34-7.27). This rise in catecholamines is attributed to hypoxaemia rather than placental transfer. TABLE 2. Clearance of [3H]L-noradrenaline and [3H]DL-adrenaline from the foetal and maternal circulation following a single injection Metabolic clearance Ewe no. 45 124 816

Gestational age (days)

rate

-

Injection L-noradrenaline DL-adrenaline L-noradrenaline

(ml. min-' kg-l)* 19-4 21.5 13.3

135 127 125

L-noradrenaline DL-adrenaline L-noradrenaline

41.2 35.5 19-6

Secretion rate (ng minkg-1)t 098 1.1 0.67

Half-life

(min)t 2-4 1.5 3.1

Foetus no. 45 124 816

2-05

1F8 1.0

093 098 3.2

* Metabolic clearance rate (M.c.R.) was calculated for M.C.R. = I/f xdt, where x is the fraction of the total injection remaining/ml. at time t (Tait & Burnstein, 1964). t Assuming a resting plasma adrenaline and noradrenaline concentration of 0-05 ng/ml. the secretion rate (S.R.) was calculated from s.R. = 005 x M.c.R. i The half-life of catecholamines, calculated from the plasma volume (V) and the M.C.R., was half-life = V log. 2/M.c.R. Plasma volume estimated from the data of Broughton Pipkin & Kirkpatrick (1973) and Christopherson & Webster (1972).

The half-life of adrenaline in the maternal and foetal circulations calculated from the infusion data is given in Table 1. Clearance of adrenaline and noradrenaline. Radioactively labelled adrenaline or noradrenaline given as a single i.v. injection to the foetus (150-200 ng) or the ewe (1500-2000 ng) had almost entirely disappeared

FOETAL CATECHOLAMINES 27 within 10 min (Fig. 7). No significant transfer of labelled catecholamine from the maternal to the foetal, or from the foetal to the maternal circulation (as measured in carotid plasma) was detected. The metabolic clearance of adrenaline and noradrenaline was calculated as described by Tate & Burstein (1964). The gross clearance of noradrenaline from the maternal circulation was 667 and 930 ml./min and from the foetal was 49 and 148 ml./min; for adrenaline the maternal clearance was 1400 ml./min, and the foetal was 149 ml./min. When expressed per unit body weight or unit plasma volume the foetal clearance of catecholamine is either equal to or as much as twice the maternal (Table 2). The half-life of adrenaline and noradrenaline (calculated from the metabolic clearance rate and total plasma volume) was 1P5-3 min in the maternal circulation and 1-4 min in the foetus. DISCUSSION

The trihydroxyindole method (Lund, 1949; Euler & Floding, 1955) has been widely used for the determination of catecholamines in tissue samples (Haggendal, 1966) but has not been sufficiently sensitive for their estimation in small volumes of plasma. The method described by Haggendal (1962) has been modified so that after separation on a cation exchange resin the catecholamines were concentrated by evaporation. This increases sensitivity five- to tenfold and allows the measurement of about 0.05 ng/ml. On 5 ml. plasma samples this is still not sensitive enough to determine the majority of resting levels. A reduction in the assay volume should increase the sensitivity of the assay still further (Renzini, Brunori & Valori, 1970). Recently other methods of comparable sensitivity have been described (McCullough, 1968; Engleman & Portnoy, 1970; Iversen & Jarrott, 1970; Renzini et al. 1970; Passon & Peuler, 1973). While our assay distinguished adrenaline and noradrenaline from their metabolites and dopamine, contributions from other catecholamines (e.g. isoprenaline, which occurs naturally (Shah, Tee, English, Redman & Bunyan, 1972) and behaves as adrenaline in our assay) cannot be excluded. Catecholamine uptake into red cells has been observed (Bain, Gaunt & Suffolk, 1937); although high concentrations were used in these studies, it is possible that red cells represent a site of storage and metabolism in the circulation. We measured the catecholamine concentration in carotid blood which had passed directly from the inferior vena cava into the left ventricle via the foramen ovale. The geometry of the foetal circulation (Dawes, 1968) is such that the catecholamine concentration in the carotid arteries may not be representative of arterial blood elsewhere. Blood passing

28 C. T. JONES AND R. 0. ROBINSON down the descending aorta will contain approximately equal proportions of superior and inferior vena cava blood which has been mixed in the right ventricle. Thus even if the catecholamines were totally cleared during passage through the head and forelimbs, the difference in concentration between the ascending and descending aorta is unlikely to be > twofold. During infusion into the jugular vein this difference is determined by clearance in the viscera, hind limbs and placenta. If the catecholamine half-life is 0-25-0-5 min (see below) and the circulation time is of the order of 10-15 sec (Reynolds, Ardran & Prichard, 1954) the difference is unlikely to be > twofold. During moderate hypoxia relatively large changes may occur in the distribution of foetal venous return to the two sides of the heart, and in the distribution of cardiac output to the peripheral tissues (Assali et al. 1962b; Campbell, Dawes, Fishman & Hyman, 1967; Dawes, Lewis, Milligan, Roche & Talner, 1968; Goodwin, 1968; Dawes, Duncan, Lewis, Merlet, Owen-Thomas & Reeves, 1969; Dunne Milligan & Thomas, 1972; Goodwin, Milligan, Thomas & Taylor, 1973); blood flow to the placenta is well maintained. Such changes may well affect the distribution of catecholamines secreted from the adrenal medulla, and their clearance. However, since it is likely that the placenta is a major site for clearance (see below), circulatory changes alone do not explain the size of the rises in catecholamines observed. The resting plasma catecholamine concentrations were < 0*05 ng/ml. in the majority of ewes and < 0 07 ng/ml. in their foetuses. This is the only data on resting values in the foetus; concentrations of about 0-05 ng/ml. have been described in adults from other species (Callingham, 1967; Engleman & Portnoy, 1970; Renzini et al. 1970). The handling of the ewe during control experiments and the small reduction in foetal blood volume due to the collection of blood samples were not associated with detectable changes in catecholamine concentration. In the three foetuses with elevated resting concentrations the stimulus for secretion did not appear to be an altered Pa,O2. During hypoxia the adrenal gland is likely to be the major source of the increased quantity of circulating catecholamines although paraganglia (Brundin, 1967) and sympathetic overflow (Herting & Axelrod, 1961; Brown, 1965; Folkow, Haggendal & Lisander, 1967) may contribute. Comline & Silver (1961), and Comline et al. (1965) demonstrated that severe asphyxia or hypoxia over several minutes caused a large increase in adrenal catecholamine secretion. This secretion, which was probably close to maximal, was dependent on the integrity of the splanchnic nerve except when the Pao, was very low (about 5 mmHg). These experiments are not directly comparable with ours because the observations were

FOETAL CATECHOLAMINES 29 made over relatively short periods of time on anaesthetized exteriorized foetuses. Combine & Silver showed that adrenaline was not secreted in significant amounts unless the Pa,02 was below 8-12 mmHg. During hypoxia we found an increase in plasma adrenaline in all foetuses, although the mean Pa,02 fell to only 18 mmHg. Comline & Silver demonstrated a large increase in adrenal adrenaline secretion (during severe hypoxia) between 120 and 140 days, while our data showed no significant change with age in the rise in plasma adrenaline concentrations during moderate hypoxaemia. In normal foetuses the range of plasma catecholamine concentrations during hypoxia was from < 0 07 ng/ml. to < 3 ng/ml. Only in sheep which were considered abnormal did the concentrations rise to > 8 ng/ml. Adrenaline infused into the foetus at 04 #,g k-1 min-' reached plasma concentrations at the upper end of the range seen in normal foetuses. In studies on isolated foetal heart muscle, catecholamine concentrations from 0'1 to as much as 100 ng/ml. have been used (McCarty, Lee & Shideman, 1960; Hall, 1957; Friedman, Lesch & Sonnenblick, 1973). Similarly in acute experiments in sheep and monkeys the foetal cardiovascular effects of catecholamines have been examined using i.v. infusion rates of 0'5-5 4ug kg-' min' (Born et al. 1956; Assali et al. 1962a; Smith et al. 1964; Adamson et al. 1971). These relatively large doses caused a rise of blood pressure as well as of heart rate, whereas we found a rise of heart rate alone. It is unlikely that an abrupt fall in Pao2 of the magnitude produced in our experiments is normally experienced by the foetus, other than during delivery. Thus the concentrations of catecholamines found during hypoxia probably do not occur during normal foetal life. However, even when the Pa,02 is within the normal range, high concentrations have been observed when the foetus has one of a number of pathological conditions. If a fall in Pao2 of the magnitude observed during hypoxia does occur in foetal life, the foetus is capable of producing and maintaining high plasma catecholamine concentrations for at least an hour. In adult ewes whose Pa, 02 had been reduced to 40-45 mmHg there was no consistent rise in plasma catecholamines, yet changes may have occurred below the limit of the method (50 pg/ml.). Maternal catecholamine infusions at 0 1-5 Pug kg-' min-' have been reported to cause a rise in maternal arterial pressure and heart rate and a fall in uterine artery blood flow (Assali et al. 1962a; Beard, 1962; Ladner, Brinkman, Weston & Assali, 1971; Adamson et al. 1971). These maternal changes were associated with a rise in foetal arterial pressure and a fall in heart rate, arterial pH and Pa,O2. We found that a maternal infusion rate of only 0-26 jug kg-' min' produced plasma levels severalfold higher than those

C. T. JONES AND R. 0. ROBINSON observed during hypoxia; the only consistent effects observed at this infusion rate was a rise in maternal heart rate and a fall in maternal and foetal arterial pH. The foetal bradyeardia seen at the onset of hypoxia is primarily the result of increased activity in parasympathetic efferent vagal fibres to the heart (Barcroft & Barron, 1945; Dawes et al. 1956). In some foetal sheep during hypoxia the heart rate at first slowed and then returned to the control value. This return was associated with high concentrations of circulating catecholamines. It is not possible to say whether the return to the normal heart rate is the result of increased sympathetic efferent activity or the direct action of circulating catecholamines on the heart (Born et al. 1956; Dawes et al. 1968). It is interesting that the clearance of catecholamines from the foetal circulation is at least as rapid as that from the maternal. As about 50 % of the cardiac output goes to the placenta either the placenta is a major site for clearance (Luschinsky & Singher, 1948) or clearance by other foetal tissues is more rapid than in the adult, which seems unlikely (Epps, 1945; Glowinski, Axelrod, Kopin & Wurtman, 1964; Bojanek, Bozkowa & Kurzepa, 1966; Iversen, de Complain, Glowinski & Axelrod, 1967; Prange, White, Lipton & Kinkead, 1967; Ignarro & Shideman, 1968; Broughton Pipkin, 1972; Mirkin, 1972). The difference in half-life values calculated from infusions and injections is probably a consequence of the non-steady state distribution of labelled catecholamines during the single injection. Thus the values calculated from the infusion data are probably closer to those in vivo. The clearance rates for the foetal and maternal circulations calculated from the infusion data are 840 and 4200 ml./min respectively. If the resting level of catecholamines in the foetal and maternal circulation is 50 pg/ml. their secretion rates would be 42 and 240 pg/min respectively. These are comparable to those reported by Comline & Silver (1961) for unstimulated adrenal glands. 30

This work was completed during the tenure of a Nuffield Foundation Research Fellowship (R. O. R.) and with the help of a grant from the Medical Research Council. We would like to thank Dr G. S. Dawes for valuable discussions and criticism and Drs E. Luther, K. Ritchie and D. Worthington for help with the animals and with the experiments. The technical assistance of A. D. Stevens, N. P. J. Green and Judy A. McCairns is gratefully acknowledged.

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REFERENCES

ADAMSON, K., MULLER-HEUBACH, E. & MEYERS, R. E. (1971). Production of

asphyxia in the Rhesus monkey by administration of catecholamines to the mother. Am. J. Obstet. Gynec. 109, 248-262. AssALI, N. S., HOLM, L. W. & SERGAL, N. (I1962 a). Regional blood flow and vascular resistance of the foetus in utero. Action of vasoactive drugs. Am. J. Obstet. Gynec. 83, 809-817. AssAil, N. S., HOLM, L. W. & SEHGAL, N. (1962 b). Hemodynamic changes in fetal lambs in utero in response to asphyxia, hypoxia and hypercapnia. Circulation Re8. 11, 423-430. BAIN, W. A., GAUNT, W. E. & SUFFOLK, S. F. (1937). Observations on the inactivation of adrenaline by blood and tissues in vitro. J. Physiol. 91, 233-253. BARCROFT, J. & BARRON, D. H. (1945). Blood pressure and pulse rate in the foetal sheep. J. exp. Biol. 22, 63-74. BEARD, R. W. (1962). The responses of the human foetal heart and maternal circulation to adrenaline and noradrenaline. Br. med. J. 1, 443-446. BoJTANEKc, J., BozK~owA, K. & KuRZEPA, ST. (1966). The 5-hydroxytryptophan decarboxylase and monoamine oxidase activities and 5.hydroxytryptamine level in subcellular fractions of the liver during development. Biologia Neonat. 9, 203-214. BORN, V. R., DAWES, G.5S. & MoTT, J. C. (1956). Oxygen lack and autonomic nervous system control of the foetal circulation in the lamb. J. Physiol. 134, 149-166. BROUGHTON P~prPKN, F. (1972). Hepatic inactivation of valb angiotensin II amide (Hypertensin), val5 angiotensin II free acid and adrenaline in immature and adult rabbits. J. Physiol. 225, 35-36P. BROUGHTON PniPKJN, F. & KIRtKPATRiciK, S. M. L. (1973). The blood volumes of foetal and newborn sheep. Q. Jl exp. Physiol. 58, 181-188. BROWN, G. L. (1965). The release and fate of the transmitter liberated by adrenergic nerves. Proc. B. Soc. B 162, 1-19. BRUNDIN, T. (1967). Studies on pre-aortal paraganglia of newborn rabbits. Acta physiol. 8cand. 70, suppl. 290, 1-54. CAIJINaGHAM, B. A. (1967). In Hormones in Blood, vol. 2, ed. GRAY, C. H. & BACHrARAcH, A. L., pp. 564-585. London, New York: Academic Press. CAMPBELL, A. G. M., DAWES, G.5S., F.TsHmAN, A. P. & HYMAN, A. I. (1967). Regional redistribution of blood flow in the mature foetal lamb. .Circulation Be&. 21, 229-235. CA~ssm, S., D)AwES, 0. S. & Ross, B. B. (1964). Pulmonary blood flow and vascular resistance in immature foetal lambs. J. Physiol. 171, 80-89. CHRISTOPHERSON, R. J. & WEB~sTER, A. J. F. (1972). Changes during eating in oxygen consumption, cardiac function and body fluids of sheep. J. Physiol. 221, 441-457. ComwNE, R. S. & SILVERt, M. (1961). The release of adrenaline and noradrenaline from the adrenal glands of the foetal sheep. J. Physiol. 156, 424-444. CommrLNE, R. S., SILVER, I. A. & SILVER, M. (1965). Factors responsible for the stimulation of the adrenal medulla during asphyxia in the foetal lamb.- J. Physiol. 178, 211-238. DAwEs, G. S. (1968). Foetal and Neonatal Physiology. Chicago: Year Book Medical Publishers. DAwEs, 0. 5., DUNCAN, S. L. B., Li~wis, B. V., MERLET, C. L., OWrEN-THOMAS, J. B. & REEvEs, J. T. (1969). Hypoxaemia and aortic chemoreceptor function in foetal lambs. J. Physiol. 201, 105-116. 2 P HY 248

32

C. T. JONES AND R. 0. ROBINSON

DAWES, G. S., Fox, H. E., LEDUC, B. M., LIGGINs, G. C. & RICHARDS, R. T. (1971). Respiratory movements and rapid eye movement sleep in the foetal lamb. J. Physiol. 220, 119-143. DAWES, G. S., LEWIS, B. V., MILLIGAN, J. E., ROCHE, M. R. & TALNER, N. S. (1968). Vasomotor responses in the hind limbs of foetal and newborn lambs to asphyxia and aortic chemoreceptor stimulation. J. Physiol. 195, 55-81. DAWES, G. S., MOTT, J. C. & RENNICK, B. R. (1956). Some effects of adrenaline, noradrenaline and acetylcholine on the foetal circulation in the lamb. J. Physiol. 134, 139-148. DUNNE, J. T., MILLIGAN, J. E. & THOMAS, B. W. (1972). Control of renal circulation in the foetus. Am. J. Obstet. Gynec. 112, 323-329. ENGLEMAN, K. & PORTNOY, B. (1970). A sensitive double-isotope derivative assay for norepinephrine and epinephrine. Circulation Res. 26, 53-57. Epps, H. M. R. (1945). The development of amine oxidase activity by human tissues after birth. Biochem. J. 39, 37-42. EULER, U. S. VON & FLODING, I. (1955). A fluorimetric method for differential estimation of adrenaline and noradrenaline. Acta physiol. scand. 33, supply. 118, 45-62. FOLKOW, B., HAGGENDAL, J. & LISANDER, B. (1967). Extent of release and elimination of noradrenaline at peripheral adrenergic nerve terminals. Acta physiol. scand. suppl. 307, 5-38. FRIEDMAN, W. F., LESCH, M. & SONNENBLICIC, E. H. (1973). Neonatal Heart Disease, pp. 21-39. New York: Grune and Stratton. FRIEDMAN, W. F., POOLE, P. E., JACOBOWITZ, D., SEAGREN, S. C. & BRAUNWALD, E. (1968). Sympathetic innervation of the developing rabbit heart. Biochemical and histochemical comparisons of fetal neonatal myocardium. Circulation Res. 23, 25-32. GLOWINSKI, J., AXELROD, J., KOPIN, I. J. & WURTMAN, R. J. (1964). Physiological disposition of 3H-norepinephrine in the developing rat. J. Pharmac. exp. Ther. 146, 48-53. GOODWIN, J. W. (1968). The impact of the umbilical circulation on the foetus. Am. J. Obstet. Gynec. 100, 461-471. GOODWIN, J. W., MILLIGAN, J. E., THOMAS, B. & TAYLOR, J. R. (1973). The effect of aortic chemoreceptor stimulation on cardiac output and umbilical blood flow in the fetal lamb. Am. J. Obstet. Gynec. 116, 48-56. HAGGENDAL, J. (1962). An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues. Acta physiol. 8cand. 59, 242-254. HAGGENDAL, J. (1966). Newer developments in catecholamine assay. Pharmac. Rev. 18, 325-329. HALL, E. K. (1957). Acetylcholine and epinephrine effects on the embryonic rat heart. J. cell. comp. Physiol. 49, 187-200. HERTING, G. & AXELROD, J. (1961). Fate of tritiated noradrenaline at sympathetic nerve endings. Nature, Lond. 192, 172-173. IGNARRO, L. J. & SHIDEMAN, F. E. (1968). Catechol-o-methyltransferase and monoamine oxidase activities in the heart and liver of the embryonic and developing chick. J. Pharmac. exp. Ther. 159, 29-37. IVERSEN, L. L., DE COMPLAIN, J., GLOWINSKI, J. & AXELROD, J. (1967). Uptake, storage and metabolism of norepinephrine in tissues of the developing rat. J. Pharmac. exp. Ther. 157, 509-516. IVERSEN, L. L. & JARROT, B. (1970). Modification of an enzyme radiochemical assay procedure for noradrenaline. Biochem. Pharmac. 19, 1841-1843.

FOETAL CATECHOLAMINES

33

JOELSSON, I., BARTON, M. D., DANIEL, S., JAMES, S. & ADAMSONS, K. (1972). The response of the unanaesthetised sheep fetus to sympathomimetic amines and adrenergic blocking agents. Am. J. Obstet. Gynec. 114, 43-50. LADNER, C., BRINKMAN, C. R., WESTON, P. V. & ASsALI, N. S. (1970). Dynamics of uterine circulation in pregnant and non-pregnant sheep. Am. J. Physiol. 218, 257-263. LUND, A. (1949). Fluorimetric determination of adrenaline in blood, I, II and III. Acta pharmac. tox. 5, 75-94, 121-128, 231-247. LusCHINSKY, H. L. & SINGHER, H. 0. (1948). Identification and assay of monoamine oxidase in the human placenta. Archs Biochem. 19, 95-107. MCCARTY, L. P., LEE, W. C. & SHIDEMAN, F. E. (1960). Measurement of the inotropic effects of drugs on the innervated and noninnervated embryonic chick heart. J. Pharmac. exp. Ther. 129, 315-321. MCCULLOUGH, H. (1968). Semi-automated method for the differential determination of plasma catecholamines. J. clin. Path. 21, 759-763. McILvAiNE, T. C. (1921). Buffer solution for colourimetric comparison. J. biol. Chem. 49, 183-186. MIRKIN, B. L. (1972). Ontogenesis of the adrenergic nervous system: functional and pharmacologic implications. Fedn Proc. 31, 65-73. MIRFIN, B. L., BROWN, D. M. & ULSTROM, R. A. (1966). Catecholamine binding protein: binding of tritium to a specific protein fraction of human plasma following in titro incubation with tritiated noradrenaline. Nature, Lond. 212, 1270-1271. PASSON, P. G. & PEULER, J. D. (1973). A simplified assay for plasma norepinephrine and epinephrine. Analyt. Biochem. 51, 618-631. PRANGE, A. J., WHITE, J. E., LIPTON, M. A. & KINKEAD, A. M. (1967). Influence of age on monoamine oxidase and catechol-o-methyltransferase in rat tissues. Life Sci. 6, 581-586. RENZINI, V., BRUNORI, C. A. & VALORI, C. (1970). A sensitive and specific fluorimetric method for determination of noradrenaline and adrenaline in human plasma. Clinica chim. Acta 30, 587-594. REYNOLDS, S. R. M., ARDRAN, G. M. &. PRICHARD, M. M. L. (1954). Observations on regional circulation times in the lamb under foetal and neonatal conditions. Publs Carnegie Instn, no. 603. Contr. Embryol. 35, 73-92. SHAH, P. P., TEE, A. R., ENGLISH, P. D., REDMAN, B. T. & BUINYAN, J. (1972). The identification of a lipid-mobilising factor from sheep midbrain. Biochem. J. 130, 467-473. SMITH, R. W., MORRIS, J. A. & ASSALI, N. S. (1964). The effects of chemical mediators on the pulmonary and ductus arteriosus circulation in the foetal lamb. Am. J. Obstet. Gynec. 89, 252-260. TATE, J. F. & BURSTEIN, S. (1964). In The Hormones, vol. v, ed. PINCUS, G., THIMANN, K. V. & ASTWOOD, E. B., pp. 441-557. New York, London: Academic Press.

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