Enzyme activities in the sheep placenta during the last three months of pregnancy

133

Biochimica et Biophysica Acta, 497 (1977) 133--143

© Elsevier/North-Holland Biomedical Press

BBA 28181 ENZYME ACTIVITIES IN THE SHEEP PLACENTA DURING THE LAST THREE MONTHS OF PREGNANCY

ELIZABETH M. EDWARDS, J.M. RATTENBURY~G. CAROLE E. VARNAM, USHA K. DHAND, MARJORIE K. JEACOCK and D.A.L. SHEPHERD Department of Physiology and Biochemistry, University of Reading, Whiteknights, Reading, RG6 2AJ (U.K.)

Summary In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and ketone body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy. The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP ÷) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do no occur in the sheep placenta. It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.

134 Introduction

Three stages are clearly discernable in the development of the sheep placenta, namely implantation, growth and maturation [1]. The implantation stage terminates at approximately the 50th day of pregnancy when the placental cotyledons have reached a fixed number. The second stage is characterised b y rapid placental growth and terminates at approximately 90 days of gestation when the placental weight has reached its maximum [2,3]. The last stage which extends from 90 days to term may be called the maturation stage because although the placenta reaches its maximum size at three months, the foetus continues to grow throughout gestation and hence the function of the placenta is greatly increased relative to its weight during the last two months of pregnancy. The placenta does not only function as a simple organ of exchange between the ewe and her foetus. Metabolism within the placenta may result in the release into the foetal circulation of substrates derived from maternal blood which have been chemically modified b y the placenta. An established example of this is the production of fructose from maternal glucose b y the ovine placenta [4] b u t other important metabolite interconversions may exist. In addition the utilization b y the placenta of substrates present in maternal blood may also prevent or limit their transfer to the foetus. Any changes in the activities of placental enzymes during pregnancy may well indicate whether particular metabolic pathways are of greater or lesser significance as pregnancy proceeds. It is generally accepted that the units of a particular enzyme activity measured in vitro reflect the amount of that particular enzymically active protein in the tissue. Changes in the amounts of certain enzyme proteins in tissues are a means whereby the rate of flux of metabolites through a metabolic pathway can be regulated. Such changes occur over relatively long periods of time and this means of metabolic regulation provides 'coarse' control of metabolism. In addition to the amount of enzyme protein present the rate of a reaction may also be regulated b y the concentration of reactants and in some cases b y the concentration of effectors. 'Fine' control of metabolic flux is believed to be brought about in this way. Even though measureable enzyme activity is b u t one factor indicating the rate of utilization of a particular substrate it has been argued that knowledge of enzyme activities may reveal clues about the metabolic potential of an organ during development [5]. In order to provide some indication of the importance of the metabolism of pyruvate, citrate, acetate, ketone bodies and certain amino acids in the placenta at different stages of pregnancy we have studied the activities of 18 enzymes concerned with their metabolism during the growth and maturation stages. Material and Methods Chemicals

Tris, sodium pyruvate, oxaloacetic acid, monosodium phosphoenolypyruvate, acetyl-CoA, ATP, ADP, NAD ÷, NADH, GSH, CoA, acetyl phosphate (dilithium salt}, carbamyl phosphate (dilithium salt) and all enzymes were obtained from

135

the Boehringer Corp. (London) Ltd. (Lewes, Sussex, U.K.); 2-mercaptoethanol, pyridoxal 5-phosphate, IDP and bovine serum albumin {crystallized and lyophilized) were from Sigma (London) Chemical Co. Ltd. (London, SW6, U.K.); o-aminobenzaldehyde was from K and K Laboratories Inc. (Plainview, Hollywood, California, U.S.A.); 5,5'-dithiobis-(2-nitrobenzoic acid) and diketene were from Ralph N. Emanuel Ltd. (Alperton, Middlesex, U.K.); diacetyl monoxime, hyamine hydroxide and dioxan-based scintillator were from Koch-Light Laboratories (Colnbrook, U.K.), and I~[1-'4C]ornithine monohydrochloride and NaH'4CO3 were from the Radiochemical Centre {Amersham, Bucks, U.K.). All other chemicals were obtained from BDH Chemicals Ltd. (Poole, Dorset, U.K.) and were of Analar grade whenever possible. Sodium acetoacetate was prepared from methyl acetoacetate b y the method of Hall [6] and was found to be at least 95% pure. Acetoacetyl-CoA was synthesised from CoA and diketene b y the method of Simon and Shemin [7] and the concentration of acetoacetyl-CoA was determined spectrophotometrically

[81. Animals and collection o f tissue Dorset Horn ewes tupped b y Suffolk rams on k n o w n dates were purchased from Reading University farms. The stage of gestation was confirmed b y radiological examination at the Grassland Research Institute, Hurley. The ewes were housed and fed as described previously [9]. Immediately after foetuses had been delivered by Caesarian section [9] cotyledons were resected and placed in ice-cold 0.15 M KC1. Wedge shaped samples for the preparation of tissue slices and homogenates were cut through the cotyledons after t h e y had been trimmed free of the cotyledonary branches of the umbilical vessels. No attempt was made to separate maternal from foetal cotyledonary tissue since it was considered that the maternal and foetal tissue together form the functional unit. Measurement of urea production Slices of placental cotyledons (0.25 mm thick, 50--150 mg wet weight), cut with a Stadie-Riggs microtome, were incubated at 37°C in 5.5 ml bicarbonate buffer [10] containing 18.2 mM ammonium chloride and 1.82 mM L-ornithine hydrochloride with 95% 02/5% CO2 as the gas phase. After 1 h, 0.5 ml 9 M perchloric acid was added to each of the incubation flasks and urea was determined in the protein-free supernatants b y condensation with diacetyl monoxime [11]. F o u r replicate incubations were performed for each cotyledon, and blank incubations were carried o u t without addition of substrate. Measurement o f acetoacetate production Samples of tissue were homogenised in a high speed mixed emulsifier (Silverson Machines Ltd., London, S.E.1.) in 20 volumes of a medium consisting of 0.25 M sucrose, 1 mM 2-mercaptoethanol and 10 mM Tris adjusted to pH 7.4 with HC1. The homogenate was then sonicated with intermittent bursts using a 9 mm probe at 6--7 kcycles, 8--9 p amplitude for a total time of 45 s (100 W ultrasonic disintegrator, Measuring and Scientific Equipment Ltd. London, S.W.1.) and centrifuged at 38 000 × g for 30 min. These procedures were carried out at 0--4 ° C. Aliquots of supernatant were incubated with an acetyl-CoA

136 generating system for 10 min at 37°C in the medium described b y Williamson et al. [12] for their assay of hydroxymethylglutaryl-CoA synthase. The acetoacetate formed was determined enzymicaUy [13].

Measurement of enzyme activities Samples of the cotyledons to be assayed for pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), isocitrate dehydrogenase (NADP +) (EC 1.1.1.42) and acetyl-CoA synthase (EC 6.2.1.1) activities were homogenised in 5--10 volumes of 0.25 M sucrose. Tissue to be assayed for 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) activity was homogenised in 10 volumes of a medium adjusted to pH 7.4 containing 10 mM succinate, 1 mM EDTA, 0.6 mM ATP and 1 mM NAD ÷. The activities of these enzymes were determined in whole homogenates which had been subjected to sonication at 0°C except for ATP citrate (pro-3S)-lyase and acetylCoA synthetase, activities of which were determined in the supernatant after the homogenates had been centrifuged at 600 X g for 15 min to remove the nuclear fraction. This supernatant was frozen and thawed three times. Tissues to be assayed for pyruvate carboxylase (EC 6.4.1.1) and phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) activities were homogenised in 5--10 volumes of a medium containing 0.2 M sucrose, 20 mM triethanolamine, 1 mM GSH and 1 mM EDTA adjusted to pH 7.5 with HC1. Aliquots of these homogenates were freeze-dried and resuspended in glass distilled water prior to assay. The preparation of the homogenates was carried out at 0--4°C and the activities of these 12 enzymes were assayed as described previously [9,14]. 3-Ketoacid CoA-transferase (EC 2.8.3.5) and a c e t y l - C o A acetyltransferase (EC 2.3.1.9) activities were assayed in tissue supernatants prepared as for the determination of the rate of acetoacetate production. The activity of 3-ketoacid CoA-transferase was determined b y measuring the decrease in extinction at 310 nm resulting from the disappearance of the acetoacetyl-CoA-Mg 2÷ complex in the presence of succinate and iodoacetamide [15]. The activity of acetyl-CoA acetyltransferase was determined by measuring the decrease in extinction at 310 nm resulting from the disappearance of the acetoacetyl-CoA-Mg 2÷ complex in the presence of CoA [12]. Tissue to be assayed for arginase (EC 3.5.3.1) activity was homogenised in 20 volumes of a medium containing 0.16 M sodium chloride and 36 mM manganous sulphate in a Vortex homogeniser (Measuring & Scientific Equipment Ltd. London, S.W.1) at 0--4°C. The tissue homogenate was activated b y incubation at 37°C for 30 min. Aliquots were then added to a solution containing 0.2 M L-arginine hydrochloride and 16 mM glycine adjusted to pH 9.45 with NaOH. The reaction was stopped by addition of perchloric acid. Any urea in the supernatant was determined by condensation with diacetyl monoxime [11]. Tissue to be assayed for ornithine carbamoyltransferase (EC 2.1.3.3) activity was homogenised in 10 volumes 3 mM cetyltrimethyl ammonium bromide in a glass-teflon Potter-Elvejhem homogeniser at 0--4°C. The homogenate was centrifuged at 4000 X g for 15 min at 0--4°C and activity was determined in the supernatant b y measuring the rate of production of citrulline in the presence of ornithine and carbamyl phos-

137 phate [16]. Any citrulline formed was determined b y the diacetyl monoxime method [11]. Tissue to be assayed for ornithine decarboxylase (EC 4.1.1.17) activity was homogenised at 0--4°C in a Potter-Elvejhem homogeniser in 5 volumes of a medium containing 25 mM Tris, 1 mM EDTA and 5 mM dithiothreitol adjusted to pH 7.5 with HC1. The homogenate was centrifuged at 20 000 X g for 10 min and the supernatant obtained recentrifuged at 105 000 X g for 60 min at 0--4°C. Activity was determined in the final supernatant fraction b y measuring the rate of production of radioactive CO2 evolved from L-[1-'4C] ornithine. 0.5 ml of the supernatant was added to 0.5 ml of a solution containing 100 mM glycylglycine, pH 7.2, 10 mM dithiothreitol, 0.4 mM pyridoxal phosphate and 2 mM L-[1-14C]ornithine (0.2 pCi) in flasks closed with subaseal stoppers from which were suspended glass vials containing 0.2 ml hyamine hydroxide (1 mol/1 methanol). The flasks were incubated for 60 min at 37°C. The reaction was stopped b y addition of 0.5 ml 2.5 M trichloracetic acid and the incubation continued for a further 30 min. The vials containing hyamine hydroxide were then placed in 10 ml dioxan-based liquid scintillator. The radioactivity was counted in a Corumatic 25 liquid scintillation counter (Tracerlab, Weybridge, Surrey, U.K.) and corrections for efficiency were made using an external standard. Tissue to be assayed for ornithine aminotransferase (EC 2.6.1.13) activity was homogenised at 0--4°C in a Potter-Elvejhem homogeniser in a medium containing 0.25 M sucrose, 0.1 M potassium dihydrogen orthophosphate and 10 mM 2-mercaptoethanol adjusted to pH 8.0 with KOH. The activity was determined by measuring the rate of formation of A-pyrroline5-carboxylate using o-aminobenzaldehyde [ 17 ]. In all cases the rates of the enzyme catalysed reactions were constant over the incubation period and proportional to the amount of homogenate added. Enzyme activities have been calculated as pmol substrate transformed/min/g wet weight cotyledon. Chemical assays

The protein contents of tissue homogenates were determined b y the method of Lowry et al. [18] using crystallized and lyophilized bovine serum albumin as a standard. The DNA contents of tissue homogenates were determined using the method described b y Burton [19]. The standard used was 2-deoxyribose and the concentration of DNA in the sample was calculated b y assuming a mean residue weight of 325 and one reacting deoxyribose molecule per two residues. Results and Discussion

The results are presented as the mean values for the last three 31 day periods of pregnancy. During the period from 55--85 days the placenta may be considered as being in the growth phase of development whilst during the two remaining periods the placenta may be considered as maturing. The results presented in Table I for the DNA content of placental cotyledons support this view since t h e y indicate that the number of cells per gram of tissue is significantly greater after 86 days gestation than in the previous month. There is no significant difference in the mean DNA content between 86--116 and 117--147

138 TABLE I THE PROTEIN CONTENT AND DEOXYRIBONUCLEIC ACID CONTENT OF PLACENTAL COTYLEDONS OF SHEEP DURING THE LAST THREE MONTHS OF PREGNANCY E x p e r i m e n t a l d e t a i l s are g i v e n in t h e t e x t . V a l u e s are g i v e n as m e a n s ± S.E. w i t h t h e n u m b e r o f a n i m a l s i n p a r e n t h e s e s . R e s u l t s are e x p r e s s e d as m g / g w e t w e i g h t . Days from conception

Protein content D e o x y r i b o n u c l e i c acid content

55--85

86--116

117--147

57 ± 3 (24) 1.72 ± 0 . 2 0 (24)

96 +- 8 (12) 2 . 3 5 +- 0 . 2 4 ( 1 2 )

118 ± 6 (28) 2.51 +- 0 . 1 6 ( 2 7 )

days of pregnancy. Since the weight of the sheep placenta is reported not to change after the third m o n t h of pregnancy [3] then it is apparent that the total number of placental cells does not alter markedly after 86 days. However, as indicated in Table I the protein content rises significantly by 67% between the third and fourth months of pregnancy and by 23% between the fourth and fifth months. The activities of 16 enzymes are shown in Table II. No activity of either phosphoenolpyruvate carboxykinase (GTP) or ornithine carbamoyltransferase could be detected at any stage studied. The constant and high activity of pyruvate kinase relative to most of the other enzymes studied suggests that the sheep placenta has a high glycolytic potential from 55 days onwards. High activity of pyruvate kinase has also been reported in the rat [20] and human [21] placentas but in these species, unlike the sheep, the activities decline as pregnancy advances. The high activity of lactate dehydrogenase in the sheep placenta will enable pyruvate formed from carbohydrate to be converted readily to lactate. Calculations based on the arterio-venous differences for lactate across the uterine and umbilical circulations [22] and the assumptions that uterine blood flow is 1.5 1/min [23] umbilical blood flow is 200 ml/kg foetus/min [24] and foetal weight near term is 3.5 kg [9] reveal that there is a release of lactate by the uterus and placenta into both the maternal and foetal circulations at rates of 105 and 112 pmol/ min respectively. The activities of pyruvate kinase and lactate dehydrogenase when related to total placental weight (assumed to be 0.45 kg) are far in excess of the rate of catalysis required if the lactate released is derived from glucose. It is probable that most of the acetyl CoA required for citric acid cycle metabolism is derived from pyruvate but no measurement of pyruvate dehydrogenase (lipoate) activity has been made in the sheep placenta. In contrast to the output of lactate by the sheep placenta calculations based on arterio-venous differences for plasma alanine [25] uterine and umbilical blood flows [23,24] and on the additional assumptions that maternal and foetal haematocrits are 30 and 35 per cent respectively [26] indicate that there is a net uptake of alanine by the placenta of 167 pmol/min. Alanine must either be utilized for protein synthesis or converted to pyruvate within placental tissue. The ability to convert alanine to pyruvate as indicated by the total activity of alanine aminotransferase in the mature placenta is 481 pmol/min and this is approximately three

Pyruvate kinase Lactate dehydrogenase Alanine aminotransferase Pyruvate carboxylase Ma]ate dehydrogenase Aspartate aminotransferase A T P citrate(pro-3S)-lyase Citrate (si)-synthase I s o c i t r a t e d e h y d r o g e n a s e ( N A D P +) Acetyl-CoA synthetase Acetyl-CoA acetyltransferase 3-Hydroxybutyrate dehydrogenase 3-Ketoacid CoA-transferase Arginase Ornithine decarboxylase Ornitihine-oxo-acid aminotransferase

Enzyme

34.8 29.1 0.51 0.034 118 2.59 0.023 3.76 2.32 0.013 0.368 0.181 0.077 0.99 0.315 0.72

55--85 37.2 53.5 1.13 0.049 103 8.39 0.024 6.41 4.33 0.011 0.869 0.327 0.203 0.97 0.216 1.37

± 6.6 (3) +- 1 8 . 8 ( 3 ) ± 0.34 (2) (1) ± 24 (3) ± 1.25 (2) ± 0.003 (6) ± 1.20 (2) ± 0.52 (2) ± 0.004 (6) ± 0.201 (6) ± 0.093 (6) ± 0.116(5) ± 0.35 (6) • 10 -3 ± 0.073 • 10 -3 (3) ± 0.26 (5)

wet weight)

86--116

transformed/min/g

+ 7.2 (5) ± 4.6 (6) + 0.09 (6) +- 0 . 0 1 3 ( 5 ) ± 40 (5) ± 0.36 (6) ± 0.001 (2) ± 0.23 (4) +- 0 . 3 1 ( 6 ) ± 0.005 (2) ± 0.160 (4) ± 0.023 (5) ± 0.065 (4) *- 0 . 2 5 ( 9 ) • 10 -3 ± 0.052 • 10 -3 (8) +- 0 . 1 3 ( 8 )

Days from conception:

Activity (pmol substrate

42.6 15.9 1.07 0.131 114 5.44 0.024 4.55 4.08 0.017 0.785 0.485 0.048 4.97 0.029 0.93

117--147 ± 4.4 (5) ± 5.8 (5) ± 0.22 (5) ± 0.017 (7) ± 26 (5) ± 1.17 (5) ± 0.004 (8) +- 0 . 3 3 ( 9 ) + 0.30 (10) ± 0.006 (8) ± 0.164 (11) ± 0.095 (14) ± 0.021 (11) ± 1.14 (14) • 10 -3 ± 0.013 • 10 -3 (8) ± 0.12 (9)

E x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e t e x t . A c t i v i t i e s a r e g i v e n a s m e a n v a l u e s + S E w i t h t h e n u m b e r o f a n i m a l s i n p a r e n t h e s e s . T h e a c t i v i t i e s o f p y r u v a t e k i n a s e , lactate dehydrogenase, malate dehydrogenase, citrate(si)-synthase and isocitrate dehydrogenase ( N A D P ÷) w e r e d e t e r m i n e d a t 2 5 ° C , w h i l s t t h e a c t i v i t i e s o f t h e o t h e r enzymes were determined at 37°C.

ACTIVITIES OF PYRUVATE KINASE, LACTATE DEHYDROGENASE, ALANINE AMINOTRANSFERASE, PYRUVATE CARBOXYLASE, MALATE DEHYDROGENASE, ASPARTATE AMINOTRANSFERASE, ATP CITRATE (pro-3S)-LYASE, C I T R A T E ( s i ) - S Y N T H A S E , ISOCITRATE DEHYDROGENASE (NADP+), ACETYL-CoA SYNTHETASE, ACETYL-CoA ACETYLTRANSFERASE, 3-HYDROXYBUTYRATE DEHYDROGENASE, 3-KETOACID CoA-TRANSFERASE, ARGINASE, ORNITHINE DECARBOXYLASE AND ORNITHINE-OXO-ACID AMINOTRANSFERASE IN PLACENTAL COTYLEDONS OF SHEEP DURING THE LAST THREE MONTHS OF PREGNANCY

TABLE II

~D

140 times the calculated alanine uptake. The activity of alanine aminotransferase per gramme of tissue is significantly less during the growth phase than that found in the mature placenta. One fate of pyruvate formed from either carbohydrate or alanine is its conversion to oxaloacetate. That this process assumes more importance as the placenta ages is indicated by the fact that the activity of pyruvate carboxylase is four times greater near term compared with that found during the third m o n t h of pregnancy. There is an uptake of aspartate from both the uterine and umbilical circulations [25]. In addition to probable roles in protein and nucleic acid synthesis, aspartate may be converted to oxaloacetate at a greater rate in the mature placenta than in the growing organ since aspartate aminotransferase activity is greater in the mature organ. Oxaloacetate formed is likely to be in equilibrium with malate at all times since the activity of malate dehydrogenase is substantiaUy higher than that of any other enzyme determined in the study. The provision of extramitochondrial oxaloacetate by way of the reaction catalysed by ATP citrate(pro-3S)lyase would not appear to be of any great quantitative significance. It is unlikely t h a t oxaloacetate derived from any source could be converted to glucose in the sheep placenta since phosphoenolpyruvate carboxykinase (GTP) activity could not be detected in this tissue in contrast to the rat [20] and h u m a n [21]. That phosphoenolpyruvate carboxykinase activity is not absent from all foetal lamb tissues is indicated by the results of our studies in foetal lamb liver in which similar methods for preparation of tissue homogenates and assay of enzyme activity were used [9]. Hence gluconeogenesis from substrates such as alanine, pyruvate and lactate is unlikely to occur in the sheep placenta. There are no changes in the activity of citrate(si)-synthase per g tissue and hence the potential for energy production by way of the citric acid cycle does not change during the maturation of the placenta when it might be expected that the energy requirements would be increased to enable active transport to the growing foetus to proceed more rapidly. The potential utilization of citrate as a source of reduced NADP ÷ by way of the isocitrate dehydrogenase reaction increases significantly during the growth phase but remains unchanged after 86 days. An important role of reduced NADP in the placenta is in the interconversion of steroid hormones resulting in the formation of progesterone and oestrogens [27]. Acetate is unlikely to be metabolised rapidly by placental tissue since acetylCoA synthetase activity is relatively low. Calculations based on the arteriovenous differences for acetate [28] and on blood flows [23,24] show that there is a net uptake of acetate by the uterus and placenta of 111 pmol/min. Assuming a mature placental weight of 0.45 kg the total placental activity of acetyl-CoA synthetase near term is not sufficient to account for the disappearance of acetate and therefore it must be assumed that acetate is being utilised by tissues other than the placenta which are drained by the uterine veins. Acetoacetate production from an acetyl-CoA generating system was found to be very variable and there were no significant differences found between the three 31 day periods studied. The mean rate of production in nineteen placentae was 0.270 + 0.128 pmol/min/g wet weight. Therefore, the rate of ketone

141 b o d y production b y a 0.45 g placenta near term would not be greater than 121 pmol/min. In the mature placenta the activity of 3 - h y d r o x y b u t y r a t e dehydrogenase is significantly higher than in the growth phase and is sufficient to convert any acetoacetate synthesised to 3-hydroxybutyrate. The calculated release of acetoacetate plus 3-hydroxyburyrate into the foetal circulation based on the studies of Morriss et al. [29] is only 6 #mol/min. If acetoacetate is synthesised in vivo at 121 pmol/min/placenta then approximately 95% might be expected to be released into the maternal circulation and hence contribute to the ketosis found in pregnant ewes. The presence of placental 3-hydroxybutyrate dehydrogenase activity may play a role in the maintenance of the low acetoacetate concentration found in the foetal circulation when the maternal acetoacetate concentration is raised b y starvation [29]. The placenta has the capacity to oxidise ketone bodies since 3-ketoacid CoA-transferase activity is present. There are no significant differences b e t w e e n the mean values during the last three months of pregnancy. The activity of this enzyme was found to be considerably less than that of acetyl-CoA acetyltransferase an enzyme believed to be concerned not only with ketone b o d y metabolism b u t also with the oxidation of long chain fatty acids. Since the activity of the acetyltransferase enzyme is only between 10--20% o f the activity of citrate synthase it seems unlikely that either ketone bodies or fatty acids make a substantial contribution to the oxidative metabolism of the placenta b y way of the citric acid cycle. The presence of the ketone b o d y oxidative pathway in the placenta suggests that the production of acetoacetate from acetyl-CoA need not proceed by way of 3-hydroxy-3-methylglutaryl-CoA. It could occur b y reversal of the oxidative pathway coupled with non-specific deacylation as discussed b y Bush and Milligan [30]. If the uptake of alanine and aspartate b y the placenta is followed b y conversion to the corresponding ketoacids, the fate of the amino group must be considered. Release of this nitrogen from the placenta in the form o f urea is extremely unlikely since urea formation could not be demonstrated in placental slices incubated with ammonium salts and ornithine. Since amino-nitrogen is not released from the placenta in the form of alanine the possibility exists that amino groups may be released in the form of glutamine provided that glutamate is available. There is an uptake of glutamate b y the sheep placenta from b o t h maternal and foetal circulations [25] but no information is available on the concentration of glutamine in either the uterine or umbilical circulations. The inability of the placenta to synthesise urea is in contrast to the finding that slices of foetal ovine liver incubated in similar conditions can synthesise urea at a mean rate of 0.310 pmol/min/g wet weight of liver at 37°C [31]. The absence of detectable ornithine carbamoyltransferase activity in placenta and its presence in foetal liver can account for these differences [32]. However, the placenta could produce urea from arginine since arginase activity is present. There is a five-fold increase in arginase activity during the last month of pregancy. From the data of Hopkins et al. [25] and the consideration of relative plasma flows it is evident that there is a net uptake of arginine b y the sheep uterus and placenta and since the activity of placental arginase far exceeds this uptake it is likely that any arginine not incorporated into protein will be converted to ornithine and urea. Ornithine is taken up from maternal and foetal circulations [25]. This orni-

142

thine together with any derived from arginine must be metabolised. The high activity of placental ornithine-oxo-acid aminotransferase compared with that found in sheep liver and kidney [31] suggests that the conversion of ornithine to A-pyrroline 5-carboxylate may be important in the placenta. This may account for the failure of raised maternal plasma ornithine concentrations to be reflected in foetal plasma [33]. In mammalian tissues A-pyrroline 5-carboxylate can be converted to either glutamate or proline. Since there is no release of glutamate it is suggested that A-pyrroline 5-carboxylate may be converted into proline and released from the placenta. It is unlikely that decarboxylation and subsequent polyamine synthesis is quantitatively an important route of ornithine metabolism since ornithine decarboxylase activity is very much lower than that of the aminotransferase. The activity of ornithine decarboxylase has been related to nucleic acid synthesis and organ growth [34]. The fact that the activity of this enzyme declines 10-fold during the maturation stage is notable since by this stage the placenta has ceased to grow and its DNA content is constant (Table I). It must be emphasised that the activities of enzymes in vivo are dependent not only on the amount of enzyme protein present but also on the concentration of substrates, products and possible allosteric effectors. Even so it might be expected that the enzyme profile of the sheep placenta would be markedly altered during the last month of pregnancy to allow placental metabolism to keep pace with the requirements of the growing foetus. The present study shows that maximal activities of most of the enzymes studied are achieved by the time placental weight has reached its maximum. Only arginase and possibly pyruvate carboxylaseaclfivity increases in proportion to foetal weight during placental maturation whilst ornithine decarboxylase activity declines during this time.

Acknowledgements This investigation was supported by a grant from the Agricultural Research Council. E.M.E., J.M.R. and G.C.E.V. were recipient of Postgraduate Studentships from the Ministry of Agriculture, Fisheries and Food. We thank Mr. I.A.N. Wilson of the Grassland Research Institute, Hurley, Berks. who carried out the radiological examination of the pregnant sheep. References 1 K u l h a n e k , J . F . , Meschia, G., M a k o w s k i , E.L. a n d B a t t a g H a , F.C. ( 1 9 7 4 ) A m . J. P h y s i o l . 2 2 6 , 1 2 5 7 - 1263 2 D a w e s , G.S. ( 1 9 6 8 ) F o e t a l a n d N e o n a t a l P h y s i o l o g y , p. 2 5 , Yeaz B o o k Medical Publishers Inc., Chicago 3 Everitt, C.G. ( 1 9 6 4 ) N a t u r e 2 0 1 , 1 3 4 1 - - 1 3 4 2 4 B r i t t o n , H . G . , H u g g e t t , A. S t . G . a n d N i x o n , D . A . ( 1 9 6 7 ) B i o c h i m . B i o p h y s . A c t a 1 3 6 , 4 2 6 - - 4 4 0 5 S t a v e , U. ( 1 9 7 0 ) P h y s i o l o g y of the P e r i n a t a l P e r i o d (Stave, U., ed.), p p . 5 5 9 - - 5 9 4 , A p p l e t o n - C e n t u r y Crofts, N e w Y o r k 6 Hall, L.M. ( 1 9 6 2 ) A n a l y t i c a l B i o c h e m i s t r y 3, 7 5 - - 8 0 7 S i m o n , E.J. a n d S h e m i n , D. ( 1 9 5 3 ) J . A m - Chem~ S o c . 75, 2 5 2 0 8 S t e r n , J . R . ( 1 9 5 6 ) J. Biol. C h e m . 2 2 1 , 3 3 - - 4 4 9 E d w a r d s , E.M., D h a n d , U.K., J e a c o c k , M.K. a n d S h e p h e r d , D . A . L . ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 399, 217--227

143 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34

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