210

Biochimica el Biophysica Acta, 630 (1980) 210--219 © Elsevier/North-Holland Biomedical Press

BBA 29286

U R E A S Y N T H E S I S IN T H E L I V E R A N D K I D N E Y OF I ) E V E L O P I N G S H E E P

J.M. RATTENBURY *, MARJORIE K. JEACOCK and D.A.L. SHEPHERD

Department o f Physiology and Biochemistry, University o f Reading, Whilek~zight~,. Reading, RG6 2AJ (U.K.t

(Received October 22nd, 1979)

Key words: Urea synthesis: Development: (Sheep)

S u m m a r y

In o rder to establ ish if the urea found in foeta l f luids in sheep could be of foe ta l origin and w h e t h e r there are changes in the abi l i ty of ovine liver to syn- thesise u rea dur ing foe ta l and pos tna ta l d e v e l o p m e n t , the rates of urea produc- t ion f r o m a m m o n i u m and b i ca rbona t e ions have been measured in liver and k idney slices f r o m animals aged f r o m 50 days c o n c e p t u a l age to 16 weeks af ter b i r th , and in p r e g n a n t and n o n - p r e g n a n t ewes. The activit ies of five e n z y m e s d i rec t ly involved in the b iosyn thes i s of u rea have also been de te rmined .

Urea was f o u n d to be synthes ised by foeta l liver f r o m at least 50 days c o n cep tua l age at ra tes similar to those observed in adul t ewes. t t ighest rates of urea synthes is per uni t weight o f liver were found i m m e d i a t e l y a f te r birth. In the liver there were s ignif icant pos i t ive cor re la t ions be tween the rates of urea synthes is by slices and the activit ies o f e a r b a m o y l p h o s p h a t e synthase (am- mon ia ) (EC 2.7.2 .5) , a rg in inosucc ina te syn the ta se (EC 6 . 3 . 4 . 5 ) a n d arginino.- succ ina te lyase EC 4.3.2 .1) . Orn i th ine c a r b a m o y l t ransferase (EC 2.1.3.3) activ- i ty was highest in the livers of r u m i n a t i n g animals . Hepa t i c arginase act ivi ty (EC 3.5 .3 .1) was highest dur ing the late foe ta l life and in the m a t u r e foetuses the ac t iv i ty was t en- fo ld grea ter t han tha t in ma te rna l liver.

Urea was n o t synthes ised f r o m a m m o n i a and b i ca rbona te in k idney slices and ne i the r o rn i th ine c a r b a m o y l t ransferase ac t iv i ty nor a rg in inosuecina te syn- the tase ac t iv i ty could be de t ec t ed . The ac t iv i ty of renal arginase was at least 70 t imes less than t ha t f o u n d in the liver and the highest act iv i ty was found in r u m i n a t i n g lambs .

The changes obse rved in the act ivi t ies o f the urea cycle e n z y m e s dur ing d e v e l o p m e n t have been con t r a s t ed with those r epo r t ed to occur in o the r spe-

* P r e s e n t a d d r e s s : D e p a r t m e n t o f P a e d i a t r i c B i o c h e m i s t r y , Rr~yal H o s p i t a l fo r S i c k C h i l d r e n , 1 R i l l b a n k C r e s c e n t , E d i n b u r g h , E H 9 1L,I , [ r .K.

211

eies. It is concluded that there is no single factor regulating the activities of the five enzymes directly concerned with urea synthesis during development. The results support the hypothesis that in mammals the ability of the liver to syn- thesise urea in foetal life is related to renal development.

In t roduct ion

Urea synthesis and excretion, adaptations which permit mammals to elimi- nate waste nitrogen in a non-aqueous environment, must be well developed at, or soon after, birth. However, the stage of prenatal development at which urea synthesis commences differs from one mammalian species to another and this has been linked with such developmental characteristics as placental permeabil- ity, matur i ty at parturi t ion and renal development [1,2]. The period at which hepatic urea synthesis first becomes evident in sheep has not been established although studies in which the rates of urea clearance across the placenta were measured have indicated that sheep foetuses of more than 130 days conceptual age produce urea [3]. The urea content of foetal sheep urine and of amniotic fluid increases with conceptual age [4] and urea clearance by foetal sheep kid- neys is measurable from 61 days conceptual age [5]. These studies suggest that urea synthesis occurs in foetal tissue at a relatively early age but the possibility remains that the urea excreted by the immature foetal kidney is of maternal origin, since the sheep placenta is freely permeable to urea [4]. It might be expected that during postnatal development the rate of ammonia released into the portal circulation will increase with the onset of rumination and that this might be accompanied by an increase in the ability of the liver to synthesise urea. It has been repor ted that in adult sheep there are adaptive changes in the activity of the enzymes concerned with urea synthesis in the liver when the sheep are fed high protein diets [61.

This paper reports the rates of urea synthesis by sheep liver slices from 50 days conceptual age to maturi ty and the activities of the enzymes specifically concerned with the synthesis of urea in liver and kidney. The results obtained enable comparisons to be made with the changes known to o(~cur in other species. A preliminary report of some of this work has been presented [7].

Materials and Methods

Chemicals. ATP and carbamoyl phosphate (dilithium salt) were obtained from the Boehringer Corp. (London) Ltd. (Lewes, Sussex, U.K.); N-acetyl glu- tamic acid, arginase, argininosuccinic acid (barium salt), bovine serum albumin (crystallized and lyophilised) and L-thyroxine (free acid) were obtained from Sigma (London) Chemical Co. Ltd. (Peele, Dorset, U.K.); diacetyl monoxime from Koch-Light Ltd. (Colnbrook, Bucks, U.K.) and 1-phenyl-l ,2 propane- dione-2-oxime from Eastman Kodak Company (Kirkby, Liverpool, U.K.). All other chemicals were obtained from BDH Chemicals Ltd. (Peele, Dorset, U.K.).

Animals and collection o f tissue. Sheep of mixed breeds were used in this study. The ewes, either Dorset Horn or Clun Forest, were tupped by Suffolk rams on known dates. The stage of gestation was confirmed after 70 days

212

pregnancy by radiological examina t ion at the Grassland Research ins t i tu te , Hur ley . Ewes were housed indoors for at least 14 days pr ior to opera t ion or lambing. The y were fed hay and concen t r a t e daily. This ra t ion provided 27 g N per day (approx . 112 g digestible c rude pro te in) for non-pregnan t ewes and for ewes dur ing the first 100 days of p regnancy , and 33 g N per (lay (approx. 138 g digestible c rude pro te in) for the lat ter par t of pregnancy. Lambs were al lowed to suckle theh" mothe r s unti l abou t 6 weeks of age, when they were weaned on to a commerc ia l pel le ted ra t ion and hay. The es t imated N intake o f 16-week-old lambs (260 days concep tua l age) was 23 g N per day.

Livers and k idneys f rom foeta l lambs were ob ta ined f rom animals slaugh- te red by cu t t ing the carot id arteries af ter del ivery by Caesarian section. Tissues f rom suckling and fa t lambs were ob ta ined f rom animals s laughtered af ter elec- trical s tunning. Samples of the cauda te lobe of ewe liver were ob ta ined by biopsy. These t echn iques have been descr ibed previously [ 81. The samples were placed immedia te ly in ice-cold 0 .154 M KCI.

Injection of thyroxine into foetal lambs. The ewes were prepared for sur- gery as for a Caesarian sect ion. The pregnant horn of the uterus was exteri- orised th rough the left flank incision and the foe tus was immobil ised by ex te r io r pressure on the uterus . T h y r o x i n e was injected into the g lu t en mass th rough the u ter ine wall. The pregnant horn was r e tu rned to the cavity and the f lank incision closed.

Thyroidectomy of foetal lambs. The pregnant horn was exposed as descr ibed above and the foeta l head and neck were exter ior i sed th rough incisions in the u te r ine wall and foeta l membranes . The thy ro id gland was exposed by blunt dissect ion and cut f r om the sur rounding tissues. The site of the gland was then cauter ised. 1 0 0 0 000 units benzylpenic i l l in were sprayed a round the wound before it was sutured. The foe tus was r e tu rned to the amnio t ic cavity and the incisions in the membranes , u terus and flank were closed separately. The foe tuses r emoved later by Caesarian sect ion appeared hea l thy and no thyro id tissue was discernible.

Urea production by tissue slices. The rates of urea p r o d u c t i o n in liver and k idney slices were de t e rmined as descr ibed previously for placental tissue [9] .

Measurement of enzyme activities. The activities of arginase (EC 3.5 .3 .1! and orn i th ine ca rbamoyl t rans fe rase (EC 2.1.3.3) were de t e rmined as descr ibed pre- viously [9] . Tissue supernants to be assayed for the activities of carbamoyl- phospha te synthase (ammonia ) (EC 2.7.2.5) , argininosuccinate synthetase (EC 6.3.4.5) and argininosuccinate lyase (EC 4.3.2.1) were prepared by homo- genisat ion of the tissue in 3 mM c e t y l t r i m e t h y l a m m o n i u m bromide solut ion and subsequen t cen t r i fuga t ion at 0--4°C [10] . The act ivi ty of ea rbamoyl phos- pha te synthase (ammonia ) was de te rmined by a m e t h o d in which ca rbamoyl phospha te synthesised f rom a m m o n i u m b icarbona te in the presence of N-ace- ty l -L-glutamate and ATP was condensed with excess orn i th ine to fo rm citrul- line in the presence of excess orn i th ine ca rbamoyl t ransferase [10] . The citrul- line f o r me d was de t e rmined color imetr ica l ly with d iacetyl m o n o x i m e [11] . Since endogenous orn i th ine ca rbamoyl t ransferase act ivi ty was found to be greater than tha t o f the synthase, it was no t necessary to add any purif ied orni- th ine ca rbamoyl t ransferase.

The act ivi ty of argininosuccinate syn the tase was de t e rmined by a m e t h o d in

213

which argininosuccinate formed from citrulline and aspartate in the presence of ATP was cleaved in the presence of endogenous argininosuccinate lyase to form arginine and fumarate [10]. Addit ion of purified argininosuccinate lyase was unnecessary, since the rate of arginine synthesis from argininosuceinate always exceeded the overall rate of arginine synthesis from citrulline and aspartate. The arginine formed was hydrolysed by addit ion of arginase, and the urea so formed measured eolorimetrically by reaction with 1-phenyl-l ,2-propane- dione-2-oxime [12]. This reagent gave negligible interfering colour with eitrul- line present in the reaction mixture.

The activity of argininosuccinate lyase was determined by a method in which argininosuceinate was cleaved to fumarate and arginine and the latter hydrol- ysed by adding arginase [10]. The urea so formed was determined colorime- trically using diacetyl monoxime [ 11].

Enzyme activities were determined at 37°C and have been expressed as pmol product formed per min.

Chemical assays. Protein and DNA contents of the tissue were determined in homogenates prepared in 0.25 M sucrose containing 0.1 M potassium phos- phate buffer, pH 8.0 [13,14].

Results and Discussion

The rates of urea synthesis in liver slices are shown in Table I. This shows that the rates of urea synthesis per unit weight of liver are similar in foetal lambs and in mature sheep whether pregnant or non-pregnant. The rates of urea product ion observed in 1-day-old lambs were approximately double those observed in foetal lambs, but by the time lambs were 3 weeks old the observed rates were not significantly different f rom values in foetal and mature ruminat- ing lambs. Increased rates of urea product ion in neonatal lambs were also evi- dent when related to either the DNA or the protein content of the tissue. Pro- tein and DNA conten t of sheep liver and kidney are shown in Table II.

Since sheep liver can synthesise urea from as early as 50 days conceptual age it would seem that the urea found in sheep foetal fluids [4,5] is of foetal origin. Studies carried out in our laboratory [15] using isolated livers perfused with an amino acid mixture show that the mean rate of urea product ion by intact livers of mature foetal lambs is 0.24 pmol/g per min compared with a mean rate of 0.23 #mol/g per min obtained in the present study for a similar age group with tissue slices. Thus the rates of urea product ion measured by two different techniques give similar values. The rates of urea product ion by the entire mature foetus may be estimated from the data repor ted here for slices, taking the mean liver weight of 83 g and the foetal weight of 3.6 kg. The value obtained is 0.32 mg/min per kg foetal weight. This may be compared with the urea excret ion rate of mature foetal lambs in vivo which has been reported to be 0.54 rag/rain per kg foetal weight [3]. The onset of rumination, which would be expected to occur between 170 and 210 days conceptual age, is not associated with any change in the rate of urea product ion, when expressed per unit weight of liver. The increased ability to de toxi fy ammonia as the animal m'atures must therefore be a funet ion of organ weight. Since the mean liver weight of the non-pregnant ewes was 984 g it is evident that the entire liver of

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15

7

±

6 (1

0)

2.1

6

± 0

.26

(1

0)

31

.8

±

1.4

(1

0)

13

9

± 1

0

(8)

4.5

2

± 0

.29

(1

0)

16

6

16

9

9,4

7

± 0

.78

(9

) 1

98

±

20

(9

) 1

55

±

1

1

(8)

1,7

3

~ 0

.34

(6

) 5

3

±

5 (9

) 1

49

±

17

(6

) 3

.13

±

0.4

1

(6)

21

7--

23

1

22

.9

+ 1

.70

(4

) 4

81

±

71

(4

) 1

70

±

14

(4

) 1

.95

±

0.1

1

(4)

93

±

11

(4

) 1

56

+

9

(4)

3.5

2

± 0

.12

(4

)

25

2

26

7

28

.5

± 1

.20

(1

1)

46

7

* 2

5

(11

) 1

71

*

7 (8

) 1

.05

±

0.0

7

(8)

96

±

5

(11

) 1

41

±

7

(8)

2.4

7

± 0

.32

(7

)

No

n-p

reg

na

nt

ew

es

65

.0

+ 3

.40

(9

) 9

84

+

70

(9

) 1

63

-~

15

(1

2)

1.5

4

± 0

.14

(1

3)

17

2

±

7 (8

) 1

28

±

1

2

(11

) 1

.90

±

0.2

0

(10

)

Pre

gn

an

t e

we

s 1

66

±

13

(1

9)

1.9

4

~ 0

.23

(2

7)

216

such animals could d e t o x i f y app rox . 29 m m o l a m m o n i a per h. Studies in vivo [16] have shown tha t the u p t a k e of a m m o n i a by the liver o f adul t sheep is 30.3 ± 8.9 m m o l per h. I t m a y be conc luded tha t the po ten t i a l o f the liver to d e t o x i f y a m m o n i a is ful ly ut i l ized in adul t sheep. I f such animals are sudden ly changed o n t o diets which resul t in rapid genera t ion of a m m o n i a in the rumen , then it is hard ly surprising tha t this f r equen t ly resul ts in dea th caused by am- m o n i a tox ic i ty .

The activit ies of the hepa t ic urea cycle e n z y m e s are shown in Table 1. The act ivi ty of a rg in inosucc ina te lyase was a lways greater than the measured values of a rg in inosucc ina te syn the tase and the re fo re the convers ion of citrull ine t:o a rg in inosucc ina te mus t be the ra te l imit ing s tep for ureogenesis in sheep as in o the r species [ 17 ]. I f the ac t iv i ty of a rg in inosucc ina te syn the t a se is t a k e n as uni ty then the activit ies o f the o the r e n z y m e s in the n o n - p r e g n a n t adul t sheep exceed this by a p p r o x . ,1-fold in the case o f a rg in inosucc ina te lyase, 4-fold in the case of c a r b a m o y l p h o s p h a t e synthase , 202- fo ld in the case of o rn i th ine c a r b a m o y l t ransferase and 582-fo ld in the case of arginase. These relative activit ies ar~, very similar to values r epo r t ed in adul t ra t liver which are 1 : 4 : 6 : 226 : 434 [171.

There were signif icant changes in the activit ies of the five e n z y m e s studied dur ing d e v e l o p m e n t and the changes were still a p p a r e n t when the activit ies were re la ted to e i ther the pro te in or the D N A c o n t e n t o f the tissm~. The highest act ivi t ies o f c a r b a m o y l p h o s p h a t e synthase , a rg in inosucc ina te synthe- tase and a rg in inosuec ina te lyase were f o u n d in 1-day-old lambs. An analysis of the mean da ta indicates tha t there were significant posi t ive cor re la t ions be tween the ra tes of urea b iosynthes is and activit ies of c a r b a m o y l p h o s p h a t e syn thase (r = 0 .75, P < 0.01), a rg in inosuce ina te syn the ta se (r = 0.58, P ' 0.051 and a rg in inosucc ina te lyase (r = 0 .76, P < 0.01). The act ivi t ies of o r n i t h m e c a r b a m o y l t ransferase and arginase were no t co r re la ted with the rates of urea synthes is in liver slices, Thus , f r o m a cons ide ra t ion of b o t h tile relative activi- ties o f the e n z y m e s and the s ignif icant cor re la t ions it appears tha t the stages involving i n c o r p o r a t i o n of n i t rogen are the m o s t l ikely to be inwHved in lh~, regula t ion of urea synthes is at all the stages o f d e v e l o p m e n t s tudied in sheep.

Orn i th ine c a r b m n o y l t ransferase act ivi t ies were highest in the rumina t ing animals . Arginase act ivi ty at 50 clays concep tua l age was twico tha t f()und in adul t ewes and activit ies rose dur ing foe ta l d e v e l o p m e n t such that lit the m a t u r e foe tuses the ac t iv i ty per unit weight o f liver was 10-fold ~n'eater than itl the i r mo the r s . The relat ive act ivi ty of arginase to a rg in inosucc ina te syn the tase in foeta l livers averages 4000 : 1, bu t fo l lowing bir th there is a progressiw~ fall in this ra t io . The high relat ive ac t iv i ty of arginase in foeta l liver suggests tha t ir arginine is available it will be rapidly hydro lysed . Thus the foetal urea o u t p u t in vivo m a y or ig inate f r o m two sources, these being the hydro lys i s of arginine k n o w n to be released f r o m the p l acen t a [18] and the func t ion ing of th¢~ urea cycle in the liver. If one- th i rd of the arginine released f rom the p lacen ta into the foe ta l c i rcu la t ion were to be d i rec t ly h y d r o l y s e d in the liver, this coul~t a c c o u n t for the d i f fe rence be tween the u rea p r o d u c t i o n rates observed in viw> [3] and the hepa t i c po ten t i a l for u rea synthesis f r o m a m m o n i a and b ica rbona te r epo r t ed here .

The synthes is o f u rea f r o m a m m o n i a and b i ca rbona t e could n o t be d e m o n - s t ra ted in slices of renal t issue f r o m animals at any age. This m a y be expla ined

217

by the fact that ornithine carbamoyl transferase activity could not be detected in sheep kidney. The absence of this enzymes is in accord with published ob- servations in the rat [19]. Argininosuccinate synthetase activity was also un- detected in sheep kidney. This contrasts with observations on rat kidney [20]. This failure to detect any argininosuccinate synthetase activity in the sheep kid- ney was surprising since the presence of this enzyme has been inferred from in vivo studies in adult sheep [21]. However in the dog and man a net renal syn- thesis of arginine does not occur [21].

The developmental changes in argininosuccinate lyase activity shown in Table III were similar to those found in the liver in that the highest activities were observed in 1-day-old lambs but activities were only approx. 10% of those found in the liver. The pat tern of arginase activity in developing sheep kidney (Table III) was different from that in the liver as the highest renal activities were found in ruminating lambs, but the activity of renal arginase was much lower than that in the liver.

Since the changes in enzyme activity with age were not the same for all five urea cycle enzymes in the liver, and there was a marked difference in the pat- tern of development of arginase between liver and kidney, it is unlikely that any common mechanism exists to control the activity of the urea cycle enzymes during development. In adult sheep the nitrogen intake has been shown to influence the activity of certain of the urea cycle enzymes, namely ornithine earbamoyl transferase, 'arginine synthetase ' (the combined activity of argininosuccinate synthetase and argininosuccinate lyase) and arginase [6,22]. Using available estimates [ 18], it can be surmised that foetal nitrogen intake in the last third of gestation is approx. 1.5 g/kg per day. In the first 3 weeks of life nitrogen intake is between 1.3 and 5 g/kg per day based on milk intake [23] and the nitrogen content of ewes milk [24] and in the present study fully weaned lambs and non-pregnant ewes received 0.79 and 0.42 g/kg per

T A B L E l I I

A C T I V I T I E S O F A R G I N I N O S U C C I N A T E L Y A S E A N D A R G I N A S E IN K I D N E Y S F R O M N O N - P R E G -

N A N T A N D D E V E L O P I N G S H E E P

Values , e x p r e s s e d as ~ m o l p r o d u c t f o r m e d / r a i n pe r g w e t w e i g h t , are m e a n s + S.E. w i t h t he n u m b e r o f

a n i m a l s in p a r e n t h e s e s . P r e g n a n c y has b e e n a s s u m e d to l a s t 1 4 7 days .

C o n c e p t u a l age E n z y m e a c t i v i t y

( d a y s ) A r g i n i n o s u c c i n a t e A r g i n a s c

lyase

4 9 - - 63 n o t d e t e r m i n e d 1 .58 ± 0 . 5 3 (4)

6 4 8 4 0 . 1 2 1 ± 0 . 0 1 1 (6) 1 .18 ± 0 . 3 2 (6)

8 5 - - 1 0 5 0 . 0 4 8 ± 0 . 0 1 5 (10 ) 0 . 4 8 + 0 . 0 3 (5)

1 0 6 126 0 . 1 1 2 ± 0 . 0 2 0 (5) 0 . 8 0 ± 0 . 2 2 (8)

1 2 7 - - 1 4 7 0 . 1 8 1 ± 0 . 0 1 3 (4) 2 .19 ± 0 . 5 9 (9)

1 4 8 - - 1 4 9 0 . 2 8 5 ± 0 . 0 1 8 (3) 2 . 7 2 ± 0 . 6 9 (5 )

1 5 1 - - 1 5 8 0 . 1 0 9 ± 0 . 0 1 2 (8) 2 . 0 0 ± 0 . 2 0 (6)

1 6 6 - - 1 6 9 0 . 1 2 6 (1) 3 . 5 5 -+ 0 . 4 4 (9)

2 1 7 - - 2 3 1 0 . 2 0 2 ± 0 . 0 2 9 (4) 8 .19 + 0 . 4 4 (4)

2 5 2 - - 2 6 7 0 . 2 3 6 ± 0 . 0 7 0 (4) 6 . 9 5 ± 0 . 8 7 (6)

N o n - p r e g n a n t ewes 0 . 1 5 9 ± 0 . 0 1 5 (6) 3 . 1 9 ± 0 . 4 2 (6)

218

day, respect ively. T h e r e f o r e in the developing sheep none of the changes ob- served in e n z y m e act ivi ty would seem to be cor re la ted with ni t rogen intake.

Cor t icos tero ids , adminis t ra t ion of which results in an increase in some urea cycle e n z y m e activities in the liver of adul t [25] and neona ta l rats [26] , may be cons idered as a possible inducer of the peak in the activities of ca rbamoyl phospha te synthase , argininosuecinate syn the tase and argininosuccinate lyase in the per inata l per iod, since tile changes in the co n cen t r a t i o n of this h o r m o n e in lamb plasma closely match the changes in these enzymes in developing sheep liver [27] . By way of con t ras t the prenata l increase in hepat ic arginase activity precedes any increase in foeta l plasma cor t i eos te ro id concen t ra t ions bu t there appears to be a close similarity be tween the changes in foeta l lamb plasma t h y r o x i n e levels [28] and hepat ic arginase act ivi ty. Since t h y ro x in e administra- t ion induces arginase act ivi ty p r ema tu re ly in foetal rat liver [29] , pre l iminary invest igat ions of a possible role of t h y r o x i n e in media t ing the increase in arginase act ivi ty in foe ta l sheep were a t t emp ted .

In fou r expe r imen t s t h y r o x i n e was injected into foe ta l lambs in u tero . When two foetuses were injected with doses higher than 0.1 mg/kg they were abor ted . Two o the r foetuses were injected with lower doses of t h y ro x in e (0.08 mg/kg es t imated b o d y weight at 85 days concep tua l age) and Caesarian sections were p e r f o r m e d 4 and 6 days later, respect ively. The hepat ic arginase act ivi ty in these animals was no greater than tha t found in un t rea ted animals of the same concep tua l age. In two foetuses which were t h y r o i d e c t o m i s e d at 88 and 98 days concep tua l age and delivered by Caesarian sect ion at 137 days and 112 days, respect ively , hepatic' arginase activity was lower by at least one standard deviat ion for the normal expec t ed value for the age. Thus a role for thyro id h o r m o n e s in inf luencing foetal arginase act ivi ty in sheep may be cons idered a possibil i ty.

The pa t t e rn o f change in the act ivi ty of the urea cycle enzymes in the liver o f sheep during d e v e l o p m e n t is marked ly d i f fe ren t f rom o ther mammalim~ species such as the rat , pig and the human [1 ,2 ,26] . In rat and human liver maximal activities are f ound in the adul t whereas in the sheep it is on ly orni- th ine ca rbamoyl t ransferase act ivi ty which is higher in the more ma tu re ani- mals. In the lamb, as in the human and the pig, considerable act ivi ty of all the urea cycle e nz ymes was f(mnd in the earlier stages of foeta l d ev e lo p m en t and this is in con t ras t to the rat where significant activities of urea cycle enzymes are de m ons t r a t e d only immedia te ly before birth. In sheep a funct ional k idney develops relat ively early in foetal life [ 5 ] and our observat ion that appreciable activity of the urea cycle enzymes occurs in the liver and tha t liver slices are able to synthesise urea fronq ammonia f rom at leasl 50 days concep tua l age is fu r the r evidence that hepat ic func t ion with respect to urea synthesis is lmkeci with renal func t ion as first suggested by Kennan and Cohen [1] . The abili ty of foe ta l sheep liver to synthesise urea may be im p o r t an t for the survival of this species. Since the abil i ty of the ewe to d e t o x i f y am m o n ia is 'almost fully uti l ized when the animals are no t pregnant added ammonia derived f rom foetal and placental metabo l i sm [ 30] during pregnancy would be toxic unless it was t , ransformed into urea in the foe tus itself.

219

Acknowledgements

J.M.R. was the recipient of a Postgraduate Studentship from the Ministry of Agriculture, Fisheries and Food. We thank Mr. I.A.N. Wilson of the Grassland Research Institute, Hurley, Berkshire, who carried out the radiological examination of the pregnant sheep.

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