Factors Affecting Oestrone Sulphate Concentrations in Milk

Rr. vet. ). ( 19!l3) . 139, 79

FACTORS AFFECTING OESTRONE SULPHATE CONCENTRATIONS IN MILK

BY R. B.

H EA P,

M.

H A M ON AN D

I. R.

FL EET

Agricultural R esearch Council lns lilule of A nimal Ph)'siology, B abraham, Cambridge CB 2 4A T

SU M ivl i\ RY

Th e co nce ntra ti on of oes tro ne sulph a te in cows' milk in creases g radu a lly d u rin g ges ta tio n . Th e pa tte rn of in c rease is c harac teri zed by epi sod es of hi g h va lu es foll owed by pe ri od s of mu ch lowe r va lu es. Ex p erim ents ca rri ed o ut in goa ts durin g early preg na n cy showed th a t the m a mm a ry ext ract ion of e H Joes tro ne s ulph a te und er s tead y s ta te co ndit io ns was 63·3 ± 8· 1% (m ea n ± SEM ). T he s pecifi c rad ioactivity of oes tro ne s ulph a te in milk (d p m / ng) durin g th ese ex pe rim ents was sig nifi ca ntl y lower th a n tha t in a rte ri a l or m a mm a ry ve no us pl as m a, wh ereas th e con ce ntrat ion of end oge no us oes tron e s ulph a te in milk (ng/ ml ) was s ig nifi ca ntl y g rea te r th a n th a t in pl as ma indi ca ting th a t the s teroid was tra nsfe rred fro m bl ood to milk aga in s t a co nce n tra tion g radi e nt. T he res ults s ugges t th a t c ha nges in th e tra ns po rt o f oes tro ne sulph a te ac ross th e m a mm a ry gla nd a nd in th e local sy nth es is a nd me ta bo lis m of oes trone sulph a te a re fac tors tha t m ay inO ue n ce th e co nce n tra ti on o f oes trone sul ph a te in milk.

I NTR O DUCTI ON

In ea rli er studi es it was fo und th a t oes tron e s ulph a te is qu a ntita tively o ne of th e m aj or oes troge ns in th e milk of pregna nt , lacta tin g co ws (H ea p & H a mo n , 19 79). Durin g ges ta tion its conce ntra tion in c reases g radu a ll y so th a t a fte r d ay 105 it is prese n t in a ll milk sa mpl es ta ken fro m preg na nt cows, wh ereas it is low or und etecta bl e in nonp reg na n t a ni ma ls (H a mon et al., 198 1) . Th ayese res ults prov id ed th e bas is for a new preg na ncy tes t whi c h co uld be used a t d ut105 o r la ter in ges tatio n to co nfirm the res ults o f a milk proges tero ne tes t carri ed o rduring th e third wee k of ges ta ti on (H ea p et al., 19 76) . A co mm e rcia l se rvi ce is now p ovi n ded in th e UK by th e Milk M a rk e ting Boa rd ; oes tro ne s ulph a te in milk is d e term i ed by a simpl e, direc t radi o immun oassay on sa mpl es ta ke n by th e fa rm e r o r practi tione r. T he tec hniqu e, d evelop ed fro m tha t of Sa b a & H at tersley ( 198 1) for th e m eas ure m e nt of oes tron e s ulph a te in pl as m a, a llows la rge numb ers of sa mpl es to be a n a lysed ra pidl y in a ce ntra l la boratory (H old sworth & C h a plin , 1982; H old sworth el al. , 1982).

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BRITISH VETE RI NA RY .JOUR NA L, 139, I

In the cow oes trone su lphate is produ ced by the g rav id uterus (R ober tso n & King, 1979; Gadsby, H ea p & Burto n , 1980), a nd is firs t d etecta bl e in peripheral p las m a by day 72 a ft e r insemination, though its con ce ntrat io n in pl as m a a nd foe ta l fluid s varies co nsid erab ly during gesta ti on (Robe rtso n & King, 1979). Th e time wh e n oestrone su lphate is first detectable in milk varies co nsid era bly between individu a ls a nd the co nce ntration in milk flu ctu a tes widely from day to d ay. The purpose o f the present s tudy was to exa min e possibl e reaso ns for these flu c tu at io ns in mi lk by a n investiga ti o n of the factors that influ ence th e transfer o f oestro ne s ulph a te from bl ood to milk in the goat .

MATERIALS AN D METHODS

Animals Milk sa mpl es were coll ected three times weekly from the In s titute's herd of J ersey cows by the procedure d escribed by H eap & H a mon ( 1979) . Blood a nd milk sa mpl es we re coll ected from the Institute's herd of Saanen goats . The goa ts were previous ly prepared with a n a utotra nspl a nted m a mm a ry g la nd a nd vesse ls (ca ro tid a rtery a nd m a mm a ry ve in ) exteriorized in skin -covered loops using the techniqu es d escribed by Lin ze ll ( 1960) a nd Pea ke r & Fleet ( 1979). Samp ling a nd infu sio ns we re ca rri ed out at known stages of gesta tio n , a nd a ll a nim a ls gave birth to live offsprin g at th e ex pected tim e of d elivery . Samples Blood sa mples were ta ke n by venepuncture o r fro m indwellin g po lyvinyl cat he ters into hepa rini zecl syr inges . Pl as m a was sepa rated by ce ntrifugat ion a nd sto red a t -I 0°C. Milk sa mpl es fro m each a nim a l were ta ken fro m th e wh o le co ll ec tion a t o ne milking o r fro m th e g la nd direc tly in th e case of m a mm a ry secret ion from non-lacta tin g goats . A fa t-de pl eted a qu eo us fraction was prepared (H ea p & Ham o n , 1979) and this is referred to herea ft er as whey ; sa mpl es were s tored at -I 0°C befo re a na lys is . Infu sion [6,7- 3 H(N)]oestrone sulphate (a mm onium salt, 144·9 mCi/ mg; New Engla nd Nuclear, Dreieich, W . Germ a ny) was infus ed close-a rteri a ll y into a transplanted m a mm a ry g la nd o r intrave nousl y into a jugul ar vein through a n ind welling polye thylen e ca th eter ( 1·2 mm o.d., 0·8 mm i.d.; Dural Pl ast ics, New South W a les, Austral ia). Th e infu sion was ca rri ed out with a m odifi ed perista lti c pump a nd a s teady state was es tabli shed within 2 h. Be fore infusion, the purity of the la bell ed s tero id was checked by eva porat in g to dry ness a n a liquot of the etha nolic solu tion con ta inin g e HJ oest ron e su lph a te a nd , after th e addition o f distill ed wa ter, th e m a terial w as extracted with di et hyl e th er ( 10 vol). Brea kdown of la be ll ed oestrone s ulphate to e th e r-solu ble co mpounds was less th a n 7% . eHJoestrone s ulph a te was infused in 2% e tha nol- steril e sa lin e (0·9% NaCI in di s till ed water) at a ra te of0·5 to 1·0 JLCi/min . Befo re the infusion a nd after s tea d y state was reached blood samples were tak en from indwelling catheters in the ca ro tid a rtery a nd transpl a nt ve in , a nd from the opposite ca ro tid a rte ry to d ete rmin e th e activity in recirc ul a tin g b lood in th e case of close-a rteri a l infu sio ns . All milk was

OESTRONE SULPHATE IN MILK

81

removed before and after the infusion using I 00 milliunits Syntocinon (Sandoz Products Ltd., Feltham, Middlesex). The volumes obtained were measured and samples taken for the determination of radioactivity. All milk samples were stored at -10°C before analysis.

Detmninations Radioimmunoassays. Oestrone su lphate in whey was measured after hydrolysis and extraction by the methods described by Heap & H a mon ( 1979) and Hamon et aL. (1981). The validation of the method for the measurement of oestrone su lphate in cows' milk has been published previously (Hamon et aL., 1981). For goats' milk (whey) sampled in early pregnancy the sensitivity of the assay was 1·8 ± 0·3 pgltube (mean± SEM); the blank value, for whey from a non-pregnant goat, was 27·1 ± 1·6 pg/tube (n == 28) and the recovery of a known amount of eH]oestrone sulphate from whey was 79·1 ± 0·9% (n == 45 ). After the addition of 50 or l 00 pg oestrone sulphate to whey, the measured concentration was 52·1 ± 3·3 and 98 ·6 ± 4··5 pg, respectively (n == 20). At a concentration of 50 ng/m l the in tra-assay coefficient of va riation was 17·1 %; at concentrations of 50 and l 00 ng/m l inter-assay coefficients of variation were 18·9 and 13·0% respective ly. Oestrone sulphate in goat plasma (100 fLl aliquots) was measured after the extraction with diethyl ether of unconjugated oestrogens (Challis, Heap & Illingworth, 1971 ). Tubes containing the remaining aqueous phase were placed on a water bath at 50°C and any residual ether was evaporated under a stream of oxygenfree nitrogen. The sample was hydrolysed and assayed by the same procedure that was used for the whey samples. The sensitivity of the assay was 3·6 ± 0·5 pgltube, the blank va lue, for plasma from a non-pregnant goat, 39·6 ± 14·2 pg/100 ,u. l (n == 5) and the recovery of a known amount of [ 3 H]oestrone sulphate, 76·0 ± 1·7% (n == 8). The measurement of two plasma pools gave va lues of0·47 ± 0·03 ng/ml and 9·48 ± 0·2 ng/ ml with coefficients ofvariation between 5·0 and 12·1%. The concentration of progesterone in cows' milk was determined by the Milk Marketing Board using the technique described by Holdsworth, Chaplin & Booth ( 1979). Extraction and purification of { 1H}oestrone suLphate. Total unconjugated oestrogens were extracted from plasma and milk samples (I ml) with seven volumes diethyl ether (peroxide free). Residual ether was removed from the remaining samp le by evaporation on a water bath at 50°C under a stream of oxygen-free nitrogen. A 500-,u. l a liquot of the rema ining sample was added to a g lass tube containing both 14 C-labell ed oestrone and oestradiol-17,8 to correct for procedural losses (approx imately 0·4 nCi/ 100 ,u.g). Conjugated oestrogens were then hydrolysed with 500 fLl dilute sulphatase enzyme a nd extracted with seven volumes of ether by the method described by Heap & Hamon ( 1979). The extract was evaporated to dryness, dissolved in I ml 70% methanol-water (v/v), and placed at -20°C for at least 18 h to precipitate remaining lipids . After centrifugation a t 600 g for 30 min at 4°C, the clear supernatant was evaporated to dryness under nitrogen, dissolved in 100 fLI methylene chloride-benzene (1:1, v/v) and chromatographed on thin-layer plates (Kiesclgel 60F 254 , Merck, Darmstadt). The plates were developed at 4°C in the solvent system methylene ch loride:diethyl ether (70:30 v/v), and the areas of the plate corresponding to

82

BRITISH VETERINARY JOURNAL, 139, I

a u then ti c oestrone were elu ted. Rad ioac tivity was determined by liqu id scinti ll at io n spectrometry using dual isotope separat ion techniques . Mamma~y bloodjlow. Mammary blood Oow was determined in the a utotran sp la nted gland by the close-arterial infusion of Evans' b lue which was subseq uently extracted from plasma by the technique of Little & Williams ( 1964). Blood fl ow determinat ions were car ri ed out after the infusion of oestrone su lph ate was co mpl e ted.

RESULTS

Difference in occurrence of oestrone suljJitate in milk of individual cows There was a considerable difference in th e time wh en significant conce ntratio ns of oestrone su lphate were first detected. In two cows (Fig. I a a nd b) values a lread y exceeded 200 pg/m l by day 40, whereas in a further three a nimal s a comparab le co ncent rat ion was not reached unti l after day 50 (e.g. Fig. I c). I n a ll fiv e cows there was a progressive in crease in oestrone su lph ate concentrat ions in milk whey throughout gestation with brief ep isodes of very hig h va lues fo ll owed by sharp decreases in co nce ntrat ion . These cha nges were not co rrelated with cha nges in m il k yield. In one cow (F ig. lc) there were s m a ll , transient increases in mi lk oestrone s ulphate concentrat ions between days 40 an d 100, and a progressive increase did not occur until after this time . Milk progesterone values ranged from abo ut 10 to 50 ng/ml an d fluctuations in concent rat ion did not fo ll ow a sim il ar pattern to th a t of oestrone su lph ate.

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Fi g. I. The concen tration of oest rone su lph ate in whey (O) a nd proges terone in mi lk (• ) of three lacta ting J ersey co ws during gestation. (a) Cow Zl4, (b) Cow Z l 3, (c) Cow Z23.

BRITISH VETERINARY .JOUR 1AL, 139, I

84

Fig. 2 shows the results in a cow that delivered a small-for-dates bull calf at normal term. Oestron e sulphate was detected in the milk at the usual stage of gestation but values in late pregnancy were in the lower range of those observed in cows carrying foetuses of normal birth weight.

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Mammary uptake of oestrone sulphate from plasma in goats In fiv e experiments on four lac tating goats in early pregnancy (60 to 88 days jJost coitum), the m a mmary extraction of oestrone sulphate was calculated using the formula ((A]-(V]/[A]) X 100, where [A] is the arterial plasma concentration and (V] is th e ma mmary vein plasma concentration. The mammary extraction of [ 3 H]oestrone sulphate from circulating blood was 63·6 ± 8·1% (range 35·8 to 82·5% ), while that of endogenous oestrone sulphate was only 15·0 ± 23·0% (range -II to 49%; Fig. 3). 1~ urther evidence for the upta ke of oestrone sulphate by the mammary gland is · indicated by the values for the endogenous steroid in milk ( 14·4 ± 4·7 ng/ml) and arterial plasma ( 1·1 ± 0·6 ng/ml) in the same experiments. This twelve-fold difference in concentration implies that oestrone sulphate is transported into milk against a concentration gradient (Fig. 4). Under steady-state conditions, the specific activity of oestrone sulphate in samples of arterial and mammary vein plasma, and of milk should be similar unless there is a local site of production . Fig. 5 shows that the specific activity (dpm/ng) decreases in the order: arterial plasma> mammary vein plasma> milk whey.

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DISCUSSION

The results in cows confirm our previous findings (Hamon et al., 1981 ) that oestrone sui ph ate concentrations in milk increase progressively after about day I 00 of gestation. A feature not previously reponed is the occurrence of brief episodes when high co ncentrat ions of oestrone sulphate are found in milk followed by transient periods with low concentrations . These episod es were first seen during the time when oestrone sulphate begins to appear in milk, namely between day 40 and I 00 of gestation, but they also occurred at later stages in gestation with low values being occasionally recorded in animals with normal pregnancies. Such results co uld give rise to false nega tives where the milk oestrone sulphate test is used in pregnancy diagnosis.

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BRIT ISH VI::TERINARY.JOURNAL, 139, I

The incidence or raise negative results with thi s tes t is clearly ve ry low (H a mon et at. , 1981) b ut the !act that s uch values occu r led us to co nsi d er th e mechanisms that influ ence oestrone su lph a te con ce ntratio ns in milk . In cows it w as lo und th at tra nsient hig h va lu es for oestro ne s ulph ate were not assoc ia ted with cha nges in milk yield, !at co ntent o r protein co n ce ntrat ion in milk ( H a mo n et al ., 198 1). Betwee n d ays 4·5 a nd 65 ol gestatio n COWS Carryi ng a lemaJe loetus tend ed tO have a hi g her co nce ritra ti o n o l oes tro ne sulphate in m ilk com pa red to those with a male loe[Us, but th e diiTe re nce was not stat ist ica ll y sig nifi ca nt (H a mon et al ., 198 1) . Episodes o l hi g h co nce ntrat ions ol oes tro ne s ulph ate in milk probably a ri se, therelore, eith er !rom flu ct ua ti o ns in oestroge n s ulph oco nj uga te sec ret ion by th e grav id ut e ru s o r !rom cha nges in th e rate ol transler or these stero id s !rom blood to milk.

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Fig. 5. The mean specifi c activ ity (±SEM) of oestrone su lph a te in samp les ofa n erial and m amma ry vein plasma a nd of milk whe)' at steady s ta te during th e infusion of f3 H )oestro ne su lph ate. The sta ti s ti ca l sign ifi ca nce of the differences was determined by Studem's I- test.

The results of Robertson & King ( 1979) showed that the concentration of oestrone sulphate in plasma and foeta l fluids varies considerably between individual animals a nd at different stages of gestation, and this supports the idea that secretion by the gravid uterus fluctuates possibly from day to day. The present experiments in goats, however, suggest that mamm a ry extraction of labelled oestrone sulphate from blood differs a ppreciably between individual anima ls, and that a mechanism exists for the concentration of endogenous oestrone sulphate in milk . One exp lanation for th e high concentration in milk would be the presence of a high-affinity binding protein but preliminary experiments have failed to show that a n oestrone sulphate-binding protein is responsible. The lowe r specific activity for oestrone sulphate found in mammary vein plasma and in milk compared to that in arterial plasma suggests that the gland synthesizes oestrone sulphate. Synthesis may involve the sulphation of circulating unconjugated oestrogens or the production of oestrone sulphate in the gland itself. In this regard it is nota bl e that other studies from this Department have demonstrated the secretion of oestradiol-17{3 by the mammary gland in the goat, cow and sheep immediately before parturition (Maule Walker & Peaker, 1978; Maule Walker, Davis & Fleet, 1983).

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BRITISH VETERINARY JOURNAL , 139 , I

Clearly, furth er st udi es arc required to co nfirm the identit y of th e oes tron e sulphate m eas ured in a rteria l pl as m a, m a mm a ry vein pl as m a a nd milk , sin ce th e co nve rsion o f oes trone s ulph ate to a rel a ted immunoreactive conjugated oest roge n would inOu cnce the va lidity of th e a bove interpretation . Notwithstanding thi s ca ve a t, th e res ults s how th a t factors which inOu encc the m a mm a ry ext rac t ion a nd synthesis o f oestrone s ulph a te a re potentially importa nt regulators of oes trone sulphate co ncentra ti o ns in milk a nd m ay inOu en ce th e duration a nd m ag nitud e of episod es ofhigh ste ro id va lu es .

AC KNO I'\' I..ED GE MENTS

V·l e g ra tefully ac kn owl ed ge th e help ofMr R . Proudfoot, Mr R. Fulto n a nd th eir s ta ff at Ba bra h a m , a nd of Mr R. J. Ho ld swo rth a nd Mi ss V . Chaplin in th e assay o f mi lk proges te ro ne . REFERENCES

CHA I.I.I S, J. R . G ., H L\1', R. B. & li.LINCWORTH, D. V . ( 197 1) . j ournal of Endocrinolog;• 5 1, 333. GADSBY, .J. E., HEAP, R. B. & BURTON, R. D. ( 1980). J ournal of RejJroduction and Fert ilil)• 60, 409. I-J ,\M O", M ., FLE ET, I. R ., HEM, R. 13 . & HOLDSWORTH , R. j. ( 198 1) . British Veterinal)' .Journn/ 137, 71. HEAP, R. 13. & I-lAM ON, M. ( 1979). British Veterinaryjoumn/135, 355. HEAP , R. B., HOI.DSII'ORTH, R. j. , GA DSBY, J. E. , LAI NG, J. A. & \VAI.TERS, D. E. ( 1976). British VeterinalJ•.Jouma/ 132, 445 . H OI.DSII'ORTH, R . .J. & CHAI'I.IN, V . M . ( 1982). British Veterinal)•} ouma/ 138, 455 . I-J OI.I)SII'ORTH , R . .J. , CHAI'I.IN, V . M . & BOOTH , .J. M . (1979) . British Veterinal)• J ournal 135, 470. HOI.DSIVORTH , R. J ., HJ:,\P, R. B., BOOTH, J. M . & HA~ !ON, tvf. ( 1982). j ournal of Endocrinolog;• 95, 7. Ll:"ZEI.I.,j. L. ( 1960). Nature , London 188, 596. LI"ITI.E, .J. M . & \>ViLI .IAMS, C. ( 1964). Proceedings of the Sociel)• of Exjmimental Biology and Medicine 115, 564 . M AULE \'VAI.KER , F. M . & PEAKER, M. ( 1978) . j ournal of PhJ>Siology 284, 71P. M AU LE W ALKER, F. M ., DAV IS, A. j. & FLEET, I. R. ( 1983). British Veterinal)• j ournal 139, in press. PEAKER, M . & FI.EF.T, I. R. ( 1979). J oumal of Dail)• Resea rch 46, 589. Ro BERTSON, H . A. & KI NG, G.]. ( 1979) . } oumal of Reproduction and Fertilil)• 55, 463. SABA, N. & HATI ERS LEY, j . P. ( 198 I ). J ournal of Reproduction and Fertility 62, 87.