Download thyroid hormones and iodide in the near

THYROIDHORMONESANDIODIDE
INTHENEAR-TERM PREGNANTRAT
Promotor:
Dr.D.vanderHeide
Hoogleraar indeAlgemene FysiologievanMensenDier
H '2
THYROID HORMONESANDIODIDE
INTHENEAR-TERMPREGNANTRAT
PetraVersloot
Proefschrift
terverkrijgingvandegraadvandoctor
opgezagvanderectormagnificus,
vandeLandbouwuniversiteit Wageningen,
Dr.C.M.Karssen,
inhetopenbaarte verdedigen
opwoensdag 1april1998
desnamiddagstevier uurindeAula
Coverdesign:Esther Leuvenink
CIP-DATAKONINKLIJKE BIBLIOTHEEK, DENHAAG
Versloot, Petra.
Thyroidhormonesandiodideinthenear-termpregnantrat
PetraVersloot
ThesisAgricultural UniversityWageningen -Withref.-WithsummaryinDutch
ISBN 90-5485-806-0
Subjectheadings:thyroidhormones/ iodide/ pregnancy
Thepresent studywasfinancially supportedbytheGebiedMedische
WetenschappenfromtheNWO,project nr.900-540 -155.
BIBLIOTHEEK
LANDBOUWUNIVERSITEIT
WAGENINGEN
Stellingen
1. Deuitwisseling vanT4uithetmaternale plasmamethetfoeto-placentairecompartiment iseenzeersnelproces.
(ditproefschrift)
2. Tijdens marginalejodiumdeficiëntiezijnzoweldebeschikbaarheidvanT4afkomstigvandemoedervoordefoetussen,alsdejodideopnamedoordefoetale
schildklier, endusdefoetale productievanT4, verminderd.
(ditproefschrift)
3. HettransportvanT4naardefoeto-placentaireeenheidresulteert indirect in
verlagingvandehoeveelheidT3indeorganenvandemoeder.
(ditproefschrift)
4. Tijdensondervoeding isdeproductievanT4doordeschildklierverlaagd. Oitis
toeteschrijvenaaneenintracellulair energietekort, resulterendineenverlaagdeopnamevanjodide.
(Schröder-vanderEistetal. (1992)Diabetes41:147-152)
5. Deladingopdeoligosaccharideketens vanglycoproteinen isbepalendvoorde
biologische activiteit.
6. Daar inNederlandbrooddevoornaamste bronvanjodiumis,ishetvoorde
ontwikkelingvanzuigelingenvangrootbelangdathunlacterendemoeders
voldoendebroodeten.
7. Borstvoeding iswereldwijddebestestartvooreenkind.
eendeskundige
8. Immunologische bepalingenhebbeninhogematelastvanstorendefactoren.
9. Deverzorgingvanproefdierendoordeonderzoekerzelfdraagt bijaanbetere
dierproeven.
10. Eencreatievewerksfeerwordtinbelangrijke matebepaalddoorjedirectecollega'sendewetenschappelijke groepdaaromheen.
Prof. Dr.T.deLange, 28februari 1998, de Volkskrant
11. Inveelgevallen isdebalanstussenontvangenengegevenonderwijsvanAIO's
verstoord.
12. Hetstructureel creërenvandeeltijdaanstellingen is,naasteengoedgeregelde
kinderopvangeenvandevoorwaardenvoorhetbevorderenvangelijkekansen
opdearbeidsmarkt voormannenenvrouwen.
13. Hetvoortdurend"updaten"vandegebruiktesoftware heeftnietaltijdeenverhogingvanhetrendementtotgevolg.
14. Indemeestegevallenzijnslaap-eneetproblemen bijkleine kinderennietwetenschappelijk aantepakken.
15. Hetschrijvenvaneenproefschrift isminstensevenzwaaraisdezwangerschap
enbevallingvaneenkind.
16.Promoverenop1aprilisnietperdefinitieeengrap.
Stellingenbehorendbijhet proefschrift:
'Thyroid hormonesandiodide inthenear-term pregnant rat"
PetraVersloot
Wageningen, 1april 1998
CONTENTS
page
Chapter1
General introduction
Chapter 2
Thyroxineand3,5,3'-triiodothyronine production,
metabolism,anddistribution inpregnantratnearterm.
Chapter3
ContributionofT3produced locallyfromT4inseveral
23
45
maternaltissuesofthenear-termpregnantrat.
Chapter4
Effectsofmarginal iodinedeficiency duringpregnancy:
iodide uptakebythematernalandfetalthyroid.
61
Chapter5
Effectsofmarginal iodinedeficiencyonthyroidhormone 79
production,distribution,andtransport innonpregnant
andnear-termpregnantrats.
Chapter6
Maternalthyroxine and3,5,3'-triiodothyronine kinetics
innear-termpregnantratsattwodifferent levelsof
hypothyroidism.
97
Chapter7
Generaldiscussionandconclusion
113
Chapter8
Summary
119
125
Samenvatting
Dankwoord
131
CurriculumVitae
133
CHAPTER1
GENERALINTRODUCTION
General Introduction
1.1.THYROIDHORMONE METABOLISM
Thyroidhormoneaction
Thyroid hormone is active throughout the whole body. Numerous biological effects
have been described. During fetal life and childhood thyroid hormone plays an
important role in development and growth. Throughout life thyroid hormones are
responsiblefor regulationofthemetabolicstate.
The thyroid produces mainly thyroxine (T4), and to a much lesser extent 3,5,3'triiodothyronine (T3). The greater part of T3 is formed in the different organs by
monodeiodination of T4. T3 has been shown to be the biologically active form of
thyroid hormone. The working mechanism of T3 is similar in all organs; T3 exerts it
action by nuclear T3 receptors. After binding of T3the T3-receptor complex interacts
with the promotor of various genes by binding to a thyroid hormone response element, resulting in regulation of transcription of the target genes and influencing the
specific mRNAsynthesisofmanytargetgenes(47).
Peripheral thyroidhormonemetabolism
In several tissues T4 and T3 are metabolized via different pathways. The most important is deiodination, especially because biologically inactive T4 is metabolized
into biologically activeT3viathis route. Intheratabout40 %ofT3issecretedbythe
thyroid, wheras about 60 % is derived from extrathyroidal conversion of T4 (50,57,
58,60).
T4 can be deiodinated inthe outer ring (in the 3' or 5' position,outer ring deiodination; ORD)orthe inner ring (inthe3or5position,inner ringdeiodination; IRD).The
ORD of T4 results inT3and isregarded astheactivatingstep.The IRDofT4results
in reverseT3(rT3),which isthe so-called inactivation step.Thedifferent deiodination
reactions are shown in Fig. 1.Three different deiodinase enzymes are known (29,
33). Type Iiodothyronine deiodinase (ID-I) isaselenocysteine containing,nonselective enzyme, which is competitively inhibited by propylthiouracil (PTU). Both the
inner andtheouter ringcanbedeiodinated bythisenzyme. It isresponsibleforboth
the production ofT3aswellastheclearance ofmainly rT3inthe liver. ID-Ihasbeen
found in liver, kidney andthyroid.rT3isthepreferredsubstrate,theconversion ofrT3
to3,3'-T2isroughly 103timesmoreefficient thanthedeiodination ofT4andT3(28).
Type IIiodothyronine deiodinase(ID-II)acts only ontheouter ringandcatalyzesthe
conversions of T4to T3and rT3to 3,3'-T2. The enzyme is not inhibited by PTU. It is
Chapter1
10
present inthe pituitary, central nervous system and brown adipose tissue. Inthese
tissues,ID-IIisresponsibleforthe localsupplyofT3(26).
Type III enzyme (ID-Ill) catalyzes the formation of rT3from T4 and the formation of
3,3'-T2 from T3. The highest levels of this enzyme arefound inthe central nervous
system,placentaandskin(63).
Other pathways for the metabolism of thyroid hormones are conjugation with either
glucuronic acid or sulfate. Conjugation of thyroid hormones increases the watersolubility andthus facilitates their excretion via bile (62). Sulfation enhances type I
deiodination of T3 (48). A relatively minor role in thyroid hormone metabolism is
playedbysidechaindeaminationanddecarboxylationaswellasether-linkcleavage
(32).
outer ring
ID-I
ID-II
inner ring
)*==^
F^
T
•Ö-Ö-
•Ö-ÖrT,3
ID-I
ID-II
3,3'-T,
Figure1:Deiodinationpathwaysofthyroidhormones.ID-I,typeIdeiodinase;ID-II,type I
deiodinase;ID-Ill,typeIII deiodinase.
General introduction
Thyroidhormonesynthesisinthethyroid
Thyroid hormone is synthesized inthethyroid gland,anendocrine organ located in
the lower part of the throat around the trachea. The functional unit of the thyroid
gland is thefollicle, a single layer of follicular cells surrounding thyroglobulin (Tg)containing colloid. T4 and T3 can be formed by coupling iodinated tyrosylresidues
(monoiodotyrosineanddiiodotyrosine) intheTgmolecule.The iodination reactionof
the tyrosyl-residues takes place on the apical membrane at the colloid site and is
catalyzed bythyroid peroxidase. The iodinatedTg,containingT4andT3, isstored in
the colloid lumen. When thyroid hormone is required the Tg molecule reenters the
follicular cell. Thyroid hormones, released by hydrolysis of theTg molecule inphagolysosomes,enterthecirculation (61).
Roleofiodideinthyroidhormonesynthesis
Iodide isanessential elementfor the synthesis ofthyroid hormone. Inorganic iodide
is transported from the blood into the thyroid follicular cell by means of Na7l" symport (34).The most important iodide-concentratingtissues invertebrates arethyroid,
placenta, salivary glands andgastric mucosa.Thethyroid isthe only organwhich is
able to organify accumulated iodide (67). The transport of iodide into the thyroid
gland is an active process (61). This process is regulated by thyroid-stimulating
hormoneorthyrotropin(TSH) (61)andthethyroidal bloodflow(2).
Regulation ofthyroidhormoneproduction
Thyroid hormone production by the thyroid gland is stimulated by TSH, which is
secreted by the anterior pituitary gland. The synthesis and secretion of TSH are
stimulated by thyrotropin-releasing hormone (TRH) from the hypothalamus. BothT4
and T3 exert a negative feedback on TSH synthesis and release, directly at the
pituitary level and indirectly at the hypothalamic level by reducing the secretion of
TRH (68). TSH binds to a TSH-receptor on the basal membrane of the thyroid cell
and stimulates thyroid hormone synthesis at many different levels. Thyroidal iodide
uptake aswellasthesynthesisandreleaseofthyroid hormones isunderthe control
of TSH (69). Since thyroid function is not abolished completely after hypophysectomy,theremustbeaninternalautoregulatory systemwithinthethyroidgland(49).
The localproductionofT3fromT4inseveralorgans isregulatedbytheactivityofthe
deiodinase enzymes andtheavailability ofthe precursor, T4. During hypothyroidism
the ID-I activity in liver and kidney is decreased, while the ID—II activity in brain
11
12
Chapter1
increases. The effects of hyperthyroidism are just in the opposite direction. Iodine
deficiency does not affect ID-I activity in liver and kidney, while ID-II activity in the
brain is increased (5, 25, 31). Inthe brain the aim of regulation is to reach homeostasisofthe intracellular T3level.
Transportofthyroidhormones
Thyroid hormones secreted into the blood are almost entirely bound by specific
binding proteins in the plasma. In humans thyroxine-binding globulin (TBG) is the
most important binding protein, while in rat albumin is the most abundant binding
proteinforthyroid hormones.Thefreehormonefraction ismetabolicallyactiveatthe
tissue level.Innormal humanserum,approximately 0.02 %oftotal T4 and0.2 %of
total T3 isfree (53). Thyroid hormones enter the cell by means of specific transport
proteins inordertoexerttheir hormonaleffects(12).
1.2. MATERNALTHYROIDHORMONEMETABOLISM DURING PREGNANCY
Thematernalthyroid
During pregnancy thyroidal activity is stimulated by TSH and human chorionic
gonadotropin (hCG) (27). In humans hCG levels are high mainly in the first half of
gestation (4),whereas slightly increased TSH levels occur duringthe secondhalfof
gestation(22).
Enlargement of the maternal thyroid is common during normal human pregnancy
(22). Theuptakeofradioiodide, expressed aspercentage dose, is increased (1, 18).
However, the availability of iodide for the maternal thyroid is decreased, due to
increased renal clearance (1) andtransport tothefeto-placentalcomplex during the
latephaseofgestation(18).Iodideistransportedactivelythroughtheplacenta(31).
Inthe rata decrease inuptake of 131lbythe maternal thyroid at the endof gestation
hasbeendescribed(16,21, 24).
Maternalthyroidhormoneconcentrations andmetabolism
Human pregnancy is accompanied by a rise in TBG and total T4 and T3 (18, 22).
However, the leveloffreeT4 isonly decreased slightly attheendofgestation (4,7,
28, 65). Plasma TSH is increased slightly inmorethan 80 %ofthe near-termpregnantwomen,eventhoughlevels remaininthenormalrange(22).
General Introduction
In rat plasma T4andT3concentrations are decreased markedly during gestation (9,
41). ThefreefractionofT4is increasedattheendofgestation (21), resulting infree
thyroid hormoneconcentrations that staywithinthenormal range,just as inhumans.
Despite the considerable decrease in plasma T4 and T3values, plasma TSH in the
ratremainsconstantor isonlyelevatedslightly attheendofgestation (9,20,30).
It has been reported that the clearance of T4from the plasma is enhanced inpregnant rats (21). Boththeextra-thyroidalT4-pool and theturnover of T4are increased
near-term.Thesecretion rateforT4isincreased markedlyjust priortotermbutequal
to control values until 1 day before parturition (35). This has also been shown in
mice. The fractional turnover of plasma T4, the volume of distribution of T4and the
T4-secretionratewereallelevated inpregnant micecomparedtovirginmice(66).
Rat studies by the Madrid group have shown that in all tissues, except T3 in the
brain, T4andT3concentrations aredecreased attheendofgestation.T3inthebrain
stayed within the normal range because of an increase inthe local production ofT3
from T4 as a result of an increase in ID-II activity (9, 38, 40, 43). No significant
alteration inmonodeiodinationinliver homogenatesfrompregnant ratsattheendof
gestationwasfound byYoshida et al. (70).However, aslight increase inhepatic ID-I
activity inthenear-termpregnant ratwasdescribedbyCalvoetal.(9).
Maternalthyroidhormonesandfetaldevelopment
It is well established that thyroid hormones play an important role during development, especially ofthe central nervous system. Until two decades ago itwasgenerally accepted that there is no transport of maternal T4 and T3 to the fetus (19).
However, convincing evidence for humans as well as rats has been presented that
there has to be a transport of maternal thyroid hormones to the fetus. Even before
the onsetoffetalthyroidfunctionT4andT3canbedetected inrat (43,44)aswellas
human embryos (6, 17).The concentrations of T4and T3 in embryonic tissues from
thyroidectomized damswere undetectable beforethe onset offetal thyroid function
and were still reduced in some tissues near-term, despite the onset offetal thyroid
function (43,55). Evenduring normal pregnancy the maternal tofetal transfer ofT4
continuesuntilterm(37,39).
In the humanfetuswithan impairedthyroid neurological damagecan be prevented
by initiating thyroid hormone treatment within a few days of birth. This implies that
during fetal development the supply of maternal thyroid hormones was sufficient to
achieveanormaldevelopment ofthecentralnervoussystem(64).
13
14
Chapter1
1.3. IODINEDEFICIENCY
Since iodine isanessential element for thyroid hormonesynthesis iodine deficiency
affects the production of thyroid hormones, which results in alterations in thyroid
hormone metabolism. Iodine intake isinsufficient inlargeareasoftheworld. Incase
of severe iodine deficiency this results in a broad spectrum of iodine deficiency
disorders, including miscarriage, stillbirth,congenital anomalies aswell asthe more
familiar goiter, cretinism, impairedbrainfunction and hypothyroidism inchildren and
infants (23). Many effects of iodine deficiency are the result of impaired maternal
thyroid functioning during pregnancy. In fact, even the effects of marginal iodine
deficiency on maternal thyroid hormone metabolism and fetal development are
unknown.
Effectsofiodinedeficiencyonthyroidhormonemetabolism
In nonpregnant rats it has been shown that during severe iodine deficiency the
weight of the thyroid increases markedly, while the iodine content of the thyroid
decreases rapidly. TheT3-to-T4ratio inthethyroid is increased (52,56). Studies of
10-day-old rats haveshownthat thethyroidal uptake of iodide, as percentagedose,
was considerably higher in iodine-deficient than in control rats (71). The plasmaT4
level is decreased and plasma TSH is increased,while circulating T3 levels remain
normal. The activities of the thyroid hormone-dependent enzymes alpha-glycerophosphatedehydrogenase and malic enzyme in liver are significantly decreased
(56), implying that the hepatic T3 levels are decreased during iodine deficiency.
Despite an increase in ID-II activity tissue T3 levels in the brain are lower during
iodine deficiency which results in cerebral hypothyroidism. This is caused by the
decreasedavailability ofT4forthelocalproductionofT3(46).
Iodinedeficiencyduringpregnancy
In areas of marginal iodine intake pregnancy constitute a goitrogenic stimulus (22).
In the pregnant rat iodine deficiency results, just as in the nonpregnant rat, in an
increase intheweight ofthethyroid andadecrease inplasmaT4.Thisdecrease in
plasmaT4isinadditiontothedecreasecausedbypregnancy. (15,45).
When iodine deficiency is severe enough to cause very low maternal plasma T4
values, embryonic and fetal tissues become deficient inT4 and T3 both before and
after the onset offetal thyroidfunction (15, 45). Despite an increase in ID-II activity
GeneralIntroduction
inthefetalbrainnormalT3valuescannotbeachieved(45).
1.4. MATERNAL HYPOTHYROIDISM
Maternal hypothyroidism can be considered a rather common problem, especially
since it is clearthat maternal thyroid hormones areofgreat importancefor anormal
development of thefetal central nervous system. Untreated or inadequately treated
hypothyroid women are less fertile and exhibit an increased risk of spontaneous
abortion (3). Forthechildrenofmotherswhowerehypothyroidduringpregnancythe
incidenceofbehavioral andneurological disorders ishigh(36).
The thyroidectomized rat isanappropiate modelformaternal hypothyroidism inrats.
Thyroidectomy results in very lowT4 and T3 levels in maternal plasma and tissues
(41, 42, 51, 55). Before the onset offetal thyroid function T4 andT3are not detectable inthe embryo (43).At the endofgestationfetal plasmaT4andT3values have
normalized (41, 51, 55).Mostfetaltissues stillexhibitdecreasedT4andT3levels on
day 20 of gestation, but the difference with respect tothe controlfetuses is already
muchsmallerthanonday 18(55).
In the thyroidectomized mother treated with methimazole, maternal as well as fetal
hypothyroidismwilloccur. InthisseveresituationithasbeenshownthatonlyT4,and
not T3, is able to mitigate T3 deficiency in the fetal brain (8, 41). This means that
maternalT4isespecially importantforthe normaldevelopment ofthefetalbrain.
1.5. OUTUNEOFTHISTHESIS
Thyroid hormones, especially T4, transported from mother to fetus, are of great
importance for the development of the central nervous system. In most studies the
point of interest is the fetal thyroid hormone status during pregnancy, especially in
the event of impaired maternal and/or fetal thyroid function or iodine deficiency.
However, much less is known about the effects of pregnancy on thyroid hormone
and iodide metabolism inthemother duringanormal,hypothyroid or iodine-deficient
situation. This information isvery important, because these conditions might restrict
theavailability ofmaternalthyroidhormoneforthefetuses.
The aim of this studywas to provide insight into maternal thyroid hormonemetabo-
15
16
Chapter1
Iism, iodide metabolism and peripheral T3 production at the end of pregnancy. For
thispurposepregnantratswerestudiedonday20ofgestation.
Thyroid hormone production, metabolism and distribution were evaluated in the
normal pregnant near-term rat (chapter 2).To study T4andT3kinetics nonpregnant
and near-term pregnant rats were given a bolus injection of [125I]T4and[131I]T3.The
physiological parameters wereestimated by means ofthethree-compartment model
ofJJ. DiStefano(10,11).
In chapter 3, the effects of pregnancy on thyroid hormone concentrations and local
conversion of JAto T3 in many maternal organs are described. For this purpose a
steady-stateexperimentwasperformed (13, 14,57-59).
In chapter4the uptakeof iodide bythethyroid gland innonpregnant and near-term
pregnant rats is compared.The uptake of iodide by thefetal thyroid glandwas also
estimated. Furthermore, the effects of marginal iodine deficiency onthyroidal iodide
uptakewerestudied;what arethe consequences for theavailability of iodidefor the
fetal thyroid ? We chose to induce an iodine deficiency which is only marginal,
because this situation closely reflects the iodine status in large populations in the
world.
Theeffects ofmarginal iodinedeficiency (chapter 5),anddifferent levelsof maternal
hypothyroidism (chapter 6)onmaternalthyroid hormone production, metabolism and
distribution were evaluated by means of a kinetic study. Special point of interest in
thesestudieswastheavailabilityofmaternalthyroidhormonesforthefetuses.
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37. Morreale deEscobar G,CalvoR, Obregon MJ,and EscobardelRey F(1990)Contribution of maternalthyroxinetofetalthyroxine pools innormal ratsnearterm. Endocrinology126:2765-2767'.
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Shulman Dl, Plenum Press, NewYork. pp.133-156.
40. Morreale deEscobar G,Obregon MJ, Calvo R, andEscobardelReyF(1993) Effects
of iodinedeficiency onthyroid hormone metabolism andthebraininfetalrats:theroleof
thematernaltransferofthyroxine.AmJClinNutrSuppl57:280S-285S.
41. Morreale de EscobarG,Obregon MJ,RuizdeOnaC,and Escobar delRey F(1988)
Transfer of thyroxine from the mother to the rat fetus near term: effects on brain
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Comparison of maternal to fetal transfer of 3,5,3'-triiodothyronine versus thyroxine in
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43. Morreale de Escobar G, Pastor R, Obregon MJ, and Escobar del Rey F (1985)
Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat
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44. Obregon MJ, Mallol J, Pastor R, Morreale de Escobar G, and Escobar del Rey F
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45. Obregon MJ,RuizdeOna C,CalvoR,Escobar delRey F,and Morreale deEscobar
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46. Obregon MJ, Santisteban P, Rodriguez-Pena A, Pascuali A, Cartagena P, RuizMarcos A, Lamas L, Escobar del ReyF,andMorrealede EscobarG(1984) Cerebral
hypothyroidisminratswithadult-onsetiodinedeficiency. Endocrinology115:614-624.
19
20
Chapter1
47. Oppenheimer JH, Schwartz HL, Mariash CN, Kinlaw WB, Wong NCW, and Freake
48.
49.
50.
51.
HC (1987)Advances inour understanding of thyroid hormone actionatthecellularlevel.
Endocrine Reviews 8: 288-308.
Otten MH, MolJA, andVisser TJ (1983) Sulfation proceeding deiodination of iodothyronines inrathepatocytes.Science221:81-83.
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Pittman CS, Chambers JB,and ReadVH (1971) The extrathyroidal conversion rate of
thyroxinetotriiodothyronineinnormalman.J ClinInvest50: 1187-1196.
Porterfield SP,and Hendrich CE (1991) The thyroidectomized pregnant rat-an animal
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53. Robbins J, and Bartalena L (1986) Plasma transport of thyroid hormones. In:HennemanG(ed)Thyroid hormone metabolism.Marcel Dekker, NewYork. pp.3-38.
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GeneralIntroduction
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71. ZeghalN,Gondran F,RedjemM,Giudicelli MD,AissouneY,andVigoureux E(1992)
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21
CHAPTER2
THYROXINEANDa.S.S'-TRIIODOTHYRONINE PRODUCTION, METABOLISM,AND DISTRIBUTION INPREGNANT RAT NEAR TERM.
P.M.Versloot,J.Gerritsen,L.Boogerd,J.P. Schröder-vander Eist,
andD.vanderHeide.
Am.J.Physiol.267(Endocrinol. Metab. 30):E860-E867,1994.
T4andT3kinetics inpregnant rat
25
ABSTRACT
Inthe pregnant rat neartermthyroxine(T4)and3,5,3'-tri-iodothyronine (T3)concentrations are lower in plasma and extrathyroidal tissues, except T3 in the brain. To
study the changes in T4 and T3 kinetics a bolus injection of [125I]T4and [131I]T3was
administered to nonpregnant controls and rats 14 and 19 days pregnant. Physiological parameters of the production, interpool transport, distribution, and metabolism
of T4 andT3were estimated by meansof athree-compartment model.Theproduction and partition of T4 remained unchanged during pregnancy. Thetotal distribution
volume of T4wasenlarged.Onday 19the plasmaclearance ratewasdoubled,and
transport to the fast pool was more than tripled. The rate of production of T3 was
slightly diminished.The plasmaclearance ratewas increased, butno changes were
found in the interpool transport rates.These results suggest that inthe pregnant rat
near term the increased transport of T4 is responsible for the distribution of the
availableT4betweenthematernal andthefetalcompartment.
Keywords:thyroidhormone,kinetics,deiodinases, pregnancy.
INTRODUCTION
Thyroid hormones are of great importance for fetal and postnatal development,
especially of the central nervous system. A deficiency of thyroid hormone during
fetal development caused by maternal hypothyroidism in early gestation results in
damagetothecentralnervoussystem(19,23).
Convincing evidence has been presented that in humans substantial amounts of
thyroxine (T4) aretransferredfrom mothertofetus.This results innormalfetaldevelopment, even in the case of congenital hypothyroidism. In such a case severe
neurological damagecanbepreventedby initiatingthyroidhormonetreatmentwithin
afewdaysofbirth(19,29).
Inhumans normalpregnancy isaccompanied byarise intheserum levelsoftotalT4
and3,5,3'-triiodothyronine (T3). However, by arise intheT4-bindingglobulinthefree
T4 and T3 levels decrease during pregnancy (11). In contrast to the findings for
humans, plasma T4 and T3 concentrations in the rat are decreased. This leads to
reduced concentrations of T4andT3 inthe tissues, except T3 inthe brain.Mainten-
26
Chapter2
ance of the T3level inthebraincanbeattributedtoincreased local productionofT3
from T4via increasedtype IIdeiodinaseactivity inthebrain(2,21). Despitethe considerable decrease inT4andT3>plasmathyroid-stimulating hormone (TSH) remains
unchangedorisonlyslightly increasedduringpregnancy (2,8, 13).
Thyroid hormonesarealready present intheratfetus before itsownthyroidstartsto
function betweendays 17and 18 ofgestation (21).Maternal T4andT3aretransferred to the fetus, as shown in experiments in which the fetal thyroid is impaired.
However, only T4 can mitigate T3deficiency inthe fetal brain (20). In normal pregnant ratsT4,butnotT3,istransportedtothefetusesattheendofgestation(6,7).
Information about secretion, distribution, and metabolism of T3 and T4 during pregnancy is lacking; nor is it known what the consequences of the lowering plasma
thyroid hormone levels during pregnancy arefor the partition of T4 andT3over the
tissues. This information may be obtained from kinetic studies based on the threecompartment modelofdistributionandmetabolismdeveloped byDiStefano (3,4).In
this modelthreecompartments canbedistinguished:(1)theplasma; (2)tissuesthat
exchange T4 and T3with plasma at a fast rate, the fast pool; and (3) the slow exchanging pool, the slowpool. Liver and kidney are considered asthe maincomponents of the fast pool, while skin, muscles and brain belong to the slow pool (3,4).
With this model it should be possible to distinguish and measure the transport of
thyroidhormonestothefetal pool.
To investigate whether the decreased T4 and T3 levels in the pregnant rat lead to
alterations inthebiologicalend-points ofthyroid hormone activitywedeterminedthe
activity ofa-glycerophosphatedehydrogenase (a-GPD; EC 1.1.2.1)inthe liver. We
have also measured hepatic type I deiodinase (ID-I) and cerebral type II and III
deiodinases (ID-IIandID-Ill).
MATERIALANDMETHODS
Animals
Three-month-old female Wistar rats (CPB/WU, IFFA CREDO, Brussels) wereused.
At the start oftheexperiment the ratsweighed 213±9 g.The ratswere individually
housedat22*C,withalternating 14-hlightand 10-hdarkperiods.Theanimalswere
fed a semisynthetic American Institute of Nutrition diet (1). The dryfoodwas mixed
with distilledwater (60%dryweight, 40%water) containing 10mg/lpotassiumiod-
T4andT3kineticsinpregnantrat
27
ide to prevent utilization of labeled iodideby thethyroid (5).This doseof potassium
iodidedoesnot influencetheT4andT3productionrates(25,26).
After tworegular estruscyclesthe ratsweremated.Thedaythatspermappeared in
the vaginal smearwastakenasday0ofgestation(21).Thegestational periodofthe
rat is22days.
Designofthestudy
Two kinetic experiments were performed with three groups of rats; nonpregnant
controls, pregnant rats on day 14and pregnant rats on day 19.In the first experiment the rats received only [125IT4(n=6,n=A,n=5 respectively), while in an second
experimenttheratsreceived[125I]T4and[131I]T3(n=6,n=6,n=5respectively).
In a separate experiment we determined the activity of a-GPD andthe deiodinases
ID-I, ID-II, and ID-Ill in liver and brain of nonpregnant controls (n=7), 14-day pregnantrats(n=6),and19-daypregnantrats(n=6).
Kineticandanalyticalprotocols
The rats received, via a cannula inserted into the right jugular vein (24), a 400 pi
bolus injection of a solution containing 10 uCi [12SI]T4and 10 uCi [131I]T3 in saline
containing heparin (0.3 U/ml; Organon, Tilburg, The Netherlands), ticarcillin (0.4
mg/ml; Ticarpen, Beecham S.A., Heppignies, Belgium) and 5% normal rat serum.
Ticarcillin is used in the solutions containing labelled iodothyronines to prevent
eventual bacterial infection and diminish artifactual deiodination. It has been used
forthat reasonsincedescribedbyVanDoorn(5).
Blood samplesof0.2 mlweretaken at 1,4.5, 10,23,44, 115,202and315 min; 0.4
ml were drawn at 465,600, 900, 1200 and 1440 min,as given bythe optimal time
scheduleaccordingtoDiStefanoetal.(3,4).
Plasma (50-100pi induplicate)was usedforcountingthetotal 131land125lactivities.
The plasma samples were extracted with ethanol/ammonia (25 %)(197:3 v/v) with
0.1 mM propylthiouracil (PTU). The dried extracts were dissolved in 0.1 ml 0.2 M
ammonia containing carrier T4,T3and potassium iodide (1 mg/10ml)andsubjected
to high-performance liquid chromatography (HPLC) to separate the iodothyronines,
accordingtothemethoddescribed bySchröder-vander EistandvanderHeide(25).
Plasmavolume incontrolsandpregnant ratswasdeterminedinanother experiment.
Rat serum albumin (Nordic Immunological Laboratories, Tilburg, The Netherlands)
was labelledwith125lbythe lactoperoxidase methodandbiologically screened (17).
28
Chapter2
A small amount of the 125l-labelled albuminwas injected via the cannula.At 1,3, 5,
10, 15,20and30minutesbloodsamplesweretaken.Theradioactivity intheplasma
was counted, and the percentage dose per milliliter was calculated. The data were
fitted to a single exponential function. The extrapolated activity at t(0) was used to
determinetheplasmavolume[100/t(0)](3).
The endogenous concentrations ofT4andT3inplasmaweremeasuredby ratradioimmunoassay (RIA) insamplestakenbeforethekineticex-perimentwasperformed.
PlasmaTSHwas measuredbythespecific RIAdevelopedfortherat bythe National
Institute of Diabetes and Digestive and Kidney Diseases of the National Institute of
Health(USA).ReferencepreparationRP-2wasusedasastandard.
Calculations
131
125
The percentage
gedoses
dosesof
of[[131I]T
l]~i33and[ I]T4permilliliter plasmawerecalculatedfrom
the total radioactivities and the [131I]T3and [i25l]T4distributions on the HPLC chromatogram.
These percentage dosesper milliliter, togetherwiththe plasmavolume att(0),were
individuallyfittedtosumsof n=1to3exponentials
Y(t)=A,exp(Vt) +A2 exp(A2*t)+A3exp(A3*t)
using the program DIMSUM, in which coefficients Aj are expressed in percentage
doseper mlandexponentsAjareexpressed permin(16).
To calculate 24 parameters of production, distribution and metabolism the program
MAMPOOL (15)wasused.Thesumofthethree exponentialfunctionswasfitted(by
weighted least-squares regression) to the data collected for each rat individually
and, together with plasma T4 and T3 levels, substituted in the three-compartment
model, accordingtothekineticT4andT3studiesof DiStefanoet al. (3,4).
Thecalculated parameters aresummarized intable 1.
All data are expressed as mean ± SE. Data were analyzed using the Statistical
Package for Social Sciences (SPSS)(28). All data were subjected to one-way analysis of variance, and statistical differences among the groups were determined
usingthemodified leastsignificant differencemethod.
T4andT3kinetics inpregnant rat
Table 1:Nomenclatureforkineticparameters ofT4andT3.
T>PR
T3-PR
T3-PAR
PCR
TRS
DRFOIDRgo
Q,,Q2andQ3
VP,V2,V3andVD
% PFand%PS
Transittime
Totalresidencetime
Rateofthyroidal productionofT4;pmol/h
Rateoftotal bodyproductionofT3;pmol/h
Rateof plasma appearance ofT3;pmol/h
Plasmaclearance rate;ml/h
Interpool transport rates between plasma andthe fast
and the slow pool, respectively in each direction;
pmol/h
Irreversible rate of disappearance from the fast and
slowtissue pools,respectively; pmol/h
Sizes of the plasma,fast andslow pools, respectively;
pmol
Plasma equivalent distribution volume of he plasma,
fast, slowandtotalbodypools, respectively; ml
Fraction of T4 or T3 in plasma transported unidirectionallytothefastandslowtissue pools, respectively
Single mean transit time for T4 and T3 molecules traversing plasma andthefast andslow pools;min
All-pass meanresidencetime intheentiresystem;min
Enzymeandprotein determinations
Liver mitochondrial a-GPD was measured by means of the method described by
Garrib andMcMurray(10).
The determinations of ID-I, ID-II, and ID-Ill activities were performed bythe method
as described by Janssen et al. (12). In short, ID-I activity was determined in liver
homogenates.Thefinal assayconditionswere: 1uMreverseT3(rT3)and[12Sl]rT3,0.1
M phosphate (pH 7.2), 2 mM EDTA and 5 mMdithiothrietol (DTT). Incubation time
was 30minat37 °C. ID-IIactivitywasdeterminedby incubationof 0.5 nM[125I]T4for
1 h at 37 °C with brain homogenate inthe presence of 1uMT3, 1mMPTU,25mM
DTT,0.1 Mphosphate(pH7.2) and2 mMEDTA. ID-Ill activity inbrain homogenates
was determined by incubating 3,5-[125l]T3for 1h at 37 °C in the presence of 1uM
rT3,0.1 mMPTU,50mMDTT,0.1 Mphosphate(pH7.2) and2mMEDTA.
All deiodinase reactions were stopped by adding 100 pi pooled human serum and
500 pi ice-coldtrichloroacetic acid. Released 1 2 5 l' was separatedfrom protein-bound
iodothyroninesbycentrifugation andcounted.
29
30
Chapter2
Protein was determined by the bicinchoninic acid method (Pierce) using bovine
serumalbuminasstandard.
RESULTS
During pregnancy wefound a significant decrease in the plasma concentrations of
T4andT3.TheTSHconcentration didnotchangesignificantly (Table2).
Table 2: Body weight and plasma thyroid hormone concentrations in nonpregnant controls
and14and 19-daypregnantrats.
Controls
Pregnant
Pregnant
day 14
day 19
274±5'
315 ± 6 * e
27.1 ±1.6
23.3± 1.8e
T 3 ,nM
211 ± 3
33.8 ±2.2
1.05 ±0.07
0.61 ±0.07*
TSH,ng/ml
0.29 ±0.05
0.33 ±0.06
0.57 ± 0.09e
0.57 ±0.05
BW,g
T4,nM
Values are means ± SE.Groups are,respectively, n=12; n=10andn=10, except for T3(n=6;
n=6;n=5).BW,bodyweight;TSH,thyroid-stimulating hormone.Statistical significance,
P<0.05:* day 14vs. controls;e day 19vs. controls;*day 19vs.day 14.
TA kinetics
FortheT4kinetics animalsfromthetwoexperimentsweretakentogether. Nosignificant differences inT4 kinetics was found between the animals which received only
[125I]T4,andthosereceiving[125I]T4and [131I]T3.
Table3showsthemeanvaluesoftheAandAvaluesandthefractionalturnoverand
transport rates (k11: k^,k^,k12k21,k13k31).Thedisappearance curvesforT4fromthe
plasma, basedonthemeanAandhvaluesforthethreegroups,areshowninFig.1.
The absolute magnitude of the slopes of the regression lines describing the
disappearance ofT4from each of the three poolswere significantly increased. This
couldimplythatT4disappeared morequicklyfromallthreepools inthepregnantrat.
The mean results of distribution volumes, pool sizes and transport rates of T4 are
summarized in Table 4. The data are expressed per 100 g body wt and per total
animal (maternal +fetal compartment). Table 5 shows the transit times of T4 inthe
threepoolsandthemeanresidencetime inthebody.
T4andT3kineticsinpregnantrat
31
Table 3:T4kinetic modelparameters:controlsandpregnantrats.
A, (%dose/ml)
X,(1/min)
Controls
Pregnant
Pregnant
(n=12)
Day 14
(n=10)
Day 19
(/7=10)
4.87 ±0.15
-0.22 ±0.03
Coefficientsandexponents
3.59 ±0.15*
-0.47 ±0.12
1.84 ±0.18
A2(%dose/ml)
2.39 ±0.18
A2(1/min)
A3(%dose/ml)
-0.017 ±0.002
-0.022±0.005
2.46± 0.07
-0.0013±0.0001
1.92 ±0.13
-0.0012 ±0.0001
A3(1/min)
k^ (1/min)
k^(1/min)
k33(1/min)
k12k21(1/min2)
2
k13k31(1/min )
2.81 ±0.15®*
-0.83±0.10®*
1.94±0.09
-0.030±0.004
1.71 ± 0.10e
-0.0016± 0.0001 e *
Fractionalturnoverandtransport rates
-0.117 ±0.0012
-0.232±0.054
-0.368±0.042 e
-0.251±0.061
-0.482±0.056 e
-0.118 ±0.013
-0.009±0.001
-0.012 ±0.003
-0.015 ±0.002
0.013 ±0.003
0.00013 ±0.0000
0.083±0.039
0.00037±0.0002
Values aremeans ±SE
Statistical significance, P<0.05: day14 vs
controls;*day 19vs.day 14.
0.183±0.035 e
0.00041 ±0.0001
controls; e day19vs.
Table5: TransittimesandtotalresidencetimeforT,
Controls
(n=12)
Transittime,min
Plasma
Fastpool
Slowpool
Total residencetime,min
9.5±0.8
9.7±1.1
127 ±15
790±30
Pregnant
Day 14
Pregnant
(n=10)
(n=10)
6.2 ±1.0
6.4±1.3
124±25
796±64
Day 19
3.4±0.8 e
2.8 ± 0.8®
96±32
631±22 e *
Values aremeans ±SE. Statistical significance, P<0.05: day 14vs. controls; ®day19 vs.
controls;*day 19vs.day 14.
Chapter 2
32
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T4andT3kineticsinpregnantrat
A.
i\
O
»
O 1
•D
--•
. .
1 11 I 11 1I 1 11I
Figure 1:T4-disappearancecurvesforcontrols and 14 and 19-day pregnant rats obtainedwith athree exponentialmodel.
Panel A: plot of log y(t) (in % dose/ml)
againsttime,withtheleast squaresregression line on the final straight part of the
curve, giving estimates of coefficient A3
andexponentA3.
Panel B: plot of log [y(t) - A3 exp(A3*t)]
against time,with the leastsquaresregression line on the curve, giving estimates of
coefficient A2andexponentA2.
Panel C: plot of log [y(t) - A3 exp(A3*t) - A2
exp(A2*t)] against time, giving estimates of
coefficientA, andexponentA,.
i ,
i j
0 240 480 720 96012001440
10i
time(min)
B.
100
200
300
400
time (min)
8'*>>•
time (min)
~*~eontr. -*- day - • - day
14
19
n=12
n=10
n=10
The T4 models for controls and 19-day pregnant ratsaregiven in Fig.2. For almost
all parameters the value on day 14of gestationwas somewhere betweenthe value
forthecontrolsandthatfor 19-daypregnantrats.
The serumT4concentration decreasedfrom33.8nMto23.3 nMonday 19 ofgestation. The production of T4 by the thyroid did not change significantly during pregnancy. The total amount ofT4 remained unchanged,asdidthe absolute massofT4
in the three pools. During pregnancy, the distribution volume of T4 increased. The
sizes of the plasma and slow pools increased by 50%, whereas the fast pool was
only25%larger.
34
Chapter 2
A.
BW = 211 gr.
T4 = 33.8 nM
100
• TR = pmol/h.
PCR = 2.91 ml/h
tot. time = 789 min.
BW = 315 gr.
T4 = 23.3 nM
118
• TR = pmol/h.
PCR = 5.10 ml/h
tot. time = 631 min.
59
Figure 2: Mean 3-pool model of T 4 kinetics in nonpregnant controls (A) and 19-day pregnant
rats (B). See table 1for nomenclature.
Many alterations inthe transport of T4occurred. On day 19of gestation the plasma
clearance rate (PCR) for T4 had increased nearly twofold.The transport to the fast
pool and vice versawas morethantripled, andtransport tothe slow pool and back
was almost doubled.Thetotal residence timeofT4decreasedfrom789to631 min.
We found a reduction inthe transit time in plasma andthe fast pool to 30%of the
controlvalue.Thetransittimeintheslowpoolwas50%lower.
T4and T3kineticsinpregnantrat
35
73 kinetics
A complete overview ofthekinetic data andstatistics ofT3 isgiven inTables6-8.
TheT3modelsforcontrols and19-daypregnant ratsaregiveninFig. 3.
The plasma T3concentration markedly decreased during pregnancy. Although all A
and Âvaluesonday19ofgestationweresignificantly different fromcontrols, onlya
fewchangesinthephysiological parameterswerefound.
Table6:T3kineticmodelparameters:controlsandpregnantrats.
A, (%dose/ml)
A,(1/min)
A2 (%dose/ml)
k2 (1/min)
A3(%dose/ml)
Controls
Pregnant
Day 14
Pregnant
Day 19
(n=6)
(17=6)
(n=5)
Coefficientsandexponents
6.63 ±0.39
8.89 ±0.31
-1.66 ±0.03
0.79±0.06
-0.048±0.004
0.212 ±0.018
A3(1/min)
-0.0020±0.0003
k^ (1/min)
k22(1/min)
-1.431 ±0.07
k33 (1/min)
k12k21(1/min2)
k13k31(1/min2)
5.88 ±0.13®
-1.71 ±0.15
-1.89 ±0.09
0.68 ±0.03
-0.052±0.005
0.195±0.14
0.62 ±0.08
-0.041±0.007
0.173 ±0.014
-0.0022±0.0002
-0.0023±0.0003
Fractionalturnoverandtransportrates
-1.514±0.14
-1.620±0.13
-0.206±0.016
-0.0093±0.002
0.239±0.020
0.0026±0.0005
-0.237±0.020
-0.247±0.030
-0.0116 ±0.001
0.293 ±0.005
-0.0096±0.001
0.344 ±0.057
0.0030±0.0004
0.0017 ±0.0004
Values aremeans±SE.Statisticalsignificance,P< 0.05: day14 vs.controls;e day19vs.
controls.
The plasma appearance rate andtheproduction rateforT3 were diminished when
expressedper100gbodywt,butnosignificant change,duetoalargevariation, was
found pertotal bodyweight. During pregnancythedistribution volumeofthe plasma
andthetwotissue poolswas enlarged.Theamount ofT3 intheplasma andthefast
pool hadnotchanged,whereas theT3 intheslow pool exhibitedasharp decrease.
PCR increased. Nosignificant differences were found inthetransport rates, transit
times,andtotalresidencetimeforT3.
36
Chapter 2
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T„ and T 3kinetics in pregnant rat
A.
BW = 211 g.
T3 = 1.05 nM
• TR = pmol/h.
PCR = 45.4 ml/h
tot. time = 607 min.
B.
BW = 211 g.
T3 = 0.57 nM
• TR = pmol/h.
PCR = 64.4 ml/h
tot. time = 490 min.
Figure 3: Mean 3-pool model of T 3 kinetics in nonpregnant controls (A) and 19-day pregnant
rats (B). See table 1for nomenclature.
Enzymeactivities
The changes inthea-GPD activity inthe liverareshown inFig.4.When expressed
per milligram of protein there is a significant decrease in the a-GPD activity, but no
change inactivity isfoundwhencalculatedpertotal liver.
37
38
Chapter2
Table 8:TransittimesandtotalresidencetimeforX,.
Controls
Pregnant
Day 14
(n=8)
(n=6)
Pregnant
Day 19
(n=5)
Transittime,min
Plasma
Fastpool
Slowpool
0.67 ±0.02
4.99±0.36
114 ±15
Totalresidencetime, min
607±76
0.69±0.07
4.38 ±0.39
94 ±14
507±48
0.63±0.05
4.30±0.56
109 ± 9
490±33
Valuesaremeans±SE.
300
r
200
100
0
per mg protein
mOD/min/mg protein
per total liver
mOD/min (thousands)
Figure 4: ^glycerophosphate dehydrogenase activity (per mg protein and per total liver) in
liver homogenates from controls (solid bars; n=7) and 14- (gray bars; n=6) and 19-day (hatchedbars;n=6) pregnantrats.Values aremeans±SE. ' P<0.05.
Figure 5 shows the changes in deiodinase activities during pregnancy. Hepatic ID-I
increased from 213 pmol/min/mg protein for controls to 270 pmol/min/mg protein on
day 19of gestation. Inthe pregnant rat near term the weight of the liver increased by
> 50%. The protein concentrations in the subcellular fractions were unchanged,
reuslting in a relatively larger increase in the ID-I activity when calculated for the
total liver. Brain ID-II had not changed on day 14butwas markedly increased on day
19 of gestation. The activity of ID-Ill in the brain remained unchanged during pregnancy. The brainweight was not significantly different inthe pregnant rats.
T4andT3kineticsinpregnantrat
39
5.04
hepatic ID-I
brain ID-II
brain ID-Ill
pmol/min/mg protein fmol/hr/mg protein fmol/min/mg protein
Figure5:Deiodinaseactivitiesintotalhomogenatesfromliver(ID-I)andbrain(ID-IIandID-Ill)
from controls (solid bars; n=7) and 14- (gray bars; n=6) and 19-day (hatched bars; n=6)
pregnantrats.Valuesaremeans±SE.*P<0.05.
DISCUSSION
In this study kinetic experiments were performed to investigate the mechanisms of
thechanges inthyroid hormoneconcentrations inthepregnant ratnearterm.
The kinetic parameters of thyroid hormone production, distribution, and metabolism
are expressed per total animal (maternal + fetal compartment) and per 100 g body
wt, becauseofthe enormous gain inbodyweight duetothefetalcompartment. After
delivery bodyweight ofthe dams isnearlythesameasthatofage-matchednonpregnant controls; noessential alterations in bodyweight ofthe damstakes place during
pregnancy. The volume of distribution of each pool increases proportionately to the
increase inbodyweight.Thiswouldmeanthatthe partition ofT4in plasma andfast
andslowpool isthe same inpregnant and nonpregnant rats.When nochanges are
found inthe proportion ofT4inthethree poolsofthe maternal compartment, thisindicatesthatthedevelopingplacentalandfetal compartments aremadeupofplasma
andfastandslowpoolssimilartothoseofthe mother.
40
Chapter2
ProductionofT4andT3
Under normal conditions a lower plasma T4 concentration will lead, by means of
stimulation of TSH release, to an increase in the T4 production rate. However, the
free fraction of T4 is increased in the pregnant rat, resulting in unchanged free T4
plasmaconcentrations (9).ThiscouldexplaintheunchangedTSHlevels.
Our experiment shows that there is no increase in thyroidal T4 production. Others
found an increasedproductionofT4(7),probably becausethey useda noncompartmentalizedmethod.Thetotal amount ofT4inthethree poolsremains unaltered; the
reducedT4concentration inplasma andtissues (2) inthe pregnant rat could bedue
totheenhanceddistributionvolume.
ThetotalbodyproductionrateofT3was unchanged.However,theT3production rate
has not increased proportionately to the increase in body weight, leading to a decreased T3production rate per 100g bodywt. This can be explained by a reduced
thyroidal T3production and/or a decreased peripheral productionfromT4. However,
thyroidal T4 production does not change essentially, and one would not expect the
thyroidal production of T3to be specifically lowered. Onthe basis of the decreased
plasma T4 andT3 levels, onewouldexpect that the pregnant rat near term ishypothyroid. Just as in hypothyroid rats (14, 27) we found an increase in cerebral ID-II
activity leading to normal tissue T3 levels (2). However, instead of the decrease
found in hypothyroid rats (14, 26), ID-I is also increased in livers of pregnant rats.
Thisfinding issupported by Calvo et al.(2). Incontrast, nosignificant differences in
hepatic andrenalT3generationfromT4havebeenobservedbyothers (9,30). From
the increased invitro ID-IandID-IIactivities itcouldbeexpectedthat the production
of T3 from T4was increased. However, from the kinetic data this appears not to be
thecase.ApossibleexplanationforthiscanbefoundintherT3.
The rT3 level in plasma of pregnant rats is lower than in nonpregnant rats (2),despite the increased placental ID-Ill activity. However, the molar ratio of rT3 to T4 is
doubled inthepregnant rat(2).Thiscanresult inadecreasedinvivoT3production,
since rT3 is the preferred substrate for ID-I,whereas the in vitro activity of ID-I is
increased.
Transportof 7"4 andT3
The T4 PCR is markedly increased. Others (9) have suggested that the increased
PCR isdue notonlytoenhanceddeiodination butalsotothemarkedincrease inthe
clearance of T4 from plasma via the gastrointestinal pathway in the pregnant rat.
T4andT3kineticsinpregnantrat
41
However, wedid notfindanysignificant changes inthe disposal ofT4fromthebody.
We think that the major cause of the elevated PCR for T4 is the result of the enlargedvolumesofplasmaandtheslowpool.
This is also the casefor the PCR for T3; both the PCR and the plasma volume are
increased by50%.From our study it isclear that gestation isassociatedwithsignificantly higher fractional T4 turnover rates for different tissue pools. This results in
enhancedtransport ratesforT4duringpregnancy, especially fortransport tothefast
pool. It isunlikely thatthe increase intransport ratecanbeattributedonlytothe liver
andkidney,whichareconsideredtobethemainorgans ofthefast pool.
Calvo et al. foundthattotal liverandkidneyfrom21daypregnant ratscontain35%
less T4 than thosefromthe nonpregnant rat (2). Inour model thetotal amount ofT4
inthefast pool remained unchanged.We suggestthat anexplanationforthisdiscrepancy couldbethat inthepregnant ratthefast poolconsists notonlyofthe liverand
kidney butalsoofanew, rapidly developed compartment (placentas plusfe- tuses).
It is possible that this placentofetal compartment represents even "faster" thyroid
hormone metabolism, butwewere not abletodistinguish afourth exponent, located
mathematically betweenplasmaandthefastpool,onourdisappearancecurve.
Inthe three-compartment model the sumofthe disposal ratesequals the production
rate in all cases. The individual values for the irreversible disposal rates from the
slow andthefast pool (DR^ and DRfc,respectively) are rough estimates,which can
vary intheory betweenzero andthe production rate. DiStefano et al. (3) havemade
the assumption that the DR^ and DRR,are both half the production rate. However,
we think that the disappearance ratefrom thefast pool is larger thanfrom the slow
pool. We based this on the threefold increased transport rate from plasma to fast
pool andthe unaltered transport to theslowpool. This could meanthat the disposal
from the slow pool is the same inthe nonpregnant and the near term pregnant rat.
For that reason we assume that the disposal from the fast pool is increasedwith at
least 18pmol/h(DRTO=50;DRfo=68onday 19).We speculate thatwhenthe disposal
from liver and kidney remained unaltered inthe pregnant rat, this 18 pmol/hdisappearsfromtheplacental andfetal compartments.
The parameters of T3transport in pregnant ratswere not significantly different from
those foundfor nonpregnant rats.This is inaccordancewiththe resultsof Duboiset
al. (6),whoshowedthatT3isnottransportedacrosstheplacenta.
42
Chapter2
Thyroidhormoneactions
Hepatic a-GPD activity per milligram protein isdecreased inthepregnant rat (2,19).
This isinagreementwiththe loweredT4andT3concentrations inthe liver. However,
only whenexpressed per total liver canthe actual enzyme activities of controls and
pregnant ratsbecompared.Thistotala-GPD activity remainedunchanged.
In summary, in the pregnant rat near term available T4 is distributed between the
maternal and the fetal compartment by very fast transport, presumably to the placenta andfetuses.The unchanged T3kinetics are inagreement withthe absence of
placental transfer ofT3inthe normal pregnant rat atthe endof gestation.Thepregnant rat does not produce morethyroid hormones to compensate for loss to thefetuses.
ACKNOWLEDGEMENTS
We thank T.J. Visser and E. Kaptein for giving us the opportunity to perform the
deiodinase assays at their laboratory in Rotterdam, and G.P. Bieger-Smith for correctionofthetext.
Reagents used for the rat TSH assay were kindly provided by the Rat Pituitary
Hormone Distribution Program of the National Institute of Diabetes and Digestive
andKidneydiseases.
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28. SPSSInc.(1990) SPSS/PC +STATISTICS4.0,McGraw-Hill,Chicago.
29. Vulsma T, Gons MH,and deVijlder JJM (1989) Maternal-fetal transfer of thyroxine in
congenital hypothyroidism due to a total organification defect of thyroid agenisis. New
Eng.J.Med. 321: 13-16.
30. Yoshida K, Suzuki M, Sakurada T, Kitaoka H, Kaise N, Kaise K, Fukazawa H,
Nomura T, Yamamoto M, Saito S, and Yoshinaga K (1984) Changes in thyroxine
monodeiodination in rat liver, kidney and placenta during pregnancy. ActaEndocrinological: 495-499.
CHAPTER3
CONTRIBUTIONOFT3PRODUCEDLOCALLYFROMT4INSEVERAL
MATERNALTISSUESOFTHENEAR-TERMPREGNANTRAT.
P.M.Versloot, J.P.Schröder-vanderEist,D.vanderHeideandL.Boogerd.
submittedEuropeanJournal of Endocrinology
T3producedlocallyinthepregnantrat
47
ABSTRACT
3,5,3'-Triodothyronine (T3) is produced bythethyroid and locally, by monodeiodination of thyroxine (T4), in the peripheral tissues. During pregnancy the thyroid hormone status inrats isaltered: plasma andtissue levels ofT4andT3aredecreased.
We investigated the effects of pregnancy on the contribution of T3 produced locally
in the maternal tissues by administering a continuous infusion of [125I]T4and[131I]T3.
The transport of T4 to almost all maternal organs diminished. Less T3 was transported fromthe plasmato brown adipose tissue (BAT), liver, kidney and pituitary. In
BAT and braintheamountof locally producedT3decreased,despite the increase in
deiodinase type IIactivity inthe brain.Inliverthe contributionoflocally produced T3
remained constant, despite an increase in deiodinase type I activity during pregnancy. Thisdiscrepancy betweendeiodinaseactivities and locally producedT3can
be explained byan insufficient availability ofT4.Thedecreased maternalT4concentration, together with the transport of T4 to the feto-placental compartment, results
indirectly inadiminishedavailability ofT3inthe maternalorgans.
INTRODUCTION
During pregnancy maternal thyroid hormones are exceedingly important for normal
development of the fetal central nervous system (7). The biologically active form of
thyroid hormone is 3,5,3'-triiodothyronine (T3) (13). T3 is produced not only by the
thyroid butalso locally inperipheral tissues bymonodeiodinationofthyroxine(T4). In
liver, kidney and the thyroid monodeiodination of T4 is catalyzed by the deiodinase
type I enzyme (ID-I), while in the pituitary, central nervous system and brownadipose tissue (BAT) deiodinase type IIenzyme (ID-II) is responsiblefor the conversion
ofT 4 toT 3 (10).
Some tissues can adapt to alterations in plasma thyroid hormone status, and regulate intracellular T3 by changing the deiodinase enzyme activities, and thus the
amount of locally derived T3. During hypothyroidism a decrease in hepatic ID-I
activity, andanincrease inbrainID-IIactivity have beendescribed (9, 18).This isin
agreement with the contribution of the local conversion found in those organs; in
liver and kidney local conversion is decreased, whereas in brain an increase has
beenfound(4).
48
Chapter3
Especially inthecentral nervoussystem localconversionofT4isthe mainsource of
intracellular T3. Therefore, when the amount of T3 available from plasma is decreased, this can be compensated by an increase in the local conversion of T4 to
someextent(4).
In the near-term pregnant rat this condition is associated with decreased T4 andT3
levels in plasma andtissues (2, 12,20). Fromearlier studies ofT4andT3kinetics it
has been suggested that in the near-term pregnant rat T4 is transported from the
plasma to the feto-placental compartment at a very fast rate (20). This leads to a
decrease intheavailability ofT4for the maternaltissues. Nochangeswerefound in
thetransportofT3.
Studies by the Madrid group have shown that T4 and T3 concentrations in maternal
tissues are decreased at the endof gestation (2, 12). However, it is not known how
pregnancy influences the amount of T3 produced locally in several tissues. An
indication about T3 produced locally in several tissues is only based on measurements of deiodinase activities. It has beenfound that inthe near-term pregnant rat
hepatic ID-I aswell as brain ID-IIactivity is increased (2,20).The aimof this study
was to compare the contribution of T3 produced locally in maternal tissues of the
non-pregnant and near-term pregnant rat. By administering a continuous simultaneous infusion of [12SI]T4and[131I]T3(4,5, 16, 17),T4andT3concentrations in maternal tissues, the relative contribution of plasma-derived vs. locally produced T3,
thyroidal T4 and T3 secretion, and the plasma-to-tissue ratios of T4 and T3 were
determined.
MATERIALANDMETHODS
Animals
Three-month-oldfemaleWistar rats (CPB/WU, IffaCredo, Brussels) wereused.The
rats were individually housed inmetabolic cagesat22 °C,withalternating 14-hlight
and 10-h dark periods. The animals werefed a semisynthetic American Institute of
Nutrition diet (1).The dryfoodwas mixed with distilledwater (60%dryweight,40%
water)containing 10mg/lpotassium iodide.
T3producedlocallyinthepregnantrat
49
Designofthestudy
Twogroups ofratswereusedinthisstudy, i.e. nonpregnant andpregnant rats.After
two regular estrus cyclesthe ratswere mated .Thedaythat spermappeared inthe
vaginal smear wastaken as day 0of gestation. At the start ofthe infusion the rats
weighed211±4g(controls) and237±3g(pregnant,day8).
The continuous intravenous infusion of [125I]T4(30 uCi/rat/day) was started onday8
of pregnancy; 4 days later [131I]T3was added to the infusion fluid. The labeled iodothyronines were administrated at a constant rate (10 ml/day), via a cannula which
was inserted intotherightjugular vein,andextendedtotherightatrium(14) atleast
4 days before the continuous infusion was started. The ratswere unrestrained, and
couldeatanddrinknormally.
Urine and feces were collected from the start of infusion. The 125l and 131l contents
were counted and expressed as a percentage ofthe daily infused radioactivity. The
animals were inisotopic equilibriumwhenthesumofradioactivity inurineandfeces
wasequaltothedailyadministereddose.
Labellediodothyronines
High specific activity [125I]T4and [131I]T3(specific activity ~2200 and ~ 3500 uCi/ug,
respectively) were prepared in our laboratory (8, 21). Na125l and Na131l were purchased from Amersham (Aylesbury, UK); L-T3 and 3,5-L-diiodothyronine, the respective substratesfor labelling,were obtainedfrom Sigma(St. Louis, MO).Purityof
the tracers was assessed by means of high performance liquid chromatography
(HPLC).All infusions consisted ofasterile 0.9 %NaCIsolutioncontaining0.2 mg/ml
ticarcillin (Ticarpen; Beecham, Heppignies, Belgium) and 0.3 U/ml heparin (Organon, OTilburg,The Netherlands). Thestock infusion solutionswerestoredat 4 °C in
the dark. The infusion flasks were protected from light to minimize artifactual deiodinationofthetracers
Analyticalprocedures
At the end of the infusion period, i.e. day20 of gestation for the pregnant rats, the
rats were bled under light ether anesthesia. Blood was collected in heparinized
tubes. Toprevent artifactual deiodination propylthiouracil (PTU)wasaddedtoafinal
concentration of 0.1 mM (11). To free the tissues of trapped blood, the rats were
perfused with 40-50 mlof a0.9 %NaCIsolution containing 3 Uheparin/ml and 0.1
mM PTU; outflow was obtained by puncturing the inferior vena cava. Maternal
50
Chapter3
tissueswerethenimmediatelyexcisedandkeptonice. Eitherwholesmall organsor
weighed portions of the bigger organs were minced and homogenized in a Potter
homogenizer (B. Braun, Melsungen, Germany) at 0 °C in methanol containing 0.1
mM PTU, carrier T4andT3 and potassium iodide (1mg/10 ml).The pituitarieswere
homogenized in1.5 mlsalinecontaining carrier andPTU.
To determinetheiodothyronine concentration ameasuredaliquotwastakenofeach
tissue homogenate and the plasma; the 125l and 131l contents were counted. The
samples were extracted with methanol-ammonia (25 %, 197:3 vol/vol) with 0.1 mM
PTU. The driedextractswere dissolved in0.1 ml0.2 Mammonia containing carrier
T4, T3 and potassium iodide (1 mg/10 ml) and subjected to HPLC to separate the
iodothyronines. The analyses with HPLC were performed according to the method
describedearlier (16).
After decay (7-8 half lives) of the 131l initially present inthe samples the concentrations of stable T4and T3 in plasma were assessed by a specific radioimmunoassay
(RIA) for rats using[131I]T4and [131I]T3astracers (6). PlasmaTSHwas measured by
the specific RIA developed for the rat by the National Institute of Diabetes and
Digestive and Kidney Diseases of the National Institutes of Health. Reference preparation RP-2wasusedasastandard.
Calculations
The levels of T4, T3, tissue T3 derived from T4 (Lc(T3(T4)) and plasma-derived T3
(P(T3)T3)werecalculated(4,5, 16,17).
Inshort:
TissueT4(pmol/gwetwt)
tissue[125I]T4(% dose/g)
=
XplasmaT4(pmol/ml;RIA)
plasma[125I]T4(% dose/ml)
Theconcentration of [125I]T4inthetissuewascorrectedfortrappedplasma(5).
LcT3(T4)wascalculatedasfollows
tissue [131I]T3(% dose/g)
Xplasma [125I]T3(%dose/ml)
131
plasma [ I]T3(% dose/ml)
tissue [125I]T3derivedfromplasma(%dose/g)
T3producedlocallyinthepregnantrat
Then
tissueLc [125I]T3(%dose/g)
= totaltissue [125I]T3(%dose/g)
- tissue [125I]T3derivedfromplasma (%dose/g)
Thus
tissue LcT3(T4)(pmol/g)
tissue Lc[125I]T3(%dose/g)
=
XplasmaT4(pmol/ml; RIA)
plasma[125I]T4(%dose/ml)
whereby [125I]T4was multiplied by 2 to correct for the loss of12SIfrom the distal
ringofT4.
The concentration ofT3derivedfrom plasma inthevarious tissueswas obtained as
follows
tissueT3derivedfromplasma (pmol/g)
tissue[131I]T3{%dose/g)
=
XplasmaT3(pmol/ml;RIA)
131
plasma [ I]T3(% dose/ml)
Thetotal levelofT3inatissue isthesumofthevaluescalculatedfortissue LcT3(T4)
andpT3(T3).
The rateof infusion of[12SI]T4and[131I]T3andtheir respective bloodlevelswere used
tocalculatetheplasmaclearance rates(PCR)forT4andT3.Iftheplasmaconcentration isexpressedasapercentageofthe infuseddose(in 1h/100gbodywt),then
PCR(ml•h"1• 100gbodywt"1)
100
%dose(h• 100gbodywt'1«ml'1 )
The production rate (PR)for T4, or the plasma appearance rate (PAR) for T3, isthe
PCRforT4orT3multipliedbytheirrespectiveplasmaconcentrations.
TheproductionofT3bythethyroid(ThPRT3)canbecalculatedasfollows:
sincetheamountofcirculatingT3derivedfromT4(T3(T4))isgivenby
%dose/ml[125I]T3
T3(T4)=
XplasmaT4(pmol/ml;RIA)
%dose/ml[125I]T4
51
52
Chapter3
and
T3(T4((pmol/ml)
X 100 %= %T3(T4)
plasmaT3(pmol/ml;RIA)
then {100-[%T3(T4)]} X PAR T3 = ThPRT3 in picomoles per hour per 100
gramsbodyweight.
All results are expressed as means ± SE. Data were analyzed using the Statistical
Package for Social Sciences (19). Statistical analysis was performed by means of
theStudent's ftest.
RESULTS
Each rat receivedthecontinuous infusion untilthe individual 1 2 5 l- and ^^-radioactivities in urine andfecesequaledthedaily inputfor at leasttwodays.Thisoccurred in
both groups after 10 days of [125I]T4 infusion and 7 days of [131I]T3 infusion. At this
time the ratswere assumedto be in isotopic equilibrium asfar asthe major pools of
T4,T3andtheir metaboliteswereconcerned.
Table1:Bodyweightand plasmathyroidhormoneconcentrationsinnonpregnant
controlsand20-daypregnantrats.
Bodyweight,g
Controls
Pregnant
(n=10)
(n=9)
229±2.7
305±4.8*
12 ± 1
# fetuses
plasmaT4,nM
30.7±3.2
18.9 ±4.8
plasmaT3,nM
0.62± 0.04
0.52 ±0.03
plasma TSH,ng/ml
0.70 ±0.04
0.80 ±0.11
Dataareexpressedasmeans±SE.T4lthyroxine;T3,3,5,3'-triiodothyronine;
TSH,thyrotropin.P< 0.01:" pregnantvs.controls.
Table 1 shows that plasma T4 decreased in pregnant rats. Plasma T3 and plasma
TSH did not change significantly. Plasma clearance rates (PCR) and production
rates (PR)forT4andT3areexpressedpertotalanimalandper 100gbodywt(Table
2). No alterations were found inthe PCR for T4 and T3 when expressed per 100 g
T3producedlocallyinthe pregnant rat
53
body wt, however the PCR for T 4 increased significantly per total animal. The production of T4 remained constant for the total animal. For T 3 the PAR decreased per
100 g body wt but remained constant per total animal. Per total animal the contribution of T 3 produced by the thyroid increased, whereas extrathyroidal T 3 production
showed a decrease.
Tabel 2: Plasmaclearance ratesforT4andT3,production rateforT4,plasma appearance rate
for T3 and T3 production bythe thyroid and local conversion from T4 in nonpregnant controls
and20-daypregnant rats.Allvaluesareexpressed pertotalanimalandper 100gbodywt.
Pertotalanimal
Per 100gbodywt
Controls
Pregnant
Controls
Pregnant
(n=10)
(n=9)
(n=10)
(n=9)
PCRT4,ml/hr
2.16 ±0.21
3.47 ±0.23*
0.95 ±0.10
1.14 ±0.07
PCRT3,ml/hr
46.7±3.6
51.7 ±2.2
20.4 ±1.6
16.9 ±0.7
PRT4,pmol/hr
63.3±6.6
64.1 ±5.8
27.7 ±2.9
21.0 ±1.8
PART3,pmol/hr
28.5 ±2.4
26.5 ±1.3
12.5 ±1.2
8.7 ±0.4*
ThPRT3, pmol/hr
7.5±2.3
13.3±1.6*
3.3 ±1.0
4.3 ±0.5
21.1 ±2.7
13.2±0.9*
9.2 ±1.2
4.4±0.3*
T3(T4), pmol/hr
Data are expressed as means ±SE. PCR, plasma clearance rate; PR, production rate;PAR,
plasma appearance rate;ThPR, production rateofT3bythethyroid;T3(T4),production rateof
T3fromlocalconversionof T4.P<0.05: * pregnantvs.controls.
The total tissue concentrations of T 4 and T 3 are shown in Fig. 1. In all tissues the T 4
concentration diminished significantly. The total T 3 concentration decreased in all
organs, except the ovary which exhibited an increase.
Figure 2 shows the contributions of plasma-derived and locally produced T 3 in the
organs. The amount of T 3 derived from plasma decreased in brown adipose tissue
(BAT), heart, kidney, liver, muscle and pituitary. The amount of T 3 produced locally
decreased in BAT and brain. In the ovary an increase was found for T 3 derived from
both plasma and local production. The percentage local conversion (that is the %T 3
locally produced from T 4 in relation to the total T3) was lower in BAT, whereas an
increase was found for the ovary. For all other organs the percentage local conversion remained constant.
54
Chapter3
«aiB.
BAT Br Cer H K Li
x10 x10
M Ov Pit
BAT Br Cer H
M Ov Pit
?10
E
C
.2 6
(0
C
a>
u
c
o
o
4
m
l-
0
2
K
Li
Figure 1: Concentrations of T4 (A) and T3 (B) in brown adipose tissue (BAT), brain (Br),
cerebellum (Cer), heart (H), kidney (K), liver (Li), muscle (M), ovary (Ov) and pituitary (Pit)
from nonpregnant (solid bars) and 20-day pregnant (hatched bars) rats. Values are mean ±
SE.* P <0.05.
Thetissue-to-plasma ratioforT4decreased inalltissues,excepttheheartandovary
(Table 3). For[131I]T3thetissue-to-plasma ratiodecreased in BAT, kidney, liver and
pituitary, whereas it increased in the ovary (Table 4). Table 5 shows the ratio of
[125l]T3-to-[125l]T4. This ratio increased for the brain, cerebellum, liver, muscle, ovary
andpituitary.
T3produced locally inthepregnantrat
55
T3
(pmol/g)
plasma
derived
2
locally
0
produced
•2
% Lc
u
,
3R
CER
H
K
LI
M
OV
C
43
53
42
2
8
13
4
P
25
45
43
2
12
15
3
5
PIT
4
3 2 * 14
Figure 2: Concentrations of T3derived from plasma and produced locally in several organs
(see figure 1)from nonpregnant (solid bars) and20-daypregnant (hatched bars) rats. Values
aremean±SE.* P<0.05.
% Lc is the percentage local conversion (i.e. the percentage T3 locally produced from T4in
relationtothetotalT3)innonpregnant (C)and20-daypregnant(P)rats.
Table3:Tissue-to-plasma ratiosforT4incontrol and20-daypregnantrats.
Controls
Pregnant
pvalue
BAT
0.144 ±0.019
0.089 ±0.011
<0.05
brain
0.091 ±0.013
0.049 ± 0.008
<0.01
cerebellum
0.160 ±0.027
0.041 ±0.007
< 0.001
heart
0.091±0.009
0.080 ± 0.006
ns
kidney
0.516 ±0.062
0.319 ±0.025
<0.05
liver
1.017 ±0.072
0.640 ± 0.035
< 0.001
< 0.001
muscle
0.066± 0.007
0.034 ± 0.002
ovary
0.283 ± 0.029
0.253 ± 0.012
ns
pituitary
0.145 ±0.023
0.052 ±0.012
< 0.005
Dataareexpressedasmeans±SE.
Chapter3
56
Tabel4:Tissue-to-plasma ratiosfor[131I]T3incontrol and20-daypregnantrats.
Controls
Pregnant
pvalue
BAT
2.76±0.30
1.92 ±0.12
<0.05
brain
1.67 ±0.11
1.80 ±0.22
ns
cerebellum
2.17 ±0.19
1.87 ±0.11
ns
heart
3.31±0.32
2.90 ±0.13
ns
kidney
11.42 ±1.13
7.75 ±0.53
<0.05
liver
7.35 ±0.43
6.07 ±0.29
<0.05
muscle
1.60 ±0.15
1.29 ±0.08
ns
ovary
2.41± 0.30
4.13 ±0.23
< 0.001
pituitary
10.14 ±0.81
7.31 ± 0.63
<0.01
Dataareexpressedasmeans±SE.
Table 5:Tissue [125l]T3-to-[125l]T„ratios incontroland20-daypregnantrats.
Controls
Pregnant
0.728 ±0.171
0.567 ± 0.075
ns
0.796 ± 0.072
1.532 ±0.201
<0.01
0.473 ± 0.056
1.916 ±0.315
< 0.001
0.594 ± 0.062
0.511 ±0.041
ns
0.390 ± 0.025
0.444 ± 0.042
ns
0.137 ±0.010
0.186 ±0.012
<0.01
0.407 ± 0.047
0.574 ± 0.060
<0.05
0.142 ±0.017
0.422 ± 0.041
<0.001
1.341 ±0.180
3.621 ± 0.837
<0.05
pvalue
Dataareexpressedasmeans±SE.
DISCUSSION
In this study we were able to determine the contribution of local conversion of T 4 to
T 3 in several tissues of the near-term pregnant rat. The method, the surgical procedure to insert the cannula and the subsequent continuous infusion of [125I]T4 and
[131I]T3 did not have a negative effect on pregnancy. This is demonstrated by the
Tjproducedlocallyinthepregnantrat
57
normal bodyweightandnumberandweight offetuses inthenear-term pregnantrats.
In accordance with previous reports pregnancy resulted inadecrease in plasmaT4.
Incontrast, plasmaT3didnotdecreasesignificantly inthisstudy (2, 12,20).
The plasma clearance rates for T4 and T3were not altered significantly. However,
when expressed pertotal animal insteadofper 100grambodywtan increase inthe
PCR for T4 was found. This is in agreement with the results of kinetic experiments
(20).
By expressing the production rates per total animal amore realistic comparison can
be made between nonpregnant and near-term pregnant rats. Inthe pregnant rat the
weight of maternal tissues is not increased in proportion to the bodyweight; the
enormous gain in bodyweight is mainly due to the feto-placental compartment.
However, this compartment does not contribute essentially to the production of
maternalT4andT3.
Despite normal plasmaTSHvalues the production ofT3bythethyroid isenhanced,
incontrast tothethyroidal productionofT4which remainedconstant. Therefore,the
T3-to-T4 ratio for thyroid hormones produced by the thyroid of the pregnant rat is
increased. This pattern is alsofound during iodine deficiency (15), diabetes mellitus
and modified fasting (17). On the other hand, the contribution of peripherally produced T3enteringthe plasma decreased.This implies that lessT3was produced,or
reached the plasma, inthose organswhich contribute to circulating T3, i.e. liver and
kidney (4).The increase inthyroidal T3production, andthedecrease in peripherally
produced T3 in the plasma together resulted in an unchanged plasma appearance
rate.
The amount of T4 decreased drastically in all organs. For some organs this had
already been reported by Calvo et al. (2). The tissue-to-plasma ratio for T4 decreased in allorgans excepttheovary andheart. Therefore, thetransport of plasma
T4 to the maternal organs diminished, resulting in a tissue T4 concentration which
was decreased even morethan the plasma T4 level. Incontrast, a marked increase
in the transport from plasma to the fast pool has been found in kinetic experiments
(20). However, in the normal situation the fast pool consists mainly of liver and
kidney (3). We have already suggested that inthe near-term pregnant rat thefetoplacental compartment alsobelongstothefast pool(20).This impliesthat lessT4is
available forthematernal organs, anhypothesiswhich isconfirmed bythe results of
this study:thetransport ofT4tothematernalorgansdecreased.
ForT3thetotaltissue concentration diminished inallorgans exceptthe ovary,which
58
Chapter3
exhibited apronounced increase intheT3concentration.Theovary istheonly organ
in which the percentageT3produced locallyfromT4had increased inrelation tothe
total T3. This could mean that the ovary is metabolically very active at the end of
gestation. Itisnotclearwhythisoccurs.
The tissue-to-plasma ratio for plasma-derived T3 ([131I]T3) decreased in BAT, liver,
kidney andpituitary,which meansthat lessT3wastransportedfromplasmatothese
organs.This isalso demonstrated bythe decreased amount of plasma-derived T3in
theseorgans.
The ratio of [125l]T3-to-[125l]T4 increased in the brain and cerebellum. Since the
[125l]T3-to-[12Sl]T4 ratio is a markerfor the activity of deiodinase enzymes, the activity
of ID-IImust haveincreased.Thiswasconfirmed bymeasurementsof ID-IIactivity in
the brainofnear-term pregnantrats (2,20).However, despitethis increase inID-IIin
the brain therewas not an increase but adecrease in locally producedT3.This has
to be explained by the decrease in T4 in the brain. In BAT the ratio of [12Sl]T3-to[125I]T4 remained unchanged, indicating that no alterations in ID-II activity occur in
BAT during pregnancy. Therefore the decrease inthe local production ofT3inBAT
hastobethe resultofadiminishedavailability ofprecursorT4.
The ratio of [125l]T3-to-[12Sl]T4also increased inthe liver, but not inthe kidney which
has to bethe result of an increase inthe activity of ID-I. It is knownthat the activity
of deiodinase type I is increased in the liver of near-term pregnant rats (2, 20),
indicating that an increased fraction of T4 is monodeiodinated to T3. In this way,
despitethedecrease inT4,a normal level of locally producedT3couldbereachedin
the liver.
In conclusion: in the near-term pregnant rat normal tissue T3 values cannot be
maintained. The tissue T4 concentrations are decreased so much that normal T3
values cannot always be reached, not even in those organs in which deiodinase
activity was elevated. Further regulation is not possible because there is not sufficient T4availablefor the local production ofT3. Therefore, the transport ofT4to the
feto-placental compartment results indirectly in a T3 deficiency in the maternal
organs.
T3 producedlocallyinthe pregnantrat
59
ACKNOWLEDGEMENTS
WethankG.P. Bieger-Smithforcorrectionofthetext.
Reagents used for the rat TSH assay were kindly provided by the Rat Pituitary
Hormone Distribution Program of the National Institute of Diabetes and Digestive
andKidneydiseases.
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10. Leonard JL, and Visser TJ (1986) Biochemistry of deiodination. In: Thyroid hormone
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11. Morreale de Escobar G, Llorente P, Jolin T, and Escobar del Rey F (1963) The
transient instability of thyroxine and its biochemical applications. Biochem.J. 88: 526530.
60
Chapter3
12. Morrealede EscobarG,CalvoR, Escobar delReyF,andObregon MJ(1994)Thyroid
hormones intissuesfromfetal andadult rats.Endocrinology 134:2410-2415.
13. Oppenheimer JH, Schwartz HL, Surks Ml, Koemer D, and Dillman WH (1976) Nuclear receptors and the initiation of thyroid hormone action. Rec.Prog. Horm.Res.32:
529-565
14. Roelfsema F, van der Heide D, and Smeenk D (1980) Orcadian rythms of urinary
electrolyteexcretioninfreelymovingrats.LifeSei27:2303-2309.
15. Santisteban P, Obregon MJ, Rodriguez-Pena A, Lamas L, Escobar del Rey F, and
Morreale de Escobar G (1982) Are iodine-deficient rats euthyroid ?Endocrinology
110:1780-1789.
16. Schröder-van der EistJP,and vander Heide D(1990) Effects of 5,5'-diphenylhydantoin on thyroxine and 3,5,3'-triiodothyronine concentrations in several tissues of the rat.
Endocrinology 126: 186-191.
17. Schröder-van der EistJP,andvan der Heide D(1992) Effects of streptozocin-induced
diabetes andfood restriction onquantities andsourceofT„andT3inrattissues.Diabetes41: 147-152.
18. Silva JE, and Larsen PR (1982) Comparison of iodothyronine 5'-deiodinase and other
thyroid-hormone-dependent enzyme activities in the cerebral cortex of hypothyroid
neonatal rat. Evidence for adaptationto hypothyroidism.J. Clin.Invest.70:1110-1123.
19. SPSSInc.(1990) SPSS/PC +STATISTICS4.0, McGraw-Hill,Chicago.
20 Versloot PM, Gerritsen J,Boogerd L, Schröder-van der EistJP,and van der Heide
D 1994) Thyroxine and 3,5,3'-triiodothyronine production, metabolism, and distribution in
pregnant ratnearterm.Am.J.Physiol. 267(Endocrinol. Metab.30):E860-867.
21. Weeke J, and Orskov K(1973) Synthesis of [125l]monolabelled T3 and T4 of maximum
specificactivityfor radioimmunoassay. Scan.J.Clin.Lab.Invest.32:357-366.
CHAPTER4
EFFECTS OF MARGINAL IODINE DEFICIENCY DURING PREGNANCY:
IODIDE UPTAKE BY THE MATERNAL AND FETAL THYROID.
P.M.Versloot, J.P.Schröder-vander Eist, D.vander HeideandL.Boogerd.
Am.J.Physiol.273(Endocrinol. Metab. 36):E1121-E1126,1997.
Iodideuptakebythematernalandfetalthyroid
63
ABSTRACT
Iodide uptake by the thyroid is an active process. Iodine deficiency and pregnancy
are known to influence thyroid hormone metabolism. The aim of this study was to
clarify theeffects ofiodinedeficiency andpregnancyoniodideuptakebythethyroid.
Radioiodide was injected intravenously into nonpregnant and 19-day pregnant rats
receiving a normal or marginally iodine-deficient diet. The uptake of radioiodide by
the thyroid was measured continuously for 4 h. The absolute iodide uptake by the
maternal and fetal thyroids at 24 hwas calculated by means of the urinary specific
activity. Pregnancy resulted in a decrease in the absolute thyroidal iodide uptake.
Marginal iodinedeficiency hadnoeffect onthe absolute iodideuptake bythe maternal thyroid. The decreased plasma inorganic iodide was compensated by an increase in thyroidal clearance. A similar compensation was not found for the fetus;
the uptake of iodide by the fetal thyroid decreased by 50% during marginal iodine
deficiency. Thiscanleadtodiminishedthyroidhormone production,whichwill have
anegativeeffectonfetaldevelopment, especially ofthebrain.
thyroxine;3,5,3'-triiodothyronine;plasma inorganiciodide;iodidekinetics.
INTRODUCTION
It is known that thyroid function is affected by various physiological conditions, for
instance, food deprivation (24), pregnancy (10, 11, 12), and iodine deficiency (19,
23). Iodide is an essential element for the production of thyroid hormones. The
uptake of iodide by the thyroid gland is an active process that is regulated by thyrotropin (TSH) (26)andthethyroidal bloodflow(3).Alterations inthethyroidal uptake
of iodidecancausechanges intheproductionofthyroidhormones.
Iodine deficiency affects the physical and mental development of humans in large
areas of the world (13). In rats it has been shownthat during iodine deficiency the
plasma thyroxine (T4) level decreased, while the 3,5,3'-triiodothyronine (T3) level
remained unchanged (23). The weight of the thyroid and the T3-to-T4 ratio in the
thyroid of rats on a low-iodine diet increased (19, 23). Studies of 10-day-old rats
have shown that the thyroidal iodide uptake was considerably higher in iodinedeficientratsthanincontrols(31).
64
Chapter4
Physiological changes in thyroid function also occur during pregnancy. Normal
pregnancy in humans is accompanied by a rise in T4-binding globulin and total T4
andT3(12). However,thefreeT4level isdecreasedattheendofgestation (29).The
availability of iodide for the maternal thyroid is decreased because of increased
renal clearance (1)andtransport tothefeto-placentalcomplex duringthe late phase
of gestation (10). The maternal thyroid is enlarged and the radioiodide uptake,
expressedasapercentageofthedose, isincreasedduring pregnancy (1, 10,13).
In the rat normal pregnancy results inadecrease inplasmatotal T4andT3concentrations (4). This is comparable to the decrease in free T4 in the third trimester of
human pregnancy (29). The thyroidal uptake of 131l isdecreased inthe pregnant rat
(9, 11, 15). Nochangeswerefound inthe urinary excretion of iodide duringthe last
days of gestation (9). Iodine deficiency induces a further decrease in plasma T4 in
the near-term pregnant rat (8). Also the weight ofthe thyroid is increased by iodine
deficiency inthepregnant rat(7).
Most studies ofthe iodide uptake in rats are performedby giving atracer injectionof
radioactive isotope of iodide (131l, 125l or 123l). After a certain period the thyroid is
removed andthe percentage ofthe injectedradioactivity iscalculated(9, 11,15,31).
In our laboratory we developed a method for measuring continuously the in vivo
uptake of 12SIbythe thyroid. By meansofthis method it is possible to study not only
the amount of iodide taken up by the thyroid, but also the kinetics of the thyroidal
uptake of iodide. We were also able to calculate the absolute iodide uptake by the
thyroid.
The aimofthis studywastoclarify the effects of pregnancy and iodinedeficiency on
iodide uptake by the thyroid. We used four groups of rats: nonpregnant and nearterm (day 19)pregnant rats receiving a normal iodine diet or a marginally iodinedeficient diet. This marginally iodine-deficient situation closely reflects the iodine
status in large populations of humans intheworld.As previously described, pregnancy aswell as iodine deficiency affects plasma thyroid hormone levels. However,
the effects on the absolute iodide uptake by the thyroid are unknown. In particular
iodine-deficient, near-term pregnant ratsrepresent an important group.What arethe
effects of iodine deficiency on iodide metabolism inthe near-term pregnant rat?Are
the low amounts of iodide available taken up totally by the maternal thyroid or is
therestill iodideavailableforthefetuses?
Iodideuptakebythematernalandfetalthyroid
65
MATERIALANDMETHODS
Animals
Three-month-oldfemale Wistar rats (CPB/WU, IFFA CREDO, Brussels) wereused.
The ratswere individually housed inmetabolic cages at22°C,withalternating 14-h
light and 10-hdark periods.TheanimalswerefedasemisyntheticAmerican Institute
of Nutrition (AIN) diet (2) mixedwith distilled water (60 %dryweight - 40 %water)
and potassium iodide: 55 ng [normal iodine dose (NID)] or 2.9 ng [marginal iodine
dose (MID)] per gram of feed. The marginally iodine-deficient groups received1%
KCI04 i n thedrinkingwater duringthefirst twodaysofthe MID,at leastfour weeks
before measurement was started. KCI04wasgiventoacceleratethyroidal depletion
oftotaliodinestores.
After two regular estrus cycles the rats were mated. Mating was confirmed by the
presence of sperm in a vaginal lavage the following morning, called day 0 of
pregnancy.
Designof thestudy
Inthisstudyfour groupsofanimalswerestudied:
1. nonpregnant ratsonanormaldiet (NID,C)
2. pregnant ratsonanormaldiet(NID,P)
3.nonpregnant ratsonamarginally iodine-deficient diet(MID,C)
4. pregnant ratsonamarginally iodine-deficientdiet(MID,P)
The pregnant ratswere assessed at the end of gestation, i.e. day 19.Fetuses are
delivered on day 21. The more frequently used day 20 was not suitable in this
experiment, because the animals were killed and bled twenty-four hours after the
injectionofradioiodide.
Dailyfeedintakeandurinaryiodideexcretionweredeterminedforallrats.Themean
feed intakewas 30 g, resulting in a daily iodide intake of 1.3 ugfor the NID groups
and66ngfortheMIDgroups.
Urinary iodidewasdeterminedasdescribedbySandellandKolthoff (22).
The experiments were approved by the University Committee on Animal Care and
UseoftheAgricultural UniversityofWageningen.
66
Chapter4
Thyroidaliodideuptake
The rat was anesthetized with xylazine (25 ul of 2 % rompunR/100 g body wt; sc),
atropine (5 |jg/100g body wt; sc) andketamine(50 ul of 10%ketamine/100gbody
wt; ip). The anesthesized rat was fixed in a bed of plaster. The body temperature
wascontrolledintherectumandmaintainedat38°Cbyawarmingjacket.
The scintillation probe[type42A. Nalcrystal 23mm, 1mmthick,connectedtominiAssay type 6-20 (Mini Instruments Ltd,Essex, UK)]was placedasclose aspossible
tothethyroidregion.
To measure the thyroidal iodide uptake in the rat, a cannula inserted into the right
venajugularis (20) anda400 ul bolusof saline containing 10uCicarrier free Na125l
(Amersham, Aylesbury, UK) was injected. The radioactivity taken up by the thyroid
was measuredautomatically everythirty secondsfor4handcalculatedasapercentage ofthe injecteddose.The percentagedoseof iodidetakenupbythethyroidwas
fitted to the equation % dose = A [1-exp(-t/v)], by non-linear regression analysis
using the program NLFIT/FIT4EXP, which is based on the Levenberger-Marquart
method.'A' representsthe maximum percentage ofdosetaken upbythethyroidand
Y isthetimeconstant(min).
Perchloratedischargetest
To investigatetheeffects of Perchlorateonradioactivity inthethyroid three animals
from each group underwent a perchlorate-discharge test (17). Four hours after the
administration of Na-12Sl 10mg/kg BWpotassium Perchloratewas administrated i.p..
Thyroidalradioactivitywasmeasuredforanother30minutes.
Iodidekineticsinplasma
To study the disappearance of the injected 1 2 5 l, plasma samples (50-100 ul blood)
were taken viathecannula inthevenajugularis 1,3, 5, 10, 16,25,40,80, 150and
240 min. after injection of the 12S I. Radioactivity in the plasma samples was expressedaspercentage doseper milliliter.
The disappearance of iodide from plasma can be described by an exponential
function. Thedatawerefitted, togetherwiththeplasmavolumeatf(0), individuallyto
sumsofn=1-3exponentials(6)
Y(t)= A,exp(A1*t)+A2exp(X2*t)+A3exp(A3*t)
using the program DIMSUM, whereby Ai coefficients are expressed as percentage
doseper milliliterandexponentsAfareexpressedperminute(16).
Iodideuptakebythematernalandfetalthyroid
67
Absoluteiodideuptakeafter24h.
After measurementtheratswereplacedinmetabolic cagestocollecturine.
Twenty-four hours after injection of the 125l the rats were killed by bleeding and
perfusion withsaline underether anesthesia.The maternalthyroid,mammarygland,
placentas andfetuseswereremoved.Thefetalthyroidwascollected byexcisingthat
partofthetracheacontainingthethyroid.
All radioactivities measured (i.e., maternalandfetalthyroid,plasma, urine,mammary
gland, placentas andfetuses minus thyroid at24 h)were expressed as percentages
ofthe injecteddose.
The assumption can bemadethat, during asteady state situation the specific activity of iodine (ratio of 125lto127l) in urine isthesameasthat intheplasmafromwhich
it originated (28). The absolute iodide uptake (AIU) by the maternal and fetal thyroids, mammary gland, placentas, and fetuses minus thyroid aswell as the plasma
inorganic iodide (Pll) can be calculated after determination of the specific activity in
urine(1).
%doseafter 24hr
AIU=
(=ng/24hr.).
%doseurine/ng iodideurine
%dose/mlplasma
Pll =
(=ng/ml).
%doseurine/ng iodideurine
Radioiodide clearance bythethyroid(CTh)andbythe kidneys(CR) iscalculatedfrom
the increment between 120 and 240 min divided by the radioactivity in a plasma
samplecollectedsimultaneously (1).
%dose/mininthyroid
CTh=
(=ml/min).
%dose/mlplasma
%doseurine
CR=
(=ml/min).
%dose/mlplasma* 1440min
68
Chapter4
Statisticalanalysis
All data are expressed as means ± SE. Data were analyzed using the Statistical
Package for Social Sciences (25). All data were subjected to one-way analysis of
variance andstatistical differences betweengroupsweredetermined usingthe least
significant differencemethod.
RESULTS
Marginal iodine deficiency hadnoeffect oneitherthe bodyweight ofthe ratsorthe
number offetuses. Nosignificant alterations werefoundfor plasma TSH.PlasmaT4
and T3 did not change significantly during marginal iodine deficiency. Pregnancy
resulted inadecrease inplasmaT4andT3inNIDaswellasMIDrats(Table1).
Table1:Bodyweightandplasmathyroidhormoneconcentrations.
Bodyweight,g
NID,C
NID,P
MID,C
MID,P
(n=13)
(n=10)
(n=8)
(n=8)
229 ± 4
300±5*
235 ± 4
304±6*
#fetuses
11 ± 1
11 ±1
T4,nM
31.4 ±0.6
21.2±1.5*
29.5±2.4
16.9±2.0*
T 3 ,nM
0.55 ±0.04
0.49 ±0.03*
0.69± 0.06
0.47 ±0.04*
TSH, ng/ml
0.51 ±0.09
0.48 ±0.07
0.75 ±0.05
0.65 ±0.09
Values are means ± SE. NID and MID, normal and marginal iodine dose, respectively;C,
controlrats;P,near-termpregnantrats;T4,thyroxine;T3,3,5,3'-triiodothyronine;TSH, thyrotropin.*P< 0.05,Pvs. C.
Feedintakeandurinaryiodideexcretion
At the start of the experiments, the marginally iodine-deficient groups received
potassium Perchlorate for 2 days. The effect of this treatment on urinary iodide
excretion is shown in Fig. 1. Potassium Perchlorate treatment from day 0 to day2
resulted in an increase in iodide excretion. Within 1wk after Perchlorate treatment
urinary iodide excretion had decreased to 0.4 ug/day; it remained constant during
the restoftheexperimentalperiod.
Iodideuptakebythematernalandfetalthyroid
69
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Kcio 4
30
day
Figure1: EffectofKCI04 treatmentonthe24-h urinaryiodideexcretioninmarginallyiodinedeficientnonpregnantrats.
The mean feed intake and urinary iodide excretion for the four groups of rats are
shown inFig.2. Noeffect ofMIDonfeed intakewasfound. Duringpregnancy feed
intakeandurinary iodideexcretion increased.
2.0
>.1-5
ra
5.1.0
o>
a
0.5
0.0
NID
C
NID
P
MID
C
MID
P
w,
#
1 11
wm
NID
C
NID
P
#*
MID MID
C
P
Figure2:Effectofmarginaliodinedeficiencyandpregnancyonthedailyfeedintake(A)and
urinaryiodideexcretion(B).P<0.05:#pregnant(P)vs.control (C);
* marginaliodinedose(MID)vs.normaliodinedose (NID).
70
Chapter4
Thyroidal
125
l uptake
Table 2 shows the data on thyroidal 125l uptake. The weight of the thyroid increased
significantly in both marginally iodine-deficient groups; pregnancy had no effect on
the weight of thethyroid.
Table 2:Weightofthyroidandparametersofuptakeof radioiodide bythyroid.
NID,C
(n=17)
Thyroid, mg
NID,P
(n=23)
MID,C
(n=15)
MID,P
(n=15)
17.3±1.8
17.4±1.0
27.3±1.5®
23.5 ±1.1®
4-h, %dose
21.2±1.7
13.4±1.1*
52.3±4.2®
35.7±3.8*®
A, %dose
26.9 ±2.2
16.0±1.2*
51.3±9.5®
42.2 ±4.4*®
166±20
139± 9
125±16
112±15
Uptake
Y,min
Values aremeans±SE.A, maximum%dosetaken upbythethyroid; v,timeconstant.
P < 0.05:* Pvs. C;® MIDvs.NID.
The 4-h 125l uptake by the thyroid increased in C and P MID rats. Pregnancy induced
a decrease in 12SI uptake by the thyroid in both NID and MID rats. The 4-h 125l uptake is a direct measurement of the thyroid; A and y are the results of fitting the data
to the one-exponential function %dose=A [1-exp(-t/Y)]. Two- and three-exponential
functions did not yield satisfactory results. The time constant (y) of the 125l uptake
was unchanged in all groups. The mean fitted thyroidal 125l uptake is given in Fig. 3,
where we also see that the thyroidal 125l uptake was lower in P rats and increased in
the MID groups.
Perchlorate discharge test
No discharge of radioiodide from the thyroid was found for any of the four groups.
The radioactivity of the thyroid remained unchanged (results not shown).
Iodide kinetics in plasma
Table 3 shows the parameters of iodide kinetics in plasma. No significant changes
were found except for A,in pregnant rats.
Iodideuptake bythematernalandfetalthyroid
NID
C
t>u
40
—
NID
P
(»30 -
o
.
-
•o
y.y'
^20
y
•
;
>
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'
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71
y
/,£.-'•""*
i
60
i
i
.
120
i
180
MID
C
MID
P
i
240
time(min)
Figure 3:Fitted4-hthyroidalradioiodide uptakebasedonmeanmaximal%dosetakenup(A)
andtime constant (y)values.
AIUafter24h.
Table4 showsthespecific activity of urineandtheresultsofcalculations oftheAIU
by the maternal thyroid, Pll, CTh and CR. The urinary specific activity was unchanged during pregnancy, but marginal iodine deficiency caused an increase in
specific activity. TheAIU bythethyroidwasdecreasedbypregnancy,while marginal
iodine deficiency had no effect on the AIU. Pll was decreased in the MID groups,
whereas pregnancy had no effect. Unidirectional iodide CTh was increased in the
MID groups. CRwas increased by pregnancy, whereas MID resulted ina decrease
in CRinCandnear-termPrats(Table4).
The AIUvalues at 24 hbythe maternal thyroid, placentas,fetalthyroids, remaining
part of the fetuses and mammary gland are shown in Fig.4. Whereas MID had no
effect ontheAIU bythe maternal thyroid,a pronounced decrease wasfoundfor the
fetal thyroids and the remaining part of the fetuses. The amount of iodide taken up
by the mammary gland was also decreased by MID, whereas no significant effect
wasfoundfortheplacentas.
Chapter 4
72
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2.0
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Figure 4:Absoluteiodideuptake bythematernalthyroidandfeto-placentalcompartment; 1.
placentas;2.fetalthyroids;3.fetuses minusthyroids;4. mammarygland (1g).*P<0.05:
NID.Pvs. MIDP.
DISCUSSION
The process by which the thyroid gland adapts to an insufficient iodine supply is to
increase the trapping of iodide. When iodine intake is low, adequate secretion of
thyroid hormones may still be achieved by marked modifications of thyroid activity.
The thyroid is stimulated by an increased thyroid bloodflow (3) and TSH (19, 23).
The T3-to-T4 ratio of iodothyronines secreted by the thyroid is increased during
iodine deficiency, especially because of a decrease in T 4 production (23).
Marginal iodine deficiency was induced in our rats byfeeding them a MID diet.
Within 2wk ofthe start of the MID diet, urinary iodide excretion remained at the
same level. So, at the moment of measurement, at least 4 wk after start of the MID
diet, the ratswere in a steady state. The fact that only a marginal iodine-deficient
statewas achieved, and not a severe iodine-deficient state, situation is demonstrated by the unchanged plasma T 4 and T 3 levels. Despite a slight increase in plasma
TSH, the weight of the thyroid had already increased by 50%.The unchanged
number offetuses during pregnancy also demonstrates that the induced iodine
deficiency was not severe (8).
Inthis study wewere able to measure thyroidal radioiodide uptake continuously. For
the kinetic analysis of radioiodide uptake inthe human thyroid a three-compart-
74
Chapter4
mental model isused(3, 14,30). Inthis model,aplasma iodidepoolandan inorganicandanorganicthyroidal iodidepoolcanbedistinguished.Wetriedtofitour
datatothis model;however, ourdatacouldonly befittedtoaone-exponential
function.Therefore,weassumethat,even ineuthyroid rats, iodidetransportfrom
plasma intothethyroid isunidirectional becauseofanextremely avid iodineorganification.This idea isconfirmed bythetotalabsenceofaPerchloratedischarge inall
rats,meaningthatthere isnoefflux of inorganic iodidefromthethyroid(17).
Thethyroidal uptakeofradioiodidewasstimulatedbymarginal iodine deficiency.
Thekineticanalysisshowsthatthetimeconstantforthyroidal radioiodide uptake
wasnotaffectedbyiodinedeficiency, meaningthatthetimeneededtoachievethe
maximumeffect remainedthesame. However, theAIUbythethyroidremained
normal.Thiswasachieved byanincreasedCThresultingfromtheincreasedthyroid
volumeandthyroidal bloodflow(3).
The urinary specificactivitywasincreased,whereastheradioiodideactivity in
plasmawas notaltered bymarginal iodinedeficiency. Thisresulted inaPllthatwas
significantly lower, suchasisfoundinhumansresiding inareasof iodine deficiency
(5) andin10-day-oldiodinedeficient rats (31).
Ourobservations emphasizetheneedforcaution ininterpretation ofresultsobtainedbystudies measuringthethyroidal radioiodide uptakeonly.Wehavedemonstratedthatadecreaseor increase inradioiodide uptakedoesnot automatically
meanthattheAIUhaschanged.
Alsoduringnormalpregnancy changes iniodidemetabolismarefound.Theurinary
iodideexcretionwas higherfor PthanCratsofthe NIDaswellasMIDgroups.This
canbeexplainedbythe increasedfeedintakeandthe increase inCR.Galton(11)
alsofoundan increased urinary iodideexcretionfor pregnantratsduringthelast
daysofgestation(11).
Attheendofgestationthepercentage radioiodidetakenupbythethyroidwas
significantly decreased.Thishasalsobeenreportedfor ratswithanormal iodine
intake(9, 11, 15).Becausetheurinary specificactivitywasnotaltered bypregnancy,thisalso resultedinadecrease intheabsolute iodideuptakebythe maternal
thyroidattheendofgestation inNIDaswellasMIDrats. Itseemsthatthethyroidof
the near-term Prat haslessiodideavailablefortheproductionofthyroidhormones.
However, nochange inthyroidal hormoneproduction isfoundattheendof gestation
(27).Thiswouldseemto implythattheuseof iodideinthethyroidofthenear-termP
rat ismoreefficient orthatthethyroglobulin storesaredepleted.
Iodideuptakebythematernalandfetalthyroid
75
Thedifference inthyroidal iodideuptakebetweenCandnear-term Prats cannotbe
attributedentirelytothe increase inurinary iodideexcretion.Wesuggestthatthe
remainingpartofthe iodide isusedbythemammaryglandsandthefeto-placental
compartment. For lactating rats ithasbeenshownthat,4hafter injection,themammaryglandscontainasmuchradioiodide asthe thyroid,and itwassuggestedthat
during pregnancy radioiodidewasalready beingtransportedtothemammaryglands
(15).The latterwascontradictedbyourmeasurements. Onegramofthe mammary
glandhadonlytakenup6.5 ng iodideat24h;this islessthan 1% ofthethyroidal
uptake.
Theplacentapossessesamechanismforactivelytransporting iodine(21).Therefore, itistobeexpectedthatacertainamountofradioiodide istransportedtothe
fetalcompartment. Ourresults,collected24hafter injection,showthatthefetal
thyroid iscapable ofconcentrating iodideonday20ofpregnancy. Thethyroidal
regionofthefetus containedasmuchiodideastherestofthefetus.However, the
total amountof iodide inplacentasandfetusescouldnotexplainthe decreased
uptakeof iodide bythematernalthyroid.Therefore,thefeto-placental compartment
andthemammaryglands arenottheonlyfactors responsibleforthedifference in
maternalthyroidal iodideuptake.
Duringmarginaliodinedeficiency theAIUofthematernalthyroidremainednormal.
However, therewasalready atendency towardashift inthyroidhormonesynthesis;
T4decreasedandT3remainednormal.Incontrasttothematernalthyroidnocompensation,byanincreasedthyroidclearanceforthedecreased Pll concentration
wasfoundforthefetalthyroid.TheAIUbythefetalthyroidwasdecreased by50%.
ThispatterncanleadtolowerT4availability forthefetus.This issupportedby
kinetic studiesofmarginally iodine-deficient, near-term pregnant ratsshowingthat
thematernaltransfer ofT4andT3tothefetuses isdecreasedduring marginal iodine
deficiency (unpublisheddata).As longasthe increase intypeIIdeiodinase issufficienttomaintainnormalT3values inthefetalbrain,problems neednotbeexpected
(18). Ifthisisnotachieved,defects inbraindevelopmentwill occur.
Inconclusion: DuringpregnancytheAIUbythematernalthyroidgland isdecreased.
Marginal iodinedeficiency doesnotaffectthematernalAIU.Thelowavailability of
iodidewascompensatedby increasedactivityofthematernalthyroid,whereasfetal
thyroidal uptakeof iodidedecreasedby50%.Thefetus isapparently notableto
regulate its iodidemetabolism incaseofmarginal iodinedeficiency.Therefore,this
levelofmarginal iodinedeficiency, despitetheneareuthyroidstatusofthe mother,
already hasaneffectontheavailability of iodideforthefetus.Thiscanmeanthat
76
Chapter4
fetalT<production ismarkedlydiminishedatatimewhenitisofeminent importance
forthenormaldevelopment ofmanyorgans,especially thebrain.
ACKNOWLEDGEMENTS
WearegratefultoJ.S.GoenseforsettingupthemeasurementsandG.van Niftrik
(GelderseVallei Hospital,Bennekom,The Netherlands)for designingthe measuring
bedforthe rats.WethanktheMsCstudent M.vanLieshoutforhercontributionto
theexperiments, T.Vietsfor performingthe iodidemeasurements,dr. R. Bakkerfor
developingthemathematical modelandG.P. Bieger-Smithforcorrectionofthetext.
Reagents usedforthe ratTSHassaywerekindly provided bytheRat Pituitary
HormoneDistribution ProgramoftheNational Institute of Diabetes and Digestive
andKidney Diseases.
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ArntzeniusAB,SmitLJ,SchipperJ, vanderHeideD,andMeindersAE(1991)
Inverserelation between iodine intakeandthyroidbloodflow:color dopplerflow imaging
ineuthyroidhumans. JournalofClin.Endocrinol, andMetab.75:1051-1055.
CalvoR,Obregon MJ, RuizdeOnaC, Ferreiro B,EscobardelReyF,and Morreale
deEscobarG(1990) Thyroid hormoneeconomy inpregnant ratsnearterm:a"physiological"animalmodelofnonthyroidal illness?Endocrinology 127:10-16.
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DeLange F(1994)Thedisordersinduced byiodine deficiency. Thyroid4:107-128.
DiStefano IIIJJ, JangM,MaloneTK,andBroutman M(1982)Comprehensive kinetics
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EscobardelReyF,MallolJ,Pastor R, andMorreale deEscobar G(1987) Effectsof
maternal iodinedeficiency onthyroidhormoneeconomyoflactatingdams andpups:
maintenanceofnormalcerebral3,5,3'-triiodo-L-thyronineconcentrations inpupsduring
majorphasesofbraindevelopment. Endocrinology 121:803-811.
Iodideuptake bythematernalandfetalthyroid
8.
EscobardelReyF, PastorR,MaliolJ, andMorrealedeEscobarG(1986) Effects of
maternal iodine deficiency onthe L-thyroxineand3,5,3'-triiodo-L-thyronine contentsof
ratembryonictissuesbeforeandafteronsetoffetalthyroidfunction. Endocrinology
118:1259-1265.
9. FeldmanJD(1958) Iodinemetabolism inpregnancy.AmJPhysiol192:273-278.
10. FisherDA(1983)Maternal-fetalthyroidfunction inpregnancy. Clinicsinperinatology
10:615-626.
11. GaltonVA(1968)Thyroxine metabolism andthyroidfunctioninthepregnantratnear
term.Endocrinology82:282-290.
12. GlinoerD,DeNayerP,Bourdoux P,LemoneM,RobynC,VanSteirteghemA,
Kinthaert J, andLejeune B(1990) Regulationofmaternalthyroidduring pregnancy. J
ClinEndocrinolMetab 71:276-287.
13. Hetzet BS(1983) Iodinedeficiency disorders (IDD)andtheir eradication.Lancet!: 11261129.
14. HilditchTE,HortonPW,andAlexanderWD(1980)Quantitation ofthyroidal bindingof
iodidebycompartmentalanalysisverifiedbyanintravenous Perchlorate dischargetest.
EurJNuclMed5:505-510.
15. linoS,andGreer MA(1961) Thyroidfunctionintheratduring pregnancyandlactation.
Endocrinology68:253-262.
16. MarinoA,DiStefanoIIIJJ, and LandawEM(1992) DIMSUM:anexpertsystemfor
multiexponentialmodeldiscrimination.AmJPhysiol262.E546-E556.
17 McCrudenDC,Hildtich TE,ConnellJMC,andAlexanderWD(1985) Kinetics of 1 2 3 liodide uptakeanddischarge byPerchlorate instudies of inhibition ofiodidebindingby
antithyroiddrugs.ActaEndocrinol(Copenh) 110:499-504.
18. Obregon MJ, RuizdeOna C,CalvoR,Escobar delReyF,and MorrealedeEscobar
G (1991)Outerringiodothyronine deiodinasesandthyroidhormoneeconomy:responsesto iodinedeficiency intheratfetusandneonate.Endocrinology129:2663-2673.
19. RiescoG,TaurogA,LarsenPR,andKrulichL(1977)Acuteandchronicresponsesto
iodinedeficiency inrats.Endocrinology 100:303-313.
20. Roelfsema F,vanderHeideD,andSmeenkD(1980)Circadian rhythms of urinary
electrolyteexcretion infreelymovingrats.LifeSei27:2303-2309.
21. RotiE,GnudiA,andBravermanLE(1983)Theplacentaltransport,synthesisand
metabolism of hormonesanddrugswhichaffectthyroidhormonefunction. Endocrine
ReviewsA:131-149.
22 SandellEB,andKolthoffIM(1937)Microdetermination ofiodinebyakatalyticmethod.
MikrochimActa1:9-25.
23. SantistebanP,ObregonMJ,Rodriguez-PenaA,LamasL,EscobardelReyF,and
MorrealedeEscobarG(1982)Areiodine-deficientratseuthyroid ? Endocrinology
110:1780-1789.
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Chapter4
24. Schröder-vanderEistJP,andvanderHeide D(1992) Effects of streptozocin-induced
diabetesandfoodrestriction onquantities andsourceofT4andT3inrattissues.DiabetesAY. 147-152.
25. Statistical PackageforSocialSciences(1990)SPSS/PC+Statistics4.0.Chicago, IL:
McGraw-Hill.
26. TaurogA(1986) Hormonesynthesis:thyroidiodine metabolism. InIngbarS.H.and L.E.
Braverman(eds.). TheThyroid. SectionB:Hormone synthesisandsecretion,pp.53-93.
5thed.Philadelphia. LippincotCo.
27. VerslootPM,GerritsenJ,Boogerd L,Schröder-van derEistJP,andvanderHeide
D(1994)Thyroxineand3,5,3'-triiodothyronineproduction,metabolism, anddistribution in
pregnant ratnearterm.Am.J.Physiol. 267:E860-E867.
28. WayneEJ, KoutrasDA,andAlexanderWD(1964)Clinicalaspectsof iodinemetabolism (IandII).Oxford:Blackwell,1964.
29. WhitworthAS,MidgleyJEM,andWilkinsTA(1982)AcomparisonoffreeT4andthe
ratiooftotalT4toT4-bindingglobulin inserumthroughpregnancy. Clin.Endocrinol. (Oxf)
17:307-313.
30. Wolff J (1964)Transport of iodideandother anions inthethyroidgland. Physiological
Reviews44:45-90.
31. ZeghalN,Gondran F,RedjemM,Giudicelli MD,AissouneY,andVigouroux E(1992)
IodideandT4kinetics inplasma,thyroidglandandskinof 10-day-oldrats:effects of
iodinedeficiency.ActaEndocrinol(Copenh) 127:425-434.
CHAPTER5
EFFECTS OF MARGINAL IODINE DEFICIENCY ON THYROID HORMONE
PRODUCTION,DISTRIBUTION AND TRANSPORT IN NONPREGNANT AND
NEAR-TERM PREGNANT RATS.
P.M.Versloot, J.P. Schröder-van der Eist, D.vander Heide,and L. Boogerd
submitted EuropeanJournalof Endocrinology.
T4andT3kineticsduringMIDinpregnantrat
81
ABSTRACT
During pregnancy maternal thyroid hormones are of great importance for normal
development of the central nervous system of the fetus. Iodine deficiency of the
mother canresult inanimpaireddevelopment ofthefetal brain.Inlargeareasofthe
world the iodine intake is moderately low. To study the effects of marginal iodine
deficiency (MID) on the production, distribution, and transport of thyroxine (T4) and
3,5,3'-triiodothyronine (T3) in nonpregnant and near-term pregnant rats we performed kinetic experiments (three-compartment analysis). Despite unchanged
plasma T4 and T3 during MID, the production and plasma clearance rates of T4
decreased, while these values for T3 increased in nonpregnant rats. Hepatic deiodinase type I activity increased during MID. It appears that during MID rats are
able to maintain their euthyroid status. The pronounced increase in transport of T4
from plasma to thefast pool observed in normal pregnant rats did not occur during
MID.Thealterations inT3metabolismduetoMIDinpregnant ratsweretheopposite
ofthosefoundfor nonpregnant rats.
Conclusion: Marginal iodine deficiency affects maternal thyroid hormone metabolism,thusinfluencingtheavailability ofmaternalT4forthefetuses.
INTRODUCTION
In large areasoftheworld iodine intake is insufficient. Severe iodinedeficiency can
result inmiscarriage, stillbirth,andcongenital anomalies aswell asthemorefamiliar
goiter, cretinism, impaired brain function and hypothyroidism in children and adults
(15).
In rats iodinedeficiency canbe inducedby prolongedadministration of a lowiodine
diet. This treatment results ina markedly increasedweight ofthethyroid.TheT3-toT4 ratio inthe thyroid aswell asthe T3-to-T4 ratio secreted by the thyroid increased
progressively with the duration of iodine deficiency (12, 23). In the iodine-deficient
rat plasmaT4isdecreasedandplasmathyrotropin(TSH) increasedwhile circulating
T3remains normal (26).SinceT3inthebrainisgeneratedmainlybylocal conversion
from T4 (8), normal circulating T3 levels, in combination with lowplasma T4, are not
sufficient tomaintaineuthyroidisminthebrain(22).
Pregnancy alsoinfluencesthethyroidhormonestatus.Inhumansnormal pregnancy
82
Chapter5
is accompanied by a rise inthe serum levels of total T4 and T3. However, due to a
rise inT4-bindingglobulin thefreeT4 andT3 levels decrease during pregnancy (11,
32). In the rat normal pregnancy also results ina decrease in plasmaT4andT3(5,
29). This leads to reduced concentrations of T4 and T3 in the maternal tissues,
exceptfor T3inthe brain(5).Theclearance ofT4fromplasma isincreased markedly
whichcanbearesultofthetransportofT4tothefetalcompartment(29).
Thyroid hormones are known to play an important role in brain maturation. Their
absence during fetal development leads to irreversible brain damage. Studies of
pregnant rats on a low iodine diet by the Madrid group revealed that when iodine
deficiency is severe enough to cause very low maternal plasma T4 levels, fetal
plasma andtissueswill suffer ashortage ofT4andT3bothbeforeandafter onsetof
fetal thyroidfunction (9,21). During normal pregnancy mainlyT4istransportedfrom
mother tofetus (4). In severely hypothyroid pregnant rats only T4 can mitigatefetal
T4 andT3deficiency (3). Inthefetal brainT4is necessary for the localgenerationof
T3fromT4bycerebraltype IIdeiodinase(ID-II)(21).
Most experimental studies are performed during severe iodine deficiency. However,
in large populations intheworldthe iodine intake isonlymarginally iodine-deficient.
Data about the effects of marginal iodine intake onthyroid hormone metabolism are
lacking. Therefore,we inducedaniodinedeficiency inratssuchmarginalthatgrowth
and reproduction outcomewerenotaffected.Theaimofthis studywastodetermine
the effects of marginal iodinedeficiency onthyroid hormone metabolism innonpregnant and near-term pregnant rats. T4 and T3 production, distribution, and transport
were examined by performing a kinetic study usingthethree-compartment modelof
distribution and metabolism developed by DiStefano etal.(6,7). Inthis modelthree
compartments canbedistinguished. 1)the plasma; 2)tissuesthat exchangeT4and
T3 with plasma atafast rate, i.e. thefast pool; and 3)the slowexchanging pool, the
slow pool. Liver and kidney are presumed to be the main components of the fast
pool,whereas skin, muscle,andbrainbelongtotheslowpool(6,7).
By means ofthis modelwe have already described the production, distribution, and
transport of T4 andT3 in near-term pregnant rats on a normal iodine diet; we suggested thatT4istransported very rapidlyfrom plasmatothefeto-placental compartment (29). In this study we will concentrate on the effects of marginal iodine deficiency on maternal thyroid hormone metabolism. Does a marginal iodine deficiency
affecttheavailability ofthyroxineforthefeto-placentalcompartment?
T4and T3kineticsduringMIDinpregnantrat
83
MATERIALANDMETHODS
Animalsanddiet
Three-month-oldfemaleWistar rats (CPB/WU, Iffa Credo, Brussels)were used.The
animals were individually housed at22 °C, with alternating 14-h light and 10-hdark
periods. The ratswerefed a semi-synthetic American Institute of Nutrition (AIN) diet
(2). The dryfeed was mixedwith distilled water (60 %dry weight- 40 %water) with
theadditionofpotassium iodide: 55ng(normal iodinediet; NID)or2.9 ng(marginal
iodine diet; MID) per gramoffeed.Theiodine-deficient groups received 1% KCI04in
their drinkingwater duringthefirst two days ofthe MID. KCI04was giventoacceleratethyroidaldepletionoftotal iodinestores.
The rats were mated after at least two regular estrus cycles. The day that sperm
appeared in the vaginal smear was taken as day 0 of gestation. The gestational
periodofthe ratis22days.
Designofthestudy
MID treatment was startedat leasttwoweeks before matingfor thepregnant groups
andminimallyfiveweeks beforemeasurementsforthenonpregnant groups. Surgery
(insertionofthecannula)wasconducted oneweekbefore measurements.
Therewerefour groupsofrats:
1. nonpregnant ratsonanormaldiet ;NID-C.
2. pregnant ratsonanormaldiet ;NID-P.
3.nonpregnant ratsonamarginal iodine-deficient diet ;MID-C.
4. pregnant ratsonamarginal iodine-deficient diet ;MID-P.
Dailyfeed intakeand urinary iodide excretionweredeterminedforall rats.Themean
feed intakewas30gram, resulting inacalculated daily iodide intakeof 1.3ugforthe
NIDgroupsand66 ngfortheMIDgroups.
Kinetic experiments were performed onday 19ofgestation.After the kineticexperimenttheanimalswerekilledandmaternalandfetalliverandbrainwerecollected.
The experiments were approved by the University Committee on Animal Care and
UseoftheAgricultural UniversityofWageningen.
84
Chapter5
Kineticandanalyticalprotocols
[125I]T4 and [131I]T3 (specific activity 80 and 130 Mbq/ug (2200 and 3500 uCi/ug),
respectively) wereprepared inour laboratory (2,18).Na125landNa131lwere obtained
from Amersham (Arlington Heights, IL); L-T3and 3,5-L-diiodothyronine, the respectivesubstratesforlabelling,werepurchasedfromSigma(St.Louis,MO).Purity ofthe
tracerswasassessed bymeansofHPLC.
Viaacannula inserted intotherightjugularvein(24)theratsreceiveda400 ulbolus
injection of a solution of 3700 Bq (10 uCi) [125I]T4and 3700 Bq (10 uCi) [131I]T3in
saline containing heparin (0.3 U/ml; Organon, Tilburg, The Netherlands), ticarcillin
(0.4 mg/ml; Ticarpen, Beecham S.A., Heppignies, Belgium) and 5 % normal rat
serum. Ticarcillin isaddedtosolutionscontaining labelled iodothyronines to prevent
eventual bacterial infections and reduce artefactual deiodination, as described by
VanDoorn(8).
Fromthe canula inthejugular vein blood samplesof0.2 mlweretakenat 1, 4.5, 10,
23, 44, 115, 202and 315 min; 0.4 ml.were drawnat465,600,900, 1200and 1440
min,beingtheoptimaltimescheduleaccordingtoDiStefanoetal.(6,7).
Fifty to hundredmicroliters plasma (induplicate)wereusedtocountthetotal 131land
125
l activities. The plasma samples were extracted with ethanol/ammonia (25
%)(197:3v/v)with0.1 mMpropylthiouracil (PTU).Thedriedextractswere dissolved
in 0.1 ml0.2 Mammoniacontaining carrier T4,T3andKl(1mg/10ml)andsubjected
to HPLC to separate the iodothyronines, according to the method described by
Schröder-vander Eistandvander Heide(27).
The endogenous concentrations ofT4andT3inplasmawere measuredbymeansof
a specific rat RIA (14) of samples taken before the kinetic experiment was performed.
PlasmaTSHwas measured bythespecific RIAdevelopedforthe ratbythe National
Institute of Diabetes and Digestive and Kidney Diseases of the National Institute of
Health(USA). Reference preparation RP-2was usedasastandard.
Calculations
The percentage doses of [131I]T3and[125I]T4per ml plasmawere calculatedfromthe
total radioactivities and the [131I]T3and [125I]T4distributions on the HPLC chromatogram. The percentage doses per ml, together with the plasma volume at t(0) (16,
29),werefitted individuallytosumsofn=1to3exponential:
Y(t)=A, exp(Vt) +A2exp(A2*t)+A3exp(A3*t)
usingtheprogram DIMSUM,wherebyA,isexpressed in%dose/ml,\ inmin'1(20).
T,andT3kineticsduringMIDinpregnantrat
85
To calculate the parameters of production, distribution and metabolism the program
MAMPOOL (19)was used.Thesumofthethreeexponentialfunctionswasfitted(by
weighted least squares regression analysis) tothe data collectedfor each individual
rat and, together with plasma J4 and T3 levels, substituted into the three-compartment model,accordingtothekineticT4andT3studiesofDiStefanoetal,(6,7).
All data are expressed as mean ± SE. Statistical analysis was performed by the
Student'sMest usingtheStatistical Packagefor SocialSciences(SPSS)(28).
Analyticaldeterminations
Urinary iodidewasdeterminedasdescribedbySandedandKolthoff(25).
Mitochondrial andcytosolicfractionsfromliver andbrainwereobtainedbydifferentialcentrifugation,accordingtotheseparationschemedescribedbyvanDoorn(8).
Liver and brain mitochondrial alpha-glycerophosphatedehydrogenase (a-GPD) was
measuredbymeansofthemethoddescribedbyGarribandMcMurray (10).
Cytosolic malic enzyme (ME) was determined for liver and brain according to the
methodofHsuandLardy(16).
The determination of type I deiodinase (ID-I) activity in liver homogenates was
performedbythemethoddescribedbyJanssenetal. (17).
Protein was determined by the bicinchoninic acid (BCA) method (Pierce Europe,
OudBeijerland) usingBSAasstandard.
RESULTS
Urinaryiodide excretion
The effect of treatment with 1 % KCI04 for two days is demonstrated in Fig. 1a.
Within aweek after Perchloratetreatment urinary iodide excretion haddecreasedto
0.4 ug/day; it remained constant during the rest of the experimental period. Fig. 1b
shows the daily urinary iodide excretion four weeks after the start of the diet. MID
resulted in a sharp decrease in urinary iodide excretion. No effect of pregnancy on
urinary iodideexcretionwaspresent inNIDandMIDrats.
Chapter5
86
B.
A.
2000
3000
>>
a 1500
•o
2000
o»
S 1000
c
o
Tj
1000
•5
5
500
NID MID
C
C
1 2 3 4 5 6 7 8 9 10
NID
P
MID
P
KCIO
Figure 1:Theeffect of KCI04treatment onthe24 hurinaryiodideexcretion(A) andthedaily
urinary iodideexcretion (B).Valuesaremeans±SEM.P<0.05,*MIDvs.NID.
Bodyweightandplasmahormones
Table 1 showsthe body weight (BW) and plasma thyroid hormone and TSH levels.
Marginal iodinedeficiency hadnosignificant effect onmaternal BWandthe number
of fetuses. During pregnancy the plasma T4andT3decreased; plasmaTSH did not
changesignificantly. Thisappliedfor NIDaswellasMIDrats. Duringmarginal iodine
deficiency plasma T4 and T3 values were unchanged in nonpregnant as well as in
pregnant rats;plasmaTSHwas increasedsignificantly innonpregnant ratsonly.
Table 1:Bodyweightandplasmathyroid hormone concentrations.
Valuesaremeans± S E M
Bodywt(g)
NID-C
MID-C
NID-P
MID-P
(n=6)
(n=9)
<n=8)
(n=6)
211 ± 6
229 ± 6
304±6'
323±9'
11 ± 1
11 ± 1
No.of fetuses
T4(nmol/l)
33.8±2.2
34.6±3.0
22.5±1.5*
17.2±1.7'
T3(nmol/l)
0.62 ±0.04
0.73 ±0.06
0.53 ±0.06
0.44 ±0.04"
TSH(ng/ml)
0.28±0.08
0.75 ±0.10®
0.49±0.06
0.65 ±0.09
P<0.05:* Pvs.C;
® MIDvs.NID.
T4andT3kineticsduring MIDinpregnantrat
87
T4kinetics
The effect of pregnancy on the fractional turnover and transport rates of T4 was
similar for NID and MID rats; T4 disappeared more quickly from all three pools in
pregnant rats. No effects of marginal iodine deficiency on the kinetic parameters of
T4werefound(datanotshown).
The mean results of distribution volumes, pool sizes, and transport rates of T4 are
summarized inTable2.
Table 2:Parametersofdistribution,volumes, poolsizes,and ratesoftransport ofT4(expressedper100gbodyweight).Valuesaremeans±S.E.M.
NID-C
PCR (ml/h)
PR (pmol/h)
1.38 ±0.06
47.2 ± 4.2
MID-C
NID-P
MID-P
0.93 ±0.07®
1.73 ±0.04*
1.79 ±0.17"
31.9 ±3.4®
38.6± 2 . 5
30.4 ±3.0
Vtotal(ml)
17.9± 0 . 6
16.1 ± 0 . 5
18.1± 0 . 8
15.9±1.5
Vp(ml)
4.9 ±0.1
4.5± 0 . 1
4.9± 0 . 1
4.7 ± 0 . 2
V2(ml)
4.3 ± 0.3
4.5 ± 0 . 3
4.0 ±0.3
3.6 ± 0 . 7
V3(ml)
8.7 ±0.6
7.1± 0 . 5
9.2 ± 0 . 5
7.6 ± 1 . 0
Qtotal (pmol)
615 ± 5 1
546± 3 4
421 ±32"
274 ± 35' ®
Qp (pmol)
166± 1 2
156 ±11
110 ± 7 *
82 ± 8*®
Q2 (pmol)
145± 1 2
152± 1 2
89± 9 "
65 ±17*
Q3 (pmol)
303 ±33
239± 1 8
207 ± 1 7 "
127 ±16"®
TRpf (pmol/h)
973 ± 1 1 4
1392± 363
2574 ±317*
1250 ±249®
TRfp (pmol/h)
952 ± 1 1 5
1376 ± 363
2563 ±313"
1233 ±250®
DRfo (pmol/h)
23.6± 2 . 1
16.0 ±1.7®
19.8 ± 1 . 3
15.4 ± 1 . 4
TRps (pmol/h)
162± 2 3
108± 2 0
188± 3 2
124± 4 4
92± 1 8
169± 3 1
109± 4 5
19.8 ± 1 . 3
15.4± 1 . 4
TRsp (pmol/h)
138± 2 1
DRso (pmol/h)
23.6± 2 . 1
16.0 ±1.7®
PCR, plasma clearance rate; PR,production rate; V, plasma equivalent distribution volume;
Q, poolquantity;TRpf,TRfp,TRps,andTRsp, ratesoftransport between plasma andthefast
and slow pools respectively, ineach direction; DRfo andDRso, disposal rate from fastand
slow pool respectively;%PFand%PS,fractionofT„inplasma transported unidirectionallyto
thefast andslowtissue poolsrespectively;p,plasma;2,fast pool;3,slowpool.
P<0.05:' Pvs. C;® MIDvs.NID.
88
Chapter5
In pregnant rats the plasma clearance rate of T4 is increased. A decreased plasma
clearance rateduetomarginal iodinedeficiencywasfoundfornonpregnant MIDrats
butnotpregnant MIDrats.
The production rate of T4 remained unchanged during pregnancy. Marginal iodine
deficiency resulted inadecrease inT4production,whichwassignificant fornonpregnant ratsonly.
The distribution volumes of T4 were not affected by pregnancy or marginal iodine
deficiency.
The total amount of T4decreased inthe pregnant groups. This decrease was found
in all three pools. Marginal iodine deficiency induced a further decrease in T4 pool
sizes for the pregnant rats; for nonpregnant rats no effect of marginal iodine deficiencywasfound.
The transport ofT4changedonly inpregnant ratsona normal iodide diet. Transport
to the fast pool and vice versa were more than doubled. This effect of pregnancy
was not found inthe MID group. The disposal of T4decreased in both nonpregnant
andpregnantrats.
The transit timesforthethree poolsandthetotal residencetimeforT4are shownin
Table 3. Inpregnant ratsthetransit timesfor T4 in plasma andthefast pool decreased. The total residence time also decreased. Marginal iodine deficiency results in
anincrease inthetotal residencetimeforT4innonpregnant ratsonly.
Table3:TransittimesandtotalresidencetimeforT..Valuesaremeans±S.E.M.
NID-C
MID-C
NID-P
MID-P
Transittime,min
Plasma
9.5 ±0.8
9.1 ±1.5
3.7 ±0.9*
4.5 ±1.1*
Fastpool
9.7 ± 1.1
10.1 ±1.8
3.5 ±1.2'
4.2±1.5*
Slowpool
Totalresidencetime ,min
127 ±15
166 ±24
99 ±23
154 ±90
789±30
1079±73®
616 ±20'
560 ±86
P<0.05:*Pvs. C;® MIDvs. NID.
T3kinetics
For T3 almost nosignificant alterationswerefoundinthe kinetic parameters. Only in
pregnant MIDrats adecreaseofthetransport ratefromplasmatothefast pool, due
tomarginal iodinedeficiency,wasfound(datanotshown).
Table4showsthedistributionvolumes, poolsizes,andtransport ratesofT3.
T„andT3kinetics duringMIDinpregnantrat
89
Table 4: Parameters of distribution volumes, pool sizes, and transport rates of T3 (expressed
per 100gbodywt). Values are means±S.E.M.
NID-C
MID-C
NID-P
PCR(ml/h)
21.7 ±2.1
36.6± 5.4 e
26.1 ±2.8
24.9±2.4
PAR(pmol/h)
13.4 ±1.5
27.6±5.0®
13.3 ±1.1
10.3±0.4* e
PR(pmol/h)
14.6 ±1.7
32.2± 6.5
e
14.6 ±1.2
11.4±0.5* e
Vtotal (ml)
209±20
278± 21®
194 ±15
224±39
Vp(ml)
4.8 ±0.1
4.8 ±0.6
4.9 ±0.1
4.8 ±0.2
V2(ml)
28.6 ±2.1
35.1 ±5.1
33.1 ±4.3
24.9 ±3.4
V3(ml)
176 ±19
238±21
180±27
194 ±37
Qtotal(pmol)
128±11
216±24 e
111 ±13
95±18*
MID-P
Qp (pmol)
3.0 ±0.2
3.4 ±0.3
2.6 ±0.3
2.1±0.2*
Q2(pmol)
17.5 ±1.0
24.4 ±2.9
17.2 ±2.5
10.6±1.4* e
Q3(pmol)
108±11
176±25 e
92 ±14
83±17*
TRpf (pmol/h)
214±15
256 ±30
229±34
100±20* e
TRfp (pmol/h)
207 ±15
242±28
222±34
95±20* e
DRfo(pmol/h)
6.7 ±0.8
13.8±2.5 e
6.6 ±0.6
5.1±0.2* e
TRps (pmol/h)
53 ±7
81 ±13
56±11
44±4*
TRsp (pmol/h)
55 ± 7
85 ±13
58 ±11
47 ±5*
8.0±0.6
6.3 ±0.3* e
DRso (pmol/h)
8.0±0.9
18.5±4.0
e
PCR, plasma clearance rate; PAR, plasma appearance rate; PR, production rate; V, plasma
equivalent distribution volume; Q, pool quantity; TRpf, TRfp, TRps, and TRsp, rates of transport between plasma and the fast and slow pools respectively, in each direction; DRfo and
DRso, disposal rate from fast and slow pool respectively; % PF and %PS, fraction of T3in
plasma transported unidirectionally to the fast and slow tissue pools respectively; p, plasma;
2, fast pool;3,slowpool.
P< 0.05: " P vs.C;e MIDvs.NID.
The plasma clearance rate of T 3 increased in the nonpregnant MID group only.
Pregnancy resulted in a decrease in T 3 production in MID rats. The effects of marginal iodine deficiency on the production of T 3 were conflicting. In nonpregnant MID
rats we found an increase, while in pregnant MID rats the T 3 production decreased
slightly.
The transport of T 3 from plasma to the fast pool and vice versa decreased in pregnant MID rats only. The disposal of T 3 increased in nonpregnant MID rats. Marginal
iodine deficiency induces in pregnant rats'even a decrease in the disposal of T3.
Chapter5
90
In pregnant MID rats the increased transit time for T 3 in plasma was the only change
intransit times andtotal residence timefoundfor T 3 (Table 5).
Table5:Transittimes andtotal residencetimeforT,.Valuesaremeans ±S.E.M.
NID-C
MID-C
MID-P
NID-P
Transittime,min
Plasma
0.67 ±0.01
0.76 ±0.22
0.57 ± 0.04
0.93±0.16®
Fastpool
4.99 ± 0.36
6.64 ±1.35
Slowpool
Totalresidencetime, min
4.78 ± 0.66
7.23 ±1.22
114± 15
122 ± 25
95 ±10
99 ±23
607 ±76
544 ± 95
513 ±52
553±94
e
P<0.05: Pvs. C; MIDvs. NID.
Enzymes
Figure 2 shows the a-GPD activity in liver and brain. The same pattern is found for
liver (2a) and brain (2b). The a-GPD activity decreased in the pregnant groups;
marginal iodine deficiency had no effect.
In the fetal liver and brain no differences in a-GPD activity were found between the
NID and MID rats (data not shown).
A
300
B.
1800
JL
1200
200
JL
600
100
NID-C MID-C NID-P
MID-P
NID-C MID-C
Figure 2:a-GPD inmaternal liver(A)andmaternal brain(B).
Valuesaremeans ±SEM.P<0.05;* Pvs.C.
NID-P
MID-P
T4andT3kineticsduring MIDinpregnantrat
91
The data on ME are shown in Fig.3. No effect of pregnancy or marginal iodine
deficiency was found in the liver. In the brain however, ME activity increased in
pregnant rats.
Pregnancy and marginal iodine deficiency both increase ID-I activity in the liver
(Fig.4); this increase is additive.
A.
B.
250
200
200
150
150
100
100
50
0
50
0
NID-C MID-C NID-P MID-P
NID-C MID-C
Figure 3: Malicenzymeinmaternalliver(A)andmaternalbrain(B).
Valuesaremeans±SEM.P<0.05; * Pvs.C.
400
c
o
*#
#
300
_,_
i
O)
E
c
Ë
ô
200 100
E
Q.
n
NID-C
MID-C
NID-P
MID-P
Figure4: Deiodinasetype Iinliver.Values aremeans ±SEM.
P<0.05;* Pvs. C,*MIDvs.NID.
NID-P
MID-P
92
Chapter5
DISCUSSION
The aim of this study was to determine whether a marginal iodine deficiency will
affect thyroid hormone production, distribution, and transport in nonpregnant aswell
as near-term pregnant rats. Duringfetal growththyroid hormones are necessary for
development ofthecentral nervous system. Ithasbeenshownthatamoderately low
iodine supply is sufficient for the daily needs of healthy adult subjects. Duringpregnancy, however, the iodine supply becomes relatively insufficient, due to enhanced
maternalrequirements (13) andincreased renalclearance(1).
Many animalstudies havebeenperformedto studytheeffect of iodinedeficiency on
the development of fetuses (9, 21), but no information is available on the effects of
iodine deficiency on maternal thyroid hormone metabolism and the availability of
maternal thyroid hormones during pregnancy. The effects of iodine deficiency on
thyroid hormone secretion have only been described for nonpregnant rats (12,23).
In most experimental studies severe iodine deficiency was induced, while in our
studythe ratswere marginally iodine-deficient. This condition ismuchmorecommon
iniodine-deficient areas.
Severe iodine deficiency is characterized by an increase in thyroid weight, low
serum T4, normal or slightly elevated serum T3 and high TSH (9, 22, 26). During
pregnancy decreased reproductive competence has been reported (9). In our
laboratory, rats receiving the sametreatment as describedfor this study showed an
increase in thyroid weight (30). Neither retarded growth nor decreased number of
fetuseswasobserved,sothe iodinedeficiency achieved musthavebeenmarginal.
During marginal iodinedeficiency plasmaT4andT3values remainedunchanged,but
plasma TSH increased sligthly. Even under these moderate conditions T4 and T3
production and their plasma clearance rates had already changed. The kinetic
experiment revealed a decreased production of T4 in nonpregnant MID rats. The
unaltered plasmaT4mightpossibly beexplainedbythedecreased plasmaclearance
rate, resulting inanincreased meanresidencetimeforT4inthebody.
The production of T3 increased, but plasma T3was not affected by marginal iodine
deficiency. Weattributethistotheincreased plasmaclearancerate.
Janssen etal.(17)found noeffect of iodine deficiency on hepatic ID-Iactivity, even
though T4 intheir animals was lower. Inthe brain an increase in ID-IIwas found in
iodine-deficient rats (17). In our study marginal iodine deficiency induced an increase in hepatic ID-Iactivity. Therefore, the increasedT3production might possibly
T„and T3kineticsduringMIDinpregnantrat
93
beexplainedbyenhancedT4toT3conversioninthe liver.
We suggest that rats are able to maintain their euthyroid status during marginal
iodine deficiency. The alterations in T4 and T3 production are compensated by
changes inthe plasma clearance rates, resulting in normal thyroid hormone values
in plasma and tissues. This is confirmed by the unchanged levels of the thyroid
hormone-sensitive enzymesa-GPD andmalicenzymeinliverandbrain.
The alterations in thyroid hormone metabolism due to pregnancy are in agreement
with previous findings for iodine-sufficient rats (29). There is a difference in the
effects of marginal iodine deficiency onthe production and metabolism ofT4 during
pregnancy andinthe nonpregnantsituation.
During pregnancy T4 production also decreased as a result of marginal iodine deficiency, but the plasma clearance rate remained unchanged. This resulted in a
decrease in the total amount of T4 in the body. Striking is the effect of marginal
iodine deficiency on the transport of T4 to the fast pool in pregnant rats. During
normal pregnancy the transport of T4 to the fast pool and vice versa is markedly
increased. We suggested that this is caused by the fetal compartment (29). However, in marginally iodine-deficient near-term pregnant rats no increase in transport
to the fast poolwasfound.This could indicate thatduring marginal iodine deficiency
thetransport ofT4tothefetal compartment isalready diminished.This isexceedingly important, because it is mainly T4that is transported from mother tofetuses. The
absence of maternal T4 during fetal development can cause damage to the central
nervous system (4). However, we did not find any effect on a-GPD activity in the
fetal brain during marginal iodine deficiency. Thismight possibly beexplained byan
increase in fetal ID-II; during severe iodine deficiency the fetal brain is protected
fromhypothyroidism byanincrease intheactivity of ID-II (21).
The alterations inT3metabolism duetomarginal iodinedeficiency found inpregnant
rats are the exact opposite of the effects in nonpregnant rats. During pregnancy
production andthe plasma clearance rate of T3decrease slightly; these parameters
increase innonpregnant rats.
Despite a decrease in plasma T4 and T3 hepatic ID-I is increased at the end of
gestation. This had already been described earlier (5, 29). Just as in nonpregnant
ratsafurther increaseofID-Iisfoundduringmarginal iodine deficiency.
The transport of T3from plasma to the fast pool and vice versa was decreased by
fifty percent in marginally iodine-deficient pregnant rats. This implies that, despite
the markedly increased ID-I activity, locally producedT3iseither decreased ordoes
94
Chapter5
not enter the circulation.
The present results demonstrate that under these conditions marginal iodine intake
influences maternal thyroid hormone metabolism. The total amount of T 4 in the
mother is decreased and less T 4 is transported to the fetuses. So, even a marginal
iodine deficiency seems to affect the availability of maternal T 4 for the fetuses.
Eventually this will cause fetal hypothyroidism, resulting in impaired fetal brain
development.
ACKNOWLEDGEMENTS
We thank the PhD students A. Luttikholt, M. van Rij and J.Verschoor for their contribution to the experiments and G.P. Bieger-Smith for correction of the text.
Reagents used for the rat TSH assay were kindly provided by the Rat Pituitary
Hormone Distribution Program of the National Institute of Diabetes and Digestive
and Kidney diseases.
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DiStefano Mi JJ, Malone TK, and Jang M (1982) Comprehensive kinetics of thyroxine
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8. DoomvanJ,vander HeideD,andRoelfsema F(1983) Sourcesandquantityof 3,5,3'triiodothyronine in several tissues of the rat. Journalof ClinicalInvestigation72: 17781792.
9. Escobar del Rey F, Pastor R, MallolJ,and Morreale de Escobar G (1986) Effects of
maternal iodine deficiency on the L-thyroxine and 3,5,3'-triiodo-L-thyronine contents of
ratembryonic tissues before and after onset offetal thyroid function.Endocrinology 118:
1259-1265.
10. GarribA,and McMurrayWC(1984)Asensitive, continuous spectrophotometricmethod
for assaying a-glycerophosphate dehydrogenase: activation by menadione. Analytical
Biochemistry139:319-321.
11. Glinoer D, De Nayer P, Bourdoux P, Lemone M, Robyn C, van Steirteghem A,
Kinthaert J, and Lejeune B (1990) Regulation of maternal thyroid during pregnancy.
JournalofClinicalEndocrinologyandMetabolism 71: 276-287.7
12. Greer MA, Grimm Y, and Studer H (1968) Qualitative changes in the secretion of
thyroid hormones induced byiodinedeficiency. Endocrinology 83:1193-1198.
13. Hainan KE (1958) The radioiodide uptake of the human thyroid in pregnancy. Clinical
Science17:281-290.
14. Heide vander D,andVisser-Ende M(1980)T4,T3and reverseT3inthe plasma of rats
duringthefirst 3monthsof life.ActaEndocrinologica 93:448-454.
15. Hetzel BS(1983) Iodinedeficiency disorders (IDD)andtheir eradications. The Lancet2:
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16. HsuRY,andLardyHA(1969) Malic Enzyme.Meth.Enzym.13:230-235.
17. Janssen PLTMK, van der Heide D, Visser TJ, Kaptein E, and Beynen AC (1994)
Thyroid function and deiodinase activities in rats with marginal iodine deficiency. Biological Trace ElementResearch 40:237-246.29
18. Kjeld JM, KukuSK, Diamant I, Fraser TR,Joplin GF,and Mashiter K(1975) Production and storage of [125l]-triiodothyronine of high specific activity ClinicalChimica Acta
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19. Lindell R, DiStefano III JJ, and Landaw EM (1988) Statistical variability of parameter
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20. Marino A, DiStefano III JJ, and Landaw EM (1992) DIMSUM: an expert system for
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Chapter5
21. Obregon MJ, RuizdeOna C,CalvoR, Escobar del Rey F,and MorrealedeEscobar
G (1991) Outer ring iodothyronine deiodinases and thyroid hormone economy: responses to iodine deficiency in the rat fetus and neonate. Endocrinology 129: 26632673.
22. Obregon MJ, Santisteban P, Rodriguez-Pena A, Pascual A, Cartagena P,
Ruiz-Marcos A, Lamas L, Escobar del Rey F, and Morreale de Escobar G (1984)
Cerebral hypothyroidism in rats with adult-onset iodine deficiency. Endocrinology115:
614-624.
23. Riesco G,TaurogA, Larsen PR,and Krulich L(1977) Acute andchronic responses to
iodinedeficiency inrats.Endocrinology 100:303-313.
24. Roelfsema F, van der Heide D, and Smeenk D (1980) Circadian rythms of urinary
electrolyteexcretion infreely movingrats.LifeScience 27:2303-2309.
25. Sandeil EB,and Kolthoff IM(1937) Microdetermination of iodine bya katalyticmethod.
MikrochimActa 1:9-25.
26. Santisteban P, Obregon MJ, Rodriguez-Pena A, Lamas L, Escobar del Rey F, and
Morreale de Escobar G (1982) Are iodine-deficient rats euthyroid ?Endocrinology110:
1780-1789.
27. Schröder-van der Eist JP, and van der Heide D (1990) Effects of
5,5'-diphenylhydantoin on thyroxine and 3,5,3'-triiodothyronine concentrations in several
tissuesoftherat.Endocrinology 126: 186-191.
28. SPSS Inc. SPSS/PC+ STATISTICS 4.0 (1990) McGraw-Hill, Chicago, IL: McGraw-Hill,
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29. Versloot PM,Gerritsen J, Boogerd L, Schröder-van der EistJP,and vander Heide
D (1994) Thyroxine and3,5,3'-triiodothyronine production,metabolism,anddistributionin
pregnant ratnearterm.Am.J.Phys.267:E860-E867.
30. Versloot PM, Schröder-van der Eist J, van der Heide D, and Boogerd L (1997)
Effects of marginal iodine deficiency during pregnancy: iodide uptake by the maternal
andfetalthyroid.Am.J.Phys.273:E1121-E1126.
31 Weeke J,and Orskov K(1973) Synthesis of [125l]monolabelled T3andT4of max-imum
specific activity for radioimmunoassay. Scandinavian Journal of ClinicalLaboratory
Investigation 32:357-366.
32. WhithworthAS, MidgleyJEM,andWilkins TA(1982) Acomparison offreeT4andthe
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17:307-313.
CHAPTER6
MATERNALTHYROXINEAND3,5,3'TRIIODOTHYRONINEKINETICSIN
NEAR-TERMPREGNANTRATSATTWODIFFERENTLEVELSOF
HYPOTHYROIDISM.
PMVersloot,D vanderHeide,JPSchröder-vanderEist,L Boogerd.
EuropeanJournalofEndocrinology138113-119,1998.
T„and T3kineticsinpregnanthypothyroidrats
99
ABSTRACT
Thyroid hormones are extremely important for development of the fetal central
nervous system. Thyroidectomy results in severe hypothyroidism. In this study two
levels of maternal hypothyroidism were reached by administration of different
amounts of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) to thyroidectomizedpregnant rats. We examined the production, distribution and transport of T4 and T3 by
performing a kinetic experiment (three-compartment analysis) with intact and thyroidectomized near-term pregnant ratswhich received either very low(Tx+lowTH) or
normal (Tx+TH) doses of T4 and T3. Despite administration of normal doses of
thyroid hormones plasma TSH was still elevated in the Tx+TH rats, meaning that
these rats were still mildly hypothyroid. The Tx+lowTH rats were markedly hypothyroid, theplasmaT4andT3levels beingvery low.Inthemildly hypothyroid ratsthe
transport ofT4fromplasmatothefast pooland viceversawasdecreased compared
with intact near-termpregnant rats.ThiscouldimplythatmuchlessT4istransported
to the feto-placental compartment. Liver type Ideiodinasewas decreased, resulting
in lowered plasma T3values. Inthe markedly hypothyroid rats all pools and ratesof
transport of T4 and T3were greatly decreased. In conclusion, even mild hypothyroidism, despite normal plasmaT4values, results insignificant changes, especially in
maternal T4transport. We suggest that even mild maternal hypothyroidism will have
a negativeeffecton the availabilityofmaternalT4forfetuses.
INTRODUCTION
Thyroid hormones are extremely important for fetal development, especially of the
central nervous system. In man, the children of mothers who were hypothyroid
during pregnancy show a high incidence of behavioral and neurological disorders
(14).
Rat studies have demonstrated that maternal hypothyroidism during pregnancy has
irreversible effects on braindevelopment, as indicated by adecrease inthe activity
of various cell marker enzymes in the brain of the progeny (8). The influence of
maternal hypothyroidism onfetal development isparticularly dramatic duringthefirst
half of the gestational period (2). However, even after the fetal thyroid starts to
function, on day 17.5-18 of gestation, maternal thyroid hormones contribute to the
100
Chapter6
fetal iodothyronine pool (16). The effects of maternal thyroidectomy, resulting in
severe hypothyroidism, onfetal development andthyroid hormone levels have been
studiedextensively bytheMadridgroup (17, 19,22).Theydemonstratedadecrease
inthe numberoffetuses andthefetalbodyweight ofthyroidectomizeddams(17,19,
22). Maternal plasmathyroxine (T4)and3,5,3'-triiodothyronine (T3) levels decreased
markedly, while thyrotropin (TSH) was highly elevated in thyroidectomized rats. T4
and T3 concentrations were decreased in both liver and brain tissue (17, 18, 20).
The concentrations of T4 and T3 in embryonic tissues from thyroidectomized dams
were undetectable before the onset of fetal thyroid function (19). At the end of
gestation, on day 21, fetal plasma TSHwas elevated (2) andthe plasma T4andT3
levels of the fetuses of thyroidectomized dams were similar (22) or even slightly
elevated (17, 20) comparedtothose of intact dams.Total thyroidal T4andT3 levels
infetusesfromthyroidectomized damshaddecreased markedly bytheendofgestation (22). The fetal liver T4 concentration was decreased, whereas such a change
was not found for hepatic T3. The deiodinase type I(ID-I) activity infetal liver had
decreased by50%(22).Noeffect ofmaternalthyroidectomy onT4andT3concentrations infetal brain on day 20 was found; in contrast, an increase inthe cerebral T4
andT3concentrations onday21wasreported(17, 19).
Euthyroidism can be restored inthyroidectomized rats by administration of bothT4
and T3 (6). Iftheamount of thyroid hormones administrated islessthantheamount
normally produced, the animal will still be hypothyroid. In the present study two
levels of thyroid hormone status were achieved by the administration of different
amountsofT4andT3tothyroidectomized pregnantrats.
The aim of ourstudywastoexaminethe effects ofamildandamarked hypothyroid
state on maternalthyroid hormone metabolism inthe near-term pregnant rat.T4and
T3 metabolism, distribution, and transport were examined by performing kinetic
studies using the three-compartmental model of distribution and metabolism developed byDiStefano (3,4). Inthismodelthreecompartments canbedistinguished:
1)the plasma;2)tissuesthat exchangeT4andT3withplasmaat afast rate,thefast
pool; and 3)tissues which exchange at a slowrate, the slow pool. Liver and kidney
are considered to be the main components of the fast pool, whereas skin, muscle
and brain belong to the slow pool (3, 4). Previously we suggested that in normal
near-term pregnant rats T4 is transported very rapidly from plasma to the feto-placental compartment (25).Inthe present studywecomparedthetransportof maternal
thyroid hormones to the feto-placental compartment during different levels of hypo-
T4andT3kinetics inpregnant hypothyroid rats
101
thyroidism. The mildly hypothyroid rats are of special interest;will there already be
an effect on maternal thyroid hormone metabolism, and isthe maternal contribution
ofthyroidhormonestothefetalcompartment affected?
MATERIALANDMETHODS
Animalsanddiet
Female Wistar rats (CPB/WU, Iffa Credo, Brussels) weighing 150 gwere rendered
hypothyroid by means of 131lthyroid ablation (a single intraperitoneal injection of 15
Mbq (0.4 mCi) Na131l). Oneweek before andone week after radiothyroidectomy the
rats received iodide-free American Institute of Nutrition (AIN) food (1). Subsequently
iodide-richAINfood(40 ugKl pergfood)wasgivenfor5daystowashthe 131loutof
the animals' system.DuringtherestoftheexperimenttheanimalsreceivedAINfood
(dry powder mixedwithdistilledwater (60%dryweight -40%water)) containing 55
ngiodide/gfood.
Intact,age-matchedcontrolsalsoconsumedAINfoodwith55ngiodide/gfood.
Hypothyroidism was demonstrated byundetectable plasmaT4 levels sixweeks after
radiation. Nosooner thantwo months after radiothyroidectomy the ratsweremated.
Until mating the rats received 0.25 ug T3 daily. Mating was confirmed by the presenceofsperminavaginal lavagethefollowingmorning,calledday0ofgestation.
Designofthestudy
Threegroupsofratswereused:
1. Intact: intact, pregnant euthyroidrats.
2. Tx+TH: pregnant radiothyroidectomized (Tx) rats on high T4 and T3 (0.25 ug T3
and2.5 ugT4perdayaddedtotheAINdiet).
3. Tx+low TH: pregnant radiothyroidectomized (Tx) rats on low T4 and T3 (0.01 ug
T3and0.1ugT4perday addedtotheAINdiet).
T4andT3werepurchasedfromSigma(St.Louis,MO,USA).
Kinetic experiments were performed onday 19ofgestation.Twenty-four hours after
the kinetic experiment the animals were killed; maternal blood, liver, brain and
pituitarywerecollected.
The experiments were approved by the University Committee on Animal Care and
UseoftheAgricultural UniversityofWageningen.
102
Chapter6
Kineticandanalyticalprotocols
[125I]T4 and [131I]T3 (specific activity 80 and 130 Mbq/|jg (2200 and 3500 (uCi/ug),
respectively) were prepared in our laboratory (11,26). Na125l and Na131l were obtained from Amersham (Arlington Heights, IL, USA); L-T3and 3,5-L-diiodothyranine,
the respective substrates for labelling, were purchased from Sigma (St. Louis,MO,
USA).PurityofthetracerswasassessedbymeansofHPLC.
Via a cannula inserted into the right jugular vein (21) the rats received a 400 pi
bolus injectionofasolution of 3700 Bq(10uCi) [125I]T4and3700 Bq(10 uCi)[131I]T3
insaline containing heparin (0.3 U/ml; Organon,Tilburg,TheNetherlands), ticarcillin
(0.4 mg/ml; Ticarpen, Beecham S.A., Heppignies, Belgium) and 5% normal rat
serum. Ticarcillin isaddedtosolutionscontaining labellediodothyroninesto prevent
eventual bacterial infections and reduce artefactual deiodination, as described by
Van Doornet al.(5).
Blood samplesof0.2 mlweretakenat 1,4.5, 10,23,44, 115,202 and315 min; 0.4
ml. were drawnat465,600,900,1200and 1440min,beingtheoptimaltimescheduleaccordingto DiStefanoefal.(3,4).
Fifty to hundred microliters plasma (in duplicate) were used to assess the total 131l
and 125l activities. The plasma samples were extracted with ethanol/ammonia
(25%)(197:3 v/v) containing 0.1 mmol/l propylthiouracil (PTU). The dried extracts
were dissolved in0.1 ml0.2 mol/lammonia containingcarrier T4,T3andKl (1mg/10
ml) and subjectedto HPLCto separate the iodothyronines, according to the method
describedbySchröder-vander Eistandvander Heide(23).
The endogenous concentrations ofT4andT3inplasmawere measured by meansof
a specific rat RIA (9) of samples taken via the canula directly before the kinetic
experimentswereperformed.
PlasmaTSHwas measured bythespecific RIAdevelopedforthe rat bythe National
Institute of Diabetes and Digestive and Kidney Diseases of the National Institute of
Health(USA). Reference preparation RP-2wasusedasastandard.
Calculations
The percentage dosesof [131I]T3and[12SI]T4per ml plasmawere calculatedfromthe
total radioactivities and the [131I]T3and [125I]T4distributions onthe HPLC chromatogram. The percentage doses per ml, together with the plasma volume at t(0) (3),
werefittedindividually tosumsofn=1to3exponential.
Y(t)=A1exp(\1*t)+A2exp(A2*t)+A3exp(A3*t)
T4andT3kinetics inpregnanthypothyroid rats
103
usingtheprogramDIMSUM,wherebyAiisexpressed in%dose/ml,Äiinmin"1(15).
To calculate the parameters of production, distribution and metabolism the program
MAMPOOL (12)wasused.Thesumofthethreeexponentialfunctionswasfitted(by
weighted least squares regression analysis) tothe data collectedfor each individual
rat and, together with the plasma T4 and T3 levels, substituted into the three-compartment model,accordingtothekineticT4andT3studies of DiStefano etal. (3,4).
All data are expressed as means ± S.E.M. Data were analyzed using the Statistical
Package for Social Sciences (SPSS)(24). All data were subjected to one-way analysis of variance, and statistical differences between groups were determined by
meansofthemodifiedleastsignificantdifferencemethod.
Analyticaldeterminations
Mitochondrial fractions from liver and brain were obtained by differential centrifugation,accordingtotheseparationschemedescribedbyvanDoom(5).
Liver and brain mitochondrial alpha-glycerophosphate dehydrogenase (a-GPD; EC
1.1.2.1)was measured by means ofthe methoddescribed by Garrib andMcMurray
(7).
The determination of type I deiodinase (ID-I) activity in liver homogenates was
performed bythemethoddescribedbyJanssenetal. (10).
Protein was determined by the bicinchoninic acid (BCA) method (Pierce Europe,
OudBeijerland,theNetherlands) usingbovine serumalbuminasstandard.
RESULTS
Bodyweightandplasma hormones
Table 1showsthe bodyweight (BW)andplasmathyroidhormone levels.Atthestart
of pregnancy all rats hada similar BW. Hypothyroidism affects the BW of the nearterm pregnant rat. Tx+lowTH rats had a significantly lower BW. The BW of the
fetuses ofTx+lowTH motherswasalsodecreased.Inthisgroupthenumberofviable
fetuseswasdecreased,whilemoredeadandresorbedfetuseswerefound.
Plasma T4 is markedly decreased intheTx+lowTH group,while intheTx+TH group
a normalT4levelwasreached.
Plasma T3 is decreased in both Tx groups; no difference was found between the
Tx+lowTHandTx+THgroups.
104
Chapter6
Plasma TSH was highly elevated in both Tx groups. In Tx+lowTH rats the plasma
TSH levelwashigherthaninTx+THrats.
Table 1: Bodyweight andplasmathyroid hormone concentrations inthe intact, Tx+THand
Tx+lowTHgroups.Valuesaremeans±S.E.M.
Intactgroup
Tx+THgroup
Tx+lowTHgroup
(n=8)
(n=7)
(1=8)
Bodywt onday0(g)
226 ± 3
229 ± 4
228 ± 5
Bodywt onday 19(g)
252 ± 5 * "
299 ± 7
300 ± 3
No.of fetuses
11 ± 1
9±1
Fetalbodywt(g)
4.24±0.07
4.14 ±0.06
T4(nmol/l)
23.6 ±1.6
30.6± 3.1
7±2
3.38 ±0.19**
4.0±0.9**
e
T3(nmol/l)
0.50± 0.06
0.28 ±0.05
TSH(ng/ml)
0.56 ±0.08
4.57 ±0.61®
0.21 ±0.01*
7.83 ±0.32**
P< 0.05:®Tx+THvs.Intact,*Tx+lowTHvs.Intact, *Tx+lowTHvs. Tx+TH
TA kinetics
The disappearance curves for T4, based on the mean A andKvalues for the three
groups, are shown in Fig. 1. The slope of the regression lines for plasma is decreased inTx+lowTH rats, meaningthat T4disappearedfrom plasma more slowly in
this group.Thefractional turnover ratesfor allthree poolswere decreased (datanot
shown). As a result the transport between plasma and the fast pool in Tx+TH rats
was 10%ofthatfor intactcontrolsandonly 1% intheTx+lowTHgroup.
ForT4,distributionvolumes,pool sizes, andtransport ratesaresummarized inTable
2. The plasma clearance rate was decreased in both Tx groups, but no additional
decreasewasfoundfor Tx+lowTH rats.The calculated rateof productionofT4inthe
Tx+TH group was similar to that found for intact rats. The lowT 4 production' inthe
Tx+lowTH group resulted inan equivalent decrease inthe amount of T4 in all three
pools.ThedistributionvolumesofT4remainedunchanged.
The rate oftransport ofT4from plasmatothefast pool and viceversawasmarkedly
decreased in both Tx groups, an additional decrease was found for Tx+lowTH rats.
The transport of T4 between plasma and the slow pool was only decreased in the
Tx+lowTH group. Italso appearedthat inthese rats an increasingfraction of T4was
transported unidirectionally fromplasmatotheslowtissuepool.
T4andT3kineticsinpregnant hypothyroidrats
105
Thetransittimes inallthreepoolsare increased inbothTxgroups, butno significant
change inthetotal residencetimewasfound(resultsnotshown).
Table 2: Parameters of distribution,volumes,poolsizes, and rates oftransport ofT4(expressed per 100g bodyweight) inthe intact, Tx+THandTX+lowTHgroups.Values are means ±
S.E.M.
Intactgroup
Tx+THgroup
Tx+lowTHgroup
PCR(ml/h)
1.64 ±0.04
1.19±0.07 e
1.26 ±0.09*
PR(pmol/h)
38.5 ±2.5
35.6±2.7
Vtotal(ml)
17.6±1.0
16.6 ±0.8
17.4±2.4
Vp(ml)
4.9 ±0.1
5.0 ±0.1
4.8 ±0.2
V2(ml)
3.8 ±0.4
3.8 ±0.7
2.4 ±0.5
10.2 ±2.6
4.7±1.0**
V3(ml)
8.9±0.6
7.8±0.3
Qtotal(pmol)
437±32
519 ±78
64 ±14**
Qp(pmol)
116±6
156 ±20
19 ± 5 * *
Q2(pmol)
88 ± 9
122±33
9±3*#
Q3(pmol)
207 ±17
239±30
36 ± 9 * *
TRpf (pmol/h)
2542±390
794±177®
33±8*
TRfp (pmol/h)
2523±389
776±177®
31±8*
DRfo (pmol/h)
19.3 ±1.2
17.8 ±1.3
2.4 ±0.5**
TRps (pmol/h)
195 ±36
143±43
16 ± 4 * *
TRsp (pmol/h)
176±35
125±43
13±4*
DRso(pmol/h)
19.3 ±1.2
17.8 ±1.3
2.4 ±0.5**
%PF
92.9±0.8
85.0±2.3
67.2±10.0*
15.0±2.3
32.8±10.0*
%PS
7.1 ±0.8
PCR, plasma clearance rate; PR, production rate; V, plasma equivalent distribution volume;
Q, poolquantity;TRpf,TRfp,TRps,andTRsp,ratesoftransport betweenplasma andthefast
and slow pools respectively, in each direction; DRfo and DRso, disposal rate from fast and
slow pool respectively; % PFand%PS,fractionof T4inplasma transported unidirectionally to
thefastandslowtissuepoolsrespectively; p,plasma;2,fastpool;3,slowpool.
P<0.05:e Tx+THvs. Intact,*Tx+lowTHvs. Intact, *Tx+lowTHvs.Tx+TH.
106
Chapter6
A.
o •:
Figure 1:
T4-disappearancecurvesobtainedwith
athreeexponentialmodel.
(A) plot of log y(t) (in % dose/ml)
against time, with the least squares
regression line on the final straight
part of the curve, giving estimates of
coefficient A3andexponentK3.
(B) plot of log [y(t) - A3 exp(A3*t)]
against time, with the least squares
regression line on the curve, giving
estimates of coefficient A2 and exponentA2.
(C) plot of log [y(t) - A3 exp(A3*t) - A2
exp(A2*t)] against time, giving estimatesofcoefficientA,andexponentA,.
•o
* * = • - - .
0
240 480 720 960 12001440
time (min)
-
1 0
B.
|
o
m
o
•o 0
I
0
I
L^
100
I
i_U
1
200
300
400
time (min)
£10
1)
: \
_i
i
i
i
i
i
i
i.«
0
10 20 30 40 50 60
time (min)
-+- Intact -*- Tx+ -•- Tx+
TH
lowTH
T3kinetics
The kinetic parameters for T3 are significantly different from intact rats only in the
Tx+lowTH group. The fractional rate of turnover of T3 in plasma and the transport
ratesfromplasmatothetissuepoolsweredecreased(data notshown).
Most changes found in the distribution, metabolism and transport of T3 (Table 3)
were similar fortheTx+TH andTx+lowTH groups.The plasma clearance rateforT3
was decreased. The plasma appearance rate and the production rate for T3 were
diminished inallTxrats.
T4andT3kineticsinpregnanthypothyroid rats
107
Table3:Parametersofdistribution,volumes, poolsizes,andratesoftransportofT3
(expressed per100g body weight) intheintact, Tx+TH andTX+lowTH groups. Valuesare
means±S.E.M.
Intact group
Tx+TH group
Tx+lowTH group
PCR (ml/h)
25.0± 2 . 4
17.6 ±1.3®
16.8 ± 1 . 9 *
PAR (pmol/h)
12.2± 1 . 2
4.9 ± 0 . 9 e
3.6 ± 0.5*
PR (pmol/h)
13.5± 1 . 1
5.5 ±1.0®
4.2 ± 0 . 8 *
Vtotal(ml)
201 ± 2 0
173± 1 3
138 ± 7 *
Vp (ml)
4.8± 0 . 1
5.4 ± 0 . 6
5.6 ± 0.5*
V2(ml)
28.3± 3 . 0
28.1± 2 . 7
V3(ml)
200 ± 37
139± 1 1
Qtotal (pmol)
115 ±19
49 ±9®
29± 2 *
Qp (pmol)
2.4± 0 . 3
1.5 ±0.2®
1.1 ± 0 . 1 *
Q2 (pmol)
13.8± 1 . 3
7.9 ±1.3®
3.6 ± 0 . 3 * *
24± 2 *
50± 6 *
Q3 (pmol)
17.3 ± 1 . 3 * *
115 ± 7 *
99± 1 9
44 ± 12®
TRpf (pmol/h)
205 ±27
115 ±21®
TRfp (pmol/h)
199± 2 7
112 ±20®
48± 6 *
DRfo (pmol/h)
6.1 ± 0 . 6
2.5 ±0.5®
1.8 ± 0 . 3 *
TRps (pmol/h)
53.3 ±13.4
16.5 ±2.6®
10.3 ± 0.8*
TRsp (pmol/h)
54.9 ±13.8
16.9 ±2.7®
10.6 ± 0 . 9 *
DRso (pmol/h)
7.4 ± 0 . 6
3.0 ± 0.6®
2.5 ± 0.5*
%PF
80.5 ±3.4
86.7± 1 . 5
81.8± 2 . 2
%PS
19.5 ± 3 . 4
13.3 ± 1 . 5
18.2 ± 2 . 2
PCR, plasma clearance rate; PAR, plasma appearance rate; PR,production rate;V,plasma
equivalent distribution volume; Q,pool quantity; TRpf, TRfp, TRps, andTRsp, rates oftransport between plasma andthefast andslow pools respectively, ineach direction; DRfoand
DRso, disposal rate from fast and slow pool respectively; %PFand%PS, fraction of T3in
plasma transported unidirectionally tothefast and slow tissue pools respectively; p,plasma;
2,fast pool;3,slowpool.
P <0.05:®Tx+THvs. Intact,*Tx+lowTHvs. Intact, *Tx+lowTHvs.Tx+TH.
The total distribution volume as well as the distribution volume of the tissue pools
was reduced intheTx+lowTH grouponly. Inall poolstheamount ofT3haddecreased inbothTxgroups. Thetransport ofT3inalldirectionswasreduced.
No significant changes, except an increase in the transit time for plasma (0.6±0.06
min and 1.2±0.15minfor intact andTx+lowTH rats respectively),werefoundfor the
Chapter6
108
transittimesandthetotalresidencetimeofT3.
Enzyme activities
The a-GPD activities in liver and brainare shown in Fig.2. Hepatic a-GPD activity
was reduced in the Tx+TH group; an even stronger decrease was found for the
Tx+lowTHgroup.Noeffect of hypothyroidism ona-GPD activity inthebraincouldbe
demonstrated.
Deiodinase type I (ID-I) activity (in mOD/min/mg protein) was diminished in all Tx
rats; 267±13, 147±16, and 73±8 for intact, Tx+TH and Tx+lowTH rats respectively.
ThusthestrongestreductionwasfoundintheTx+lowTHrats.
a-GPD in liver
Z
500
a-GPD in brain
~2000
a 400
O)
I 300
E 1000
"H" 200
1 100
Intact Tx +
TH
Tx +
lowTH
Intact Tx +
TH
Tx +
lowTH
Figure 2: a-GPD in maternal liver and maternal brain in the three groups studied (intact,
Tx+TH,Tx+lowTH).P< 0.05;sTx+THvs.intact;*Tx+lowTHvs.intact;*Tx+lowTHvs. Tx+TH.
DISCUSSION
Thyroidectomy resulted ina lower increase in bodyweight during pregnancy. This is
due not onlytothediminished weight ofthe mother but alsoto reduction ofthefetal
compartment. Treatment of the Tx animals with T4 and T3 (Tx+TH) normalized the
bodyweights of mother andfetus.
Since the plasmathyroid hormoneandTSH levels are not normal,theTx+THgroup
is certainly not euthyroid;we describe themasmildly hypothyroid. Txanimalswhich
received a very low dose of T4 and T3 (Tx+lowTH) were markedly hypothyroid, the
levelsofplasmaT4andT3beingverylow.
T4andT3kineticsinpregnanthypothyroidrats
109
Hypothyroidism is known to influence thyroid hormone metabolism. Inthis studywe
concentrated onthe effects of hypothyroidism onmaternal thyroid hormonemetabolism. The most important information about the effects of hypothyroidism on the
availability ofmaternal thyroid hormonesforfetuseswill beobtainedfromthe mildly
hypothyroid group; does this state already have an effect on maternal-fetal transport?
Despite normal plasma T4values inthe mildly hypothyroid group the transport ofT4
from plasma to the fast pool, compared to intact near-term pregnant rats, was already strongly reduced; in markedly hypothyroid rats almost no T4was transported
tothefast pool. Previouslywe suggestedthat inpregnant ratsthefast poolconsists
of the liver, the kidney (3, 4), and the feto-placental compartment. In intact rats
pregnancy results inapronounced increase inthetransport ofT4fromplasmatothe
fast pool(25).
It is unlikely that the total decrease in the transport of T4 to the fast pool in mildly
hypothyroid rats can be explained solely by a decrease inthe transport of T4to the
liver and kidney. This implies, therefore, that even in a mildly hypothyroid situation
the transport of maternal T4 to the placenta can also be diminished. This is very
important, because T4 plays a major role in the development of the fetal central
nervous system. Only maternal T4 is able to mitigate fetal cerebral T4 and T3 deficiency beforethefetalthyroidstartstofunction(18).
The plasma T3level in the mildly hypothyroid groupwas not normal. The amount of
T3 added was less than the amount of T3 produced daily in intact rats (0.25 ug vs.
0.60 ug per day). However, since the plasma T4 level was normal, it was to be
expected that T3 would also be produced locally. T3 produced locally in the liver
contributes in particular to the plasma T3 level (5). However, in the hypothyroid rat
the local production of T3 is decreased in the liver (5). This was also demonstrated
by the decrease in ID-I activity in the liver; this can possibly be explained by the
regulation of hepatic ID-I activity by T3(13). Therefore, even ina mildly hypothyroid
condition with normal plasmaT4 levels,theT3 level inthe liver will be reduced;this
hypothesis issupportedbythedecrease inhepatica-GPDactivity.
For non-pregnant thyroidectomized rats receiving an infusion of T4 and T3 it has
been reportedthat intheevent ofnormal plasmaT4levelsandareducedplasmaT3,
hepatic ID-I and cerebral ID-II activities will be normal (6). However, despite the
administration ofT4 inanamountwhichequals theamount normally produced, local
T3 production was decreased in our near-term pregnant rats. It appears, therefore,
110
Chapter6
that in near-term pregnant rats the effect of mild hypothyroidism is stronger than in
non-pregnant rats.
Marked hypothyroidism in both non-pregnant and near-term pregnant rats results in
highly reduced pools and diminished rates of transport of T4 andT3.
In conclusion, even mild hypothyroidism results in significant alterations in maternal
thyroid hormone metabolism, despite normal plasma T 4 levels. In particular the
transport of T 4 from plasma to the fast pool will be reduced. We speculate that this
results in a decrease in the availability of maternal T 4 for the fetuses. This could
affect the development of the fetal brain, which depends mainly on maternal T 4 for
normal T 3 values.
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D (1994) Thyroxine and3,5,3'-triiodothyronine production,metabolism, anddistribution in
pregnant ratnearterm.AmJPhysiol 267:860-867.
26. Weeke J,and Orskov K (1973) Synthesis of [125l]monolabelled T3andT4 of maximum
specific activity for radioimmunoassay. Scandinavian Journal of Clinical Laboratory
Investigation32:357-366.
CHAPTER 7
GENERAL DISCUSSION
ANDCONCLUSION
Generaldiscussion andconclusion
115
GENERALDISCUSSIONANDCONCLUSION
Thyroid hormone metabolism is influenced by several (patho)physiological conditions. During pregnancy thyroid hormone metabolism isalso altered.The aim ofthis
study was to investigate the effects of pregnancy on maternal thyroid hormone
metabolism, i.e. -T4andT3secretionbythethyroid
- peripheral productionofT3
-T4andT3concentrations inplasmaandtissues
-distribution ofT4andT3
-interpooltransportofT4andT3
Since iodine is an essential element of the synthesis of thyroid hormones we also
studiedchanges iniodide uptakebythethyroidduringnormalpregnancy andduring
marginal iodinedeficiency.
7.1. NORMAL PREGNANCY
Iodide uptake by the thyroid is regulated by TSH. In the near-term pregnant rat the
plasma TSH level remains unchanged or is slightly elevated (3, 4, 6). During pregnancy the iodide uptake,which isslightly decreased,doesnot seemto be regulated
by TSH only. Another factor which affects iodide uptake by the thyroid is the thyroidal bloodflow(1).Wedidnotmeasurethis parameter, butnochangeswerefound
in either the clearance of iodide from plasma by the thyroid or plasma inorganic
iodide. We suggest that less iodide is taken up by the maternal thyroid simply because less iodide is available: at the end of gestation the mother has to compete
with the fetuses for her iodine supply. Iodine can be transported through the placenta from mother to fetuses (7); on day 19of gestation the fetal thyroid is capable
of concentrating iodine. Thedecreased availability of iodidefor the maternal thyroid
is not so dramatic that thyroid hormone production by the thyroid is affected. The
thyroid isstillabletomaintainthyroidhormonesynthesis atanormallevel.
Despite the unaltered thyroidal production of T4, the plasma T4 concentration is
decreased at the end of gestation which demonstrates that altered plasmaconcentrations are not always the result of changes in production rate. In the pregnant rat
the transport of T4 from plasma increases drastically. Most likely T4 is also transported to the feto-placental compartment, which is a developing compartment that
116
Chapter7
must be filled at the end of gestation. With the three-compartment model it is not
possible to distinguish between maternal tissues and the feto-placental compartment. Therefore, we could only speculate about changes in transport to the fetoplacental compartment in our kinetic studies. However, for the normal pregnant rat
we also have datafrom steady-state experiments. These data showthatthe tissueto-plasma ratioforT4decreased inallmaternaltissues inthe near-term pregnant rat.
Since plasma T4 decreased, the amount of T4 inthe tissues diminished even more.
In the kinetic study the amount of T4 in the tissue pools of near-term pregnant rats
remained constant. This implies that one part of the tissue pools consists of nonmaternal organs, i.e. placentas plus fetuses. Therefore, despite a normal thyroidal
production less T4 is available for the maternal organs which results in decreased
tissueT4concentrations, aswellasadecrease inthetissueT3concentration inmost
organs. Even an increase in deiodinase activity, ID-I aswell as ID-II (3), would not
lead to normal tissue T3 levels, as has been demonstrated by the steady-state
experiments.
7.2. MARGINALIODINEDEFICIENCY DURING PREGNANCY
In the normal situation the mother shares the available iodide and T4 with her fetuses, at the cost of her own supply. However, during marginal iodine deficiency
(MID) the amount of available iodide is reduced. By means of an increase in thyroidal clearance, resultingfromanincrease inthyroidvolumeandthyroidal bloodflow
(1), the mother is able to maintain a normal absolute iodide uptake by the thyroid.
Just as during normal pregnancy these processes do not seem to be regulated by
TSH, because plasma TSH remains unchanged. In the fetuses the absolute iodide
uptake is decreased during MID; the fetal thyroid is not able to compensate for the
decrease inplasma inorganic iodide.Thiscan possibly result inadiminished thyroid
hormone production by the fetal thyroid. During maternal hypothyroidism fetal plasmaTSHiselevated(2). However, theeffect ofMIDonfetalTSHisunknown.
Boththe productionofT4bythematernalthyroidandtheamountofT4inplasma and
the tissue pools are only slightly decreased in the near-term pregnant rat. The
mother is able to maintaintheeuthyroid stateduring MID.Therefore, itappearsthat
MIDwill notaffecttheavailability ofT4forthefetuses. However, thetransportofT4is
seriously altered during MID. In particular interpool transport between plasma and
Generaldiscussionandconclusion
117
thefast poolisdecreased.Thiscouldmeanthat lessT4istransportedtothefetuses.
Since data onfetal thyroid hormones are not available it is difficult to speculate on
the consequences for fetal development. The fetal brain is able to avoid T3 deficiency by increasing ID-II activity (6). However, if there is insufficient substrate, i.e.
T4, this will not result in normal T3 levels. This might be the situation during MID,
because boththematernaland fetalsuppliesofT4aredecreased.
7.3. MATERNAL HYPOTHYROIDISM
During maternal hypothyroidism only the maternal thyroid function is affected. The
availability of iodide for the fetuses is normal or perhaps even increased. We have
described a moderate situation, meaning that T4 and T3 are available, but normal
plasma concentrations had not yet been reached; plasma T4was normal, whereas
plasma T3wasdecreased. Despite the normal plasma T4, the transport of T4to the
fast pool, including the feto-placental compartment, was reduced to 30 % of that
found for intact animals. This meansthat even ina mild hypothyroid statethe availability ofmaternalT4forthefetuses isdiminished.
7.4. INCONCLUSION
During normal pregnancy the production of T4 by the thyroid remains unchanged.
However, this T4 has to be distributed between the mother and her fetuses. This
results inadecreased availability ofT4for the maternal organs, leading indirectly to
adeficiency ofT3inthematernalorgans.
During marginal iodinedeficiency andmaternal hypothyroidism themother maintains
the same thyroidal status inall organs as during normal pregnancy. This occurs at
the expense ofthe availability of iodideandT4for thefetuses,which inthe long run
couldleadtoimpaireddevelopment ofthefetalcentralnervoussystem.
118
Chapter7
7.5. REFERENCES
1.
2.
3
4.
5.
6.
7.
Arntzenius AB, Smit LJ, Schipper J, van der Heide D, and Meinders AE (1991)
Inverse relation between iodine intake andthyroid bloodflow: color dopplerflow imaging
ineuthyroid humans.JClinEndocrinolMetab75: 1051-1055.
Bonet B, and Herrera E (1988) Different response to maternal hypothyroidism during
thefirstandsecond half ofgestation intherat.Endocrinology 122:450-455.
Calvo R, Obregon MJ, Ruiz deOna C, Ferreiro B, Escobar del Rey F,and Morreale
de Escobar G(1990) Thyroidhormoneeconomyinpregnant ratsnearterm:A"physiological"animalmodelof nonthyroidal illness?.Endocrinology 127:10-16.
Fukuda H,Oshima K, MoriM,Kobayashe I,andGreer MA(1980) Sequential changes
in the pituitary-thyroid axisduring pregnancy and lactation inthe rat. Endocrinology 107:
1711-1716.
Kojima A, Hersman JM, Azukizawa M, and DiStefano III JJ (1974) Quantification of
thepituitary-thyroid axisinpregnant rats.Endocrinology95:599-605.
Obregon MJ, Ruizde Ona C,Calvo R, Escobar delRey F,and Morreale de Escobar
G (1991) Outer ring iodothyronine deiodinases and thyroid hormone economy: responsesto iodinedeficiency inthe ratfetus andneonate.Endocrinology 129:2663-2673.
Roti E, Gnudi A, and Braverman LE (1983) The placental transport, synthesis and
metabolism of hormones and drugs which affect thyroid hormone function.Endocrine
Reviews4:131-149.
CHAPTER8
SUMMARY
SAMENVATTING
Summary
SUMMARY
Thyroid hormones, thyroxine (T4) and 3,5,3'-triiodothyronine (T3), are produced by
the thyroid gland. To synthesize thyroid hormones the thyroid needs iodide. The
uptake of iodide aswell as the production and secretion of T4andT3by the thyroid
gland isregulated bythyrotropin (TSH),which isproduced bythepituitary. However,
most ofthe biologically activeform,T3, is producedfromT4via monodeiodinationin
peripheral tissues.This reaction iscatalyzedbythedeiodinases,typeI(ID-I) in liver
and kidney, and type II (ID-II) in the central nervous system and brown adipose
tissue (BAT). T4andT3concentrations differ inthevarioustissues, likethecontribution of T3 produced locally from T4. A large portion of the T3 produced in the liver
enters the circulation, whereas T3 produced in the brain and cerebellum is mainly
used locally.
The production, distribution and transport of thyroid hormones are influenced by
several (patho)physiological conditions. Inthis studyweconcentrated onthe effects
of pregnancy on maternal thyroid hormone metabolism. It iswell known that thyroid
hormones are very important for normalfetal development, especially of the central
nervous system. During development thyroid hormones produced by the mother,
mainlyT4,contributetothefetalthyroidhormonepoolsbeforeandalsoafteronsetof
fetal thyroid function. Insufficient production of maternal thyroid hormones during
pregnancy can result in permanent brain damage in the offspring. At the end of
gestation the concentrations of T4 and T3 in maternal plasma and tissues have
decreased. Inorder togainmore insight intotheeffects ofpregnancy ontheproduction, distribution, and transport of thyroid hormones in the mother we performed
kinetic experiments with T4 and T3 using nonpregnant and near-term pregnant rats
(chapter 2). A bolus injection of [12SI]T4and [131I]T3was given, and blood samples
were taken at regular times during the next twenty-four hours. Physiological parameters of the production, interpool transport, distribution and metabolism of T4 and
T3were estimated by meansof athree-compartment model.According tothis model
three compartments can bedistinguished: 1.the plasma;2.thefast pool; and3.the
slow pool. Liver and kidney are considered to be the main components of the fast
pool, whereas skin, muscles and brain belong to the slow pool. In the near-term
pregnant rat the production and distribution of T4 remained unchanged. The transport of T4 from plasma tothe fast poolwas morethantripled,whereas transport to
the slow pool remained constant. We suggest that in the near-term pregnant rat
121
122
Chapter8
available T4wasdistributed betweenthe maternalandfetalcompartments bymeans
of veryfast transport. This hypothesis isbasedonthefactthat itseemsunlikely that
the transport ofT4tomaternal liverandkidney,whichareconsideredtobethemain
components ofthefastpool,willhaveincreasedthat muchinthenear-term pregnant
rat. This was confirmed by the results of steady-state, double isotopic experiments
using nonpregnant and near-term pregnant rats (chapter 3). In this study, the rats
received acontinuous simultaneous infusionof[12SI]T4and[131I]T3inordertoachieve
equilibrium inall tissues.With this method itwas possibletocalculate theT4andT3
concentrations, the relative contributions of plasma-derived vs. locally producedT3,
the thyroidalT4andT3secretion rates, andtheplasma-to-tissue ratiosforT4andT3.
Indeed, the transport of T4to liver and kidney, aswell as almost all other maternal
organs,wasdiminished. Sincetheproduction ofT4remainedunchangedthis implies
that T4 is transported to another compartment, i.e. the feto-placental compartment.
This compartment was not measured inthesestudies.The plasmaappearance rate
for T3remainedconstant inthenear-termpregnantrat.Thiswasaccomplishedbyan
increase inthe secretion of T3bythe thyroidandadecrease inlocally producedT3.
Less T3was transportedfrom plasmato liver, kidney, BATand pituitary. ID-I activity
in liver, andID-IIactivity inthebrainbothincreasedduringpregnancy. However,this
did not result inanincrease inthe localconversionofT4toT3inthesetissues. Inthe
liver the contribution of T3 produced locally remained constant, while in the brain
even a decreasewasfound.The insufficient availability ofT4inmaternaltissues,as
demonstrated bythelowerT4concentrations, mightexplainthediscrepancy between
deiodinase activities and the local production of T3. The transport of T4 to thefetoplacental compartment resulted indirectly in a deficiency of T3 in the maternal organs. We can conclude that pregnancy affects maternal thyroid hormone metabolism. The mother hastosharetheavailable thyroid hormones, especially T4,withthe
fetuses.
Iodide is anessential elementforthesynthesisofthyroidhormones. Inratsthefetal
thyroid is capable of producing thyroid hormones on day 18 of gestation. Iodide is
transported acrossthe placenta fromthe maternal tothefetal circulation. Inchapter
4 we assessediodideuptakebythematernalthyroid,whilethe iodide uptakebythe
fetal thyroid was estimated. We measured the in vivo uptake of 12SI by the thyroid
continuously. By using the specific activity of iodide in the urine we were able to
calculatetheabsolute iodideuptake inthethyroid.Pregnancyresultedinadecrease
in the absolute thyroidal iodide uptake. On day 20 of pregnancy the fetal thyroid is
Summary
already capable of concentrating iodide. However, the difference in absolute iodide
uptake by the maternal thyroid, compared to nonpregnant controls, cannot fully be
explained by the transport of iodide to the fetal compartment and/or the mammary
glands.Thedecrease iniodide uptakebythe maternalthyroid has noimpact onthe
thyroidal productionofthyroidhormones.
Iodine deficiency can lead to disturbed physical and mental development. In large
populations in the world iodine intake is marginally deficient. For this reason a
marginal iodine deficiency, instead of the more common severe iodine deficiency,
was induced in our rats. We used this model to study the effects of marginal iodine
deficiency on iodide metabolism (thyroidal iodide uptake; chapter 4) and thyroid
hormone metabolism (kinetic experiments; chapter 5) in near-term pregnant rats.
The absolute iodide uptake by the maternal thyroid was not affected by marginal
iodine deficiency. The decreased plasma inorganic iodide was compensated by an
increase inthyroidal clearance. A similar compensation was notfoundfor thefetus;
the uptake of iodide by thefetal thyroid decreased by 50 %during marginal iodine
deficiency. During this marginal iodine deficiency plasma T4 and T3 remained constant innonpregnant aswellasnear-term pregnant rats.Theproductionrateandthe
plasma clearance rate for T4 were both decreased. No effects of marginal iodine
deficiency onpoolsizesandtransport rateswerefoundfor nonpregnant rats. Inthe
near-term pregnant rat marginal iodine deficiency resulted in a marked decrease in
the transport ofT4fromplasmatothefast pool. ForT3an increase inthe production
rate and plasma clearance rate was found for nonpregnant, marginally iodinedeficient rats,whiletheseparameterswereslightly decreased innear-term pregnant
rats. Marginal iodine deficiency induced a 50 %decrease in the interpool transport
ratesofT3betweenplasmaandthefastpoolinnear-termpregnantrats.Thehepatic
activity of ID-I was increased as a result of marginal iodine deficiency in nonpregnantaswellasnear-termpregnantrats.
Onthebasisoftheresultsofthyroid hormonestudies innormalpregnant rats(chapter 2 and 3) we suggest that during marginal iodine deficiency less maternal T4 is
available for the fetal compartment. Together withthe lower uptake of iodide by the
fetal thyroid this can lead to diminished levels of thyroid hormone of maternal and
fetal origin in the fetal organs. In this case, marginal iodine deficiency will have a
negative effectonfetaldevelopment, especially ofthebrain.
Another situation which irreversibly affects fetal brain development is maternal
hypothyroidism. Two different levels of hypothyroidism were induced infemalerats,
123
124
Chapter8
by giving thyroidectomized rats two different doses of T4 and T3. The effects of
hypothyroidism on maternal thyroid hormone metabolism in near-term pregnant rats
(kinetic experiment, chapter6)were studied. PlasmaT4andT3levelswere very low
severely hypothyroid animals,whereas only plasma T3was decreased inthe mildly
hypothyroid group. Even during this mild hypothyroidism profound alterations inthe
transport rates of T4were found compared to intact, pregnant rats. Thetransport of
T4 from plasma to the fast pool was decreased. Therefore, it appears that even
during mild hypothyroidism the transport of T4 to the feto-placental compartment is
affected.
In conclusion: Pregnancy seriously affects the maternal thyroid hormone status.
Despite an unchanged thyroidal production of T4, all maternal T4 tissue levels are
decreased. Less T4 is available for the mother because ofthe transport of T4to the
feto-placental compartment. Indirectlythis results inaT3-deficiency inmost maternal
organs. Duringmarginal iodinedeficiency andmaternal hypothyroidismthetransport
of maternal T4 to the feto-placental compartment is diminished, whereas during
marginal iodine deficiency the availability of iodine for fetal thyroid hormone synthesis is also decreased. Eventually this can result in impaired development of the
fetalcentralnervoussystem.
Samenvatting
SAMENVATTING
Schildklierhormonen, thyroxine (T4) en 3,5,3'-triiodothyronine (T3), wordengeproduceerddoordeschildklier.Voordevormingvanschildklierhormoon heeftde schildklier
Jodide nodig. Zowel de opname van jodide als de productie en uitscheiding van
schildklierhormoon door de schildklier worden gereguleerd door het schildklier
stimulerend hormoon (TSH). Dit hormoon wordt geproduceerd door de hypofyse.
Echter, hetgrootstedeelvande biologischactievevormvanschildklierhormoon, het
T3lwordt,door middelvaneenmonodejoderingsreactie,uitT4gevormdindeperifere
weefsels. Voor deze reactie zijn dejoderende enzymen verantwoordelijk; type I
monodejodase (ID-I) in de lever en nieren, en type II monodejodase (ID-II) in het
centraalzenuwstelsel enbruinvetweefsel. Deconcentratie vanT4enT3indediverse
weefselsverschilt,evenalsdebijdragevandehoeveelheidT3welke lokaal isgevormd
uitT4.EengrootdeelvandeT3,welke lokaal indeleverisgevormd,wordt afgegeven
aan de circulatie, terwijl lokaal gevormd T3 in de hersenen en het cerebellum
voornamelijk lokaalgebruiktwordt.
Deproductie,deverdelingenhettransportvanschildklierhormoonwordenbeïïnvloed
doorverschillende(patho)fysiologischeomstandigheden.Indezestudiehebbenweons
geconcentreerd op de effecten van zwangerschap op het schildklierhormoonmetabolismevandemoeder.Hetisalgemeenbekenddatschildklierhormoon vangroot
belang is voor een normale ontwikkeling van de foetus, met name van het centraal
zenuwstelsel. Tijdens deze ontwikkeling levert schildklierhormoon van de moeder,
zowelvooralsnadestartvandefoetaleschildklierfunctie, eenbijdrageaandefoetale
schildklierhormoonpools. Eenontoereikendeproductievanschildklierhormoon doorde
moeder tijdens dezwangerschap kan resulteren inpermanente hersenbeschadiging
van het nageslacht. Inde ratzijndeconcentraties vanT4enT3 in het plasma ende
weefsels van de moeder aan het einde vande dracht verlaagd. Ommeer inzicht te
verkrijgen in de effecten van zwangerschap op de productie, de verdeling en het
transportvanT4enT3indemoeder, hebbenwe inniet-drachtige rattenenrattenaan
heteindevandedraagtijdexperimentenuitgevoerdwaarbijhetkinetische gedragvan
T4 en T3 is bepaald (hoofdstuk 2). De ratten ontvingen een eenmalige injectie van
[125I]T4en[131I]T3, waarnagedurende24uur, opvastgestelde tijdstippen bloedmonsters
werden genomen. Fysiologische parameters van productie, transport, verdeling en
metabolisme van T4 en T3 werden geschat met behulp van een drie-kompartimentenmodel.
125
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Chapter8
Inditmodelkunnendriekompartimentenwordenonderscheiden: 1. hetplasma;2.een
snel kompartiment; en 3. een langzaam kompartiment. Lever en nieren worden
beschouwdalsdebelangrijkstecomponentenvanhetsnellekompartiment, terwijlde
huid,spierenenhersenentothet langzamekompartiment behoren.Indedrachtigerat
zijn,aanheteindevandedracht,deproductie vandeverdelingvanT4nietveranderd.
Het transport van T4 van het plasma naar het snelle kompartiment was meer dan
verdrievoudigd,terwijl hettransport vanhetplasmanaar hetlangzame kompartiment
constantgeblevenwas.Wijveronderstellen datinderat,aanheteindevandedracht,
hetbeschikbareT4,doormiddelvaneenzeersneltransport,wordtverdeeldtussende
moeder en de foetussen. Deze hypothese is gebaseerd op het feit dat het
onwaarschijnlijk lijkt dat inde drachtige rat hettransport vanT4naar de lever ende
nieren van de moeder, welke als belangrijkste componenten van het snelle
kompartiment beschouwdworden, indeze mate istoegenomen. Ditwordt bevestigd
doorderesultatenvanstudieswaarbijrattencontinueeninfuus met[125I]T4en [131I]T3
ontvingentotdatinalleorganeneenevenwichtssituatie bereiktwas. Dithoudt indatde
verhoudingradioactiefhormoonenendogeenhormooninhethelelichaamgelijk is.Met
behulpvandezemethodeishetmogelijkomdeconcentratiesvanT4enT3,de relatieve
bijdrage van T3 afkomstig uit het plasma ten opzichte van lokaal gevormd T3, de
sekretievanT4enT3doordeschildklierendeplasma/weefselratiosindeverschillende
weefsels te berekenen. En inderdaad, het transport vanT4 naar lever en nieren, en
bijnaalleandereorganenwasverminderd.DeonveranderdeproductievanT4wijsterop
datT4wordtgetransporteerdnaareenanderkompartiment. Wijgaanervanuitdatdit
de placentas plusdefoetussen zijn. Inonzeexperimenten kondit kompartiment niet
gemetenworden.DesnelheidwaarmeeT3inhetplasmaverschijnt blijftconstant inde
drachtigerat.Ditwordtbereiktdoormiddelvaneentoename indesekretievanT3door
deschildkliereneenafnameindehoeveelheid lokaalgevormdT3.DehoeveelheidT3
die vanuit het plasma naar de lever, nieren, bruinvet en hypofyse getransporteerd
wordt is afgenomen. Zowel de ID-I activiteit in de lever als de ID-II activiteit in de
hersenenistoegenomentijdensdedracht.Echter,ditheeftgeentoenamevandelokale
omzettingvanT4naarT3indezeweefselstotresultaat. Indeleverblijftdebijdragevan
lokaalgevormdT3gelijk,terwijlindehersenenzelfseenverlagingwordtgevonden.Dit
kanverklaardwordendoordatdebeschikbaarheidvanT4indeweefsels vandemoeder
ontoereikend is. Het transport van T4 naar de placentas plus foetussen resulteert
indirekt ineentekortaanT3indeorganenvandemoeder.
Samenvatting
Wekunnenconcluderendatzwangerschaphetschildklierhormoonmetabolisme vande
moeder beïnvloedt. Demoeder moetdebeschikbareschildklierhormonen, vooralT4,
metdefoetussendelen.
Voordesynthesevanschildklierhormoon isjodideeenonmisbaarelement. Inderatis
de foetale schildklier vanaf dag 18 van de dracht in staat om schildklierhormoon te
produceren.Jodidewordtviadeplacentavandebloedsomloopvandemoeder naar
defoetale bloedsomloop getransporteerd. Inhoofdstuk4 hebbenwedeopnamevan
jodidedoordeschildkliervandemoedergemeten,terwijleenschatting isgemaakt van
deopnamevanjodidedoordefoetale schildklier. Deinvivo opnamevan125ldoorde
schildklierwerdgedurendevieruuronafgebrokengemeten. Doorgebruiktemakenvan
de specifieke activiteit vanjodide inde urinewaren we instaat omde hoeveelheid
jodide opgenomen door de schildklier te berekenen. Zwangerschap had een kleine
afname van de hoeveelheid jodide welke door de schildklier van de moeder werd
opgenomentot resultaat. Opdag20vandedrachtwasdefoetaleschildklier instaat
omjodide te concentreren. Het verschil in opgenomen hoeveelheid jodide door de
schildklier vande moeder, vergeleken metde niet-drachtige situatie, kanechter niet
volledigwordentoegeschrevenaanhettransportvanjodidenaardefoetussenen/ofde
melkklieren.Dedalingindeopnamevanjodidedoordeschildklier vandemoeder heeft
geengevolgenvoordeproductievanschildklierhormoon doordeschildklier.
Jodiumdeficiëntie kan een verstoorde fysische en mentale ontwikkeling tot gevolg
hebben. Ingrotepopulaties indewereld isdeinnamevanjodiummarginaal.Daarom
is in onze experimenten gekozen voor het toebrengen van een marginalejodiumdeficiëntie,inplaatsvandeveelvakergebruikteernstigejodiumdeficiëntie. Wehebben
de effectenvanmarginalejodiumdeficiëntie ophetmetabolismevanjodide (opname
vanjodidedoordeschildklier; hoofdstuk4)enhetmetabolismevanT4enT3(kinetiek
experimenten; hoofstuk 5) in de drachtige rat, aan het einde van de draagtijd,
bestudeerd.Dehoeveelheidjodideopgenomendoordeschildkliervandemoederwerd
nietbeïnvloeddoormarginalejodiumdeficiëntie.Deafnameindejodideconcentratie in
het plasmawerdgecompenseerd dooreentoename indeklaringdoordeschildklier.
Eendergelijkecompensatiewerdnietgevondenindefoetussen; deopnamevanjodide
door defoetale schildklier wasgehalveerdtijdens marginalejodiumdeficiëntie.Zowel
indrachtigealsniet-drachtigerattenblevendeconcentratiesvanT4enT3inhetplasma
onveranderdtijdens marginalejodiumdeficiëntie.ZoweldesnelheidwaarmeeT4werd
geproduceerdalsdesnelheidwaarmeeT4uithetplasmawerdgeklaard namenaf.
127
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Chapter8
In niet-drachtige rattenwerden geen effecten van marginalejodiumdeficiëntie opde
hoeveelheidT4indediverse kompartimentenendesnelhedenvanhettransport van
T4gevonden.Marginalejodiumdeficiëntie resulteerde indedrachtigerat ineensterke
dalingvanhettransportvanT4vanhetplasmanaar hetsnellekompartiment.VoorT3
werd een toename in de snelheid van productie en de snelheid van klaring uit het
plasma gevonden in niet-drachtige, maginaal jodiumdeficiënte rat, terwijl deze
parameterslichtelijkwarenafgenomen indedrachtigerat. Marginale jodiumdeficiëntie
hadeenafnamevan50%indesnelheidvantransportvanT3tussenhetplasmaenhet
snellekompartimentindedrachtigerattotgevolg.Deactiviteitvan ID-Iindeleverwas
toegenomentijdensmarginalejodiumdeficiëntie inzowelniet-drachtige als drachtige
ratten.
Gebaseerd op de resultaten vande schildklierhormoonstudies in normale, drachtige
ratten(hoofdstuk2en3)veronderstellenwijdattijdens marginalejodiumdeficiëntie de
hoeveelheidT4vandemoeder,welkebeschikbaar isvoordefoetussen, verminderdis.
Samenmetdedalingindeopnamevanjodidedoordefoetaleschildklier, kandit leiden
totverlaagdeschildklierhormoonniveaus, afkomstigvanzowelmoederalsfoetus,inde
foetaleorganen.Inditgevalzalmarginalejodiumdeficiëntie negatieveeffecten hebben
opdeontwikkelingvandefoetussen, inhetbijzonder vandehersenen.
Hypothyreoïdie vande moeder iseenanderesituatiewaarbijdeontwikkeling vande
foetale hersenen irreversibel beïnvloed wordt. Door vrouwelijke ratten waarvan de
schildkliermetbehulpvan131lkapotisgestraaldtweeverschillende dosissenT4enT3
toetedienen,werdentweeverschillendeniveausvanhypothyreoïdie geïnduceerd. Het
effect vanhypothyreoïdie op het metabolismevanT4enT3indedrachtige ratwordt
beschreveninhoofdstuk6.Indediephypothyreote dierenwarendeconcentraties van
T4 en T3 in het plasma erg laag, terwijl in de milde hypothyreote dieren alleen de
concentratie van T3 in het plasma verlaagd was. Zelfs tijdens deze milde vorm van
hypothyreoïdie werden, in vergelijking met intacte, drachtige dieren, verregaande
veranderingen indesnelheden vantransportvanT4gevonden. Hettransport vanT4
van het plasma naar het snelle kompartiment wasafgenomen. Dus,het lijkteropdat
zelfs tijdens een milde hypothyreoïdie het transport van T4 naar de placentas plus
foetussen isaangetast.
Concluderend:zwangerschapheeftveranderingen indeschildklierhormoonstatus van
demoedertotgevolg.Ondankseenonveranderde productievanT4doordeschildklier
isinalleorganen vandemoederdeconcentratievanT4verlaagd. Door hettransport
vanT4naardeplacentasplusfoetussenisminderT4beschikbaar voordemoederzelf.
Samenvatting
Ditresulteert indirect ineentekort aanT3indemeesteorganen.Zowel bij marginale
jodiumdeficiëntie als bij milde hypothyreoïdievan de moeder is het transport vanT4
naar de placentas plusfoetussen verminderd.Tijdens marginalejodiumdeficiëntie is
ook de beschikbaarheid van jodide voor synthese van schildklierhormoon door de
foetale schildklier verlaagd. Dezeveranderingen kunnen indefoetusuiteindelijk een
aantastingvandeontwikkelingvanhetcentralezenuwstelsel totgevolghebben.
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131
Dankwoord
Het is er dan eindelijk toch van gekomen,vele proeven, ratten entwee bevallingen
na het beginvan mijnaanstelling, het proefschrift is af !! Maar het is niet eerlijk om
te suggereren dat ik dit helemaal in mijn eentje had kunnen bewerkstelligen. Zowel
op het werk als in de privésfeer zijner natuurlijk vele mensen die hun bijdrage aan
dit boekjegeleverdhebben.
Om te beginnen mijn directe begeleider en promotor, prof. dr. D. van der Heide.
Daan, zonder jouw was dit onderzoek en dus dit boekje er niet geweest. Samen
hebben wij in Wageningen alles opnieuw op moeten zetten. Hierbij konden wij
gelukkig terugvallenopdehulpuit Leiden.Vooraltijdens decontinue infusieexperimentenwasjouwhulp vaak nodig.Verder hebje megeleerd omalles ineen breder
verband te zien, met nametijdens de discussies over de resultaten. Ik hoop dat dit
eengoedewetenschappelijke basisisvoordetoekomst.
Janny, ik denk datjij voor mijn proefschrift bijna net zo belangrijk bent geweest. In
eerste instantie hebje meop het practische vlak erg geholpen. Ineen later stadium
hebikookheelveelgehadaanjekritische blikopmijnartikelen.
Leslie, zonderjouwas het onmogelijk geweest om mijn experimenten uit tevoeren.
Bedankt voor alle bepalingen en eindeloze series testen met de HPLC en RIA's die
jij hebt uitgevoerd. Maar vooral ook bedankt voor alle gezelligheid, ik hoop dat we
noglangcontactzullenhouden.
En dan de studenten, Vincent, Jeanet, Anita, Monique, Machteld en Jan, die ieder
op een eigen deel vanhetproefschrift eenbelangrijke bijdrage hebbengeleverd,en
diebovendienophetlabaltijdwat levenindebrouwerijbrachten.
Irma en Marjolijn, ik ben heel blij dat wij met zijn drieën een kamer hebben mogen
delen. Het was altijd erg gezellig, enwe hebben ook veel steun aan elkaar gehad.
En uiteindelijk hebben we toch alledrie een mooi boekje en een schare kinderen
afgeleverd. Ikgaervanuitdatwenoglangvriendinnenzullenblijven.
Ook de andere AIO's en de rest van de vakgroep wil ik bedanken voor de steun
tijdensmijnonderzoekendegezelligetijdopFMD.
Esther, bedankt dat ikgebruik heb mogen maken vanjouwartistieke talent. Zo ziet
ookdevoorkant vanhetboekje er mooiuit.
132
Mijn ouders, verdere familie en vrienden wil ik bedanken voor hun belangstelling
voor mijn onderzoek. Ik hebjullie geduld lang op de proef gesteld, maar nu kunnen
jullie eindelijkhetresultaat bewonderen.
Lieve Richard, bedankt voor alles.Voordevelestressvolle urenachter decomputer,
maar vooral dat jij mijzo gelukkig maakt. Lieve Renske en Mirne,jullie maken ons
gelukcompleet.
Tètr
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CurriculumVitae
Petronella Maria (Petra) Versloot is geboren op 5 maart 1968 te Wymbritseradeel,
Friesland. In1986behaaldezijhaarVWOdiplomaaanhetHanFortmann Collegete
Heerhugowaard en begon haar studie Biologie aan de Universiteit vanAmsterdam.
Na het propedeatisch examen (cum laude) werd gekozen voor de bovenbouw studierichting Medische Biologie. Specialisaties tijdens de studie waren fysiologie en
endocrinologie. Ook werd de artikel 9 bevoegdheid voor het werken met proefdieren behaald. Tijdens de doctoraal fase zijn afstudeervakken uitgevoerd bij de
vakgroepen Experimentele Kindergeneeskunde en Anatomie en Embryologie van
het Academisch Medisch Centrum te Amsterdam. Het doctoraal examen werd behaald in februari 1991,waarna zij werkzaam is geweest als Onderzoeker in Opleiding bij de vakgroep Fysiologie van Mens en Dier van de Landbouwuniversiteit te
Wageningen.Hetonderzoek bijdezevakgroep heeftgeleidtotdit proefschrift.