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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. REFERENCES 1. Aboul-Khair SA, Crooks J, Turnbull AC, and Hytten FE (1964) The physiological changesinthyroidfunctionduringpregnancy. ClinSei27:195-207. 2. Amtzenius AB, Smit LJ, Schipper J, van der Heide D, and Meinders AE (1991) Inverse relationbetween iodine intake andthyroidbloodflow: color dopplerflow imaging ineuthyroidhumans.J ClinEndocrinolMetab75:1051-1055. Balen AH,and Kurtz AB (1990) Succesful outcome of pregnancywith severe hypothyroidism.Casereportandliteraturereview. BrJObstetGynecol97:536-539. 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Morreale deEscobarG,CalvoR, EscobardelReyF,andObregonMJ(1994)Thyroid hormonesintissuesfromfetalandadultrats.Endocrinology134:2410-2415. 39. Morreale deEscobarG,Obregon MJ, CalvoR,andEscobardelReyF(1991) Maternalthyroid hormonesduring pregnancy: effects onthefetusincongenital hypothyroidism and in iodine deficiency. In: Advances in perinatal thyroidology, Ed. Bereu BB and 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 3,5,3'-triiodothyroninedeficiency. Endocrinology122: 1521-1531. 42. Morreale de EscobarG,Obregon MJ, RuizdeOnaC,and Escobar delReyF(1989) Comparison of maternal to fetal transfer of 3,5,3'-triiodothyronine versus thyroxine in rats, as assessed from 3,5,3'-triiodothyronine levels in fetal tissues. ActaEndocrinologies(Copenh.) 120: 20-30. 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 embryonic tissues, before and after onset of fetal thyroid function. Endocrinology117: 1890-1900. 44. Obregon MJ, Mallol J, Pastor R, Morreale de Escobar G, and Escobar del Rey F (1984) L-thyroxine and 3,5,3'-triiodothyronine in ratembryos before onset of fetal thyroid function.EndocrinologyA14: 305-307. 45. Obregon MJ,RuizdeOna C,CalvoR,Escobar delRey F,and Morreale deEscobar G (1991) Outer ring iodothyronine deiodinase andthyroid hormone economy: responses to iodinedeficiency intheratfetusandneonate.Endocrinology129:2663-2673. 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. Pisarev MA(1985) Thyroidautoregulation.J.Endocrinol. Invest.8:475-484. 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 modelto studyfetal effects of maternal hypothyroidism.AdvExpMedBiol299:107-132. 52. Riesco G, Taurog A, Reed Larsen P, and Krulich L (1977) Acute and chronic responsesto iodinedeficiency inrats.Endocrinology100:303-313. 53. Robbins J, and Bartalena L (1986) Plasma transport of thyroid hormones. In:HennemanG(ed)Thyroid hormone metabolism.Marcel Dekker, NewYork. pp.3-38. 54. 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. 55. Ruiz de Ona C, Morreale de Escobar G,Calvo R, Escobar del Rey F, and Obregon MJ(1991) Thyroid hormones and 5'-deiodinases inthe ratfetus late ingestation: effects of maternal hypothyroidism. Endocrinology 128: 422-432. 56. Santisteban P, Obregon MJ, Rodriguez-Pena A, Lamas L, Escobar del Rey F, and Morreale deEscobarG (1982)Are iodine-deficient rats euthyroid ?Endocrinology 110: 1780-1789. 57. Schröder-van der EistJP, and van der Heide D (1990) Effects of 5,5-diphenylhydantoin onthyroxine and 3,5,3'-triiodothyronine concentrations in several tissues of the rat. Endocrinology 126: 186-191. 58. Schröder-van der Eist JP, and van der Heide D (1990) Thyroxine, 3,5,3'-triiodothyronine, and 3,3',5-triiodothyronine concentrations in several tissues of the rat: effects of amiodarone and desethylamiodarone on thyroid hormone metabolism.Endocrinology 127:1656-1664. 59. Schröder-van der Eist JP, and van der Heide D (1992) Effects of streptozocin induced diabetes andfood restriction onquantities and source of T4andT3in rat tissues. Diabetes 41: 147-152. 60. Schwartz HL, Surks Ml, and Oppenheimer JH (1971) Quantitation of extrathyroidal conversion of L-thyroxine to 3,5,3'-triiodothyronine inthe rat.JClinInvest50:1124-1130. 61. Taurog A(1996) In: Werner and lngbar"sTheThyroid(Braverman LE& Utiger RDEds) 7thedJB Lippincott-Raven,Philadelphia, NewYork, pp47-80. GeneralIntroduction 62. Visser TJ (1990) Importance of deiodination and conjugation inthe hepatic metabolism of thyroid hormone. In: Greer (ed). Thethyroid gland. Raven Press, New York, pp285306. 63. Visser TJ, and Schoenmaker CHH (1992) Characteristics of type III iodothyronine deiodinase.ActaMedAustriaca19: 18-21. 64. 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 EnglandJournalofMedicine 321: 13-16. 65. Whitworth AS, Midgley JEM,and Wilkins TA (1982) A comparison of free T4 andthe ratio of total T4toT4-bindingglobulin inserum through pregnancy. ClinEndocrinol (Oxf) 17:307-313. 66. Willis PI, and Schindler J (1970) Radiothyroxine turnover studies in mice: effect of temperature, diet,sexandpregnancy. Endocrinology86:1272-1280. 67. Wolff J (1964) Transport of iodide and other anions in the thyroid gland. Physiological Reviews44:45-90. 68. Wondisford FE, Magner JA, and Weintraub BD (1996) In: Werner and Ingbar's The Thyroid (Braverman LE& Utiger RDEds) 7thedJB Lippincott-Raven, Philadelphia, New York,pp190-206. 69. Rapoport B, and Spaulding SW (1996) In:Werner and Ingbar's The Thyroid (Braverman LE & Utiger RDEds) 7th edJB Lippincott-Raven, Philadelphia, NewYork, pp207219. 70. Yoshida K, Suzuki M,Sakurada T,Kitaoka H, KaiseN,KaiseK, Fukazawa H,Nomura T, Yamamoto M, Saito S,and Yoshinaga K (1984) Changes inthyroxine monodeiodination in rat liver, kidney and placenta during pregnancy. Acta Endocrinologica 107: 495-499. 71. ZeghalN,Gondran F,RedjemM,Giudicelli MD,AissouneY,andVigoureux E(1992) Iodide and T4 kinetics in plasma, thyroid gland and skin of 10-day-old rats: effects of iodinedeficiency.ActaEndocrinologica (Copenhagen) 127:425-434. 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. 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Q) 0) 3 3 eu 33 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 I H o - j - j O D D O < : < : < 33 Z) 70 73 <i y - 11 o I o 3 51 £ S •o .< H .H S V ? ? - /° • 0 - D - 0 - 0 3 3 3 1 - - 3 3 3 3 0 O 0 . I o o_ o o — •o 3 o 3=5 5= 3= 3s 3= CD 3 1* ft -J "5. S o (A DJ ï1 M M N> CO w - Kco 4k _ IO 4k IO -k CD CO co Ui -ok o -si CO o i b i b o c o c o s ^ H-Kn- H- HI- H- H- H- H- H - H - H - H - H - H - l + H ^ ^ ^ M U a ^ o M I O M O I O U I O l O O - k co - k 00 IO co co ?co en4k *. Ol 5>' aa- 3 II O) c »-^ 5' < o c » c n O — 4k H- H-> œ ö c n ? > IO - J H- Hc» o 'bo r^ co co r o c o H O H- Hro ro -Ni CO o o 0 , w ' H- H- HS -k O * ro r o O, 0 1 IO NJ CO _ CO _ > l. 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O co eo eo 4k. oo en en 00 en b en en H- H- H- H- H- H- H- H- HO ro CO 4k o CO en en ro 0> b CO 00 00 CD 4k O O Il *< "O 3 - * a CT O Q. 3 a. en ro ^ ^ ro 4k o oo co CD co en to én b> H- H- HH- Hr o c o c o - k - k - » . c o c D - k o> o> t o r o ro o -k © co en K. ^ oo Hco « oo -k en 4k CO 0> co en ro O en 4v ro en ö 4k 4k H- H- H- HH- Hco co co 4k -k o J en u _k CD ro © CD O b «> 3 0) 3' CD 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. REFERENCES 1. Ad Hoc Committee on Standards for Nutritional Studies (1977) Report of the AmericanInstituteof Nutrition.J.Nutr.7:1340-1348. 2. Calvo R, Obregon MJ, Ruizde Ona C, Ferreiro B, Escobar del Rey F,andMorreale deEscobar G(1990)Thyroidhormone economy inpregnantrats nearterm:A"physiological"animalmodelof nonthyroidal illness?.Endocrinology127: 10-16. 3. DiStefano IIIJJ, Jang M, Malone TK,andBroutmanM(1982) Comprehensive kinetics of triiodothyronine production, distribution, and metabolism in blood andtissue poolsof the ratusingoptimizedblood-sampling protocols.Endocrinology 110: 198-213. T4andT3kinetics inpregnantrat 43 4. DiStefano III JJ, Malone TK, and Jang M(1982) Comprehensive kinetics of thyroxine distribution and metabolism in blood and tissue pools of the rat from only six blood samples: dominance of large, slowly exchanging tissue pools. Endocrinology111: 108117. 5. DoornvanJ,vander Heide D,andRoelfsema F(1983)Sourcesandquantityof 3,5,3'triiodothyronine inseveraltissuesoftherat.J. Clin.Invest.72:1778-1792. 6. DuboisJD, CloutierA,Walker P,and DussauKJH (1977)Absence of placentaltransfer of L-triiodothyronine (T3) intherat.Pediat.Res.11: 116-119. 7. Dussault JH,and Coulombe P (1980) Minimal placental transfer of L-thyroxine (T4) in therat.Pediat.Res.14:228-231. 8. Fukuda H, OshimaK, Mori M,Kobayashe I,andGreer MA(1980) Sequential changes in the pituitary-thyroid axis during pregnancy and lactation inthe rat. Endocrinology 107: 1711-1716. 9. Galton VA (1968) Thyroxine metabolism and thyroid function in the pregnant rat near term. Endocrinology82:282-290. 10. GarribA,and McMurray WC(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. J. Clin.Endocrinol. Metab.71: 276-287. 12. 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. 13. Kojima A, Hersman JM, Azukizawa M, and DiStefano III JJ (1974) Quantification of thepituitary-thyroid axis inpregnant rats.Endocrinology95:599-605. 14. Leonard JL, Kaplan MM,Visser TJ, Silva JE, and Larsen PR (1981) Cerebral cortex responds rapidlytothyroidhormones.Science214:571-573. 15. Lindell R, DiStefano III JJ, and Landaw EM (1988) Statistical variability of parameter bounds for n-poolunidentifiable mammillaryadn catenary compartmental models.Math. Biosciences91:175-199. 16. Marino A, DiStefano III JJ, and Landaw EM (1992) DIMSUM: an expert system for multiexponentialmodeldiscrimination.Am.J.Physiol.End.Metab. 262:546-556,1992. 17. McFarlaneAS(1963) Catabolismof plasma proteins.Lancet~\: 131-134. 18. Morreale deEscobar G,CalvoR,Obregon MJ,and Escobar del Rey F(1990)Contribution of maternal thyroxine tofetalthyroxine pools innormal rats nearterm.Endocrinology 126:2765-2767. 19. Morreale deEscobar G, Obregon MJ, and Escobar del Rey F(1987) Fetalandmaternalthyroidhormones. Hormone Res. 26:12-27. 44 Chapter2 20. Morreale de Escobar G,Obregon MJ,RuizdeOna C,and Escobar delRey F(1989) Comparison of maternal to fetal transfer of 3,5,3'-triiodothyronine versus thyroxine in rats, as assessed from 3,5,3'-triiodothyronine levels infetal tissues. Acta Endocrinologica(Copenh.) 120:20-30. 21. 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 embryonic tissues, before and after onset of fetal thyroid function. Endocrinology117: 1890-1900. 22. Newham AP, Krieger K, Maly IP, and Sasse 0 (1991) Changes in activity and intraacinar distribution of glucose-6-phosphate dehydrogenase and malic enzyme during pregnancy inratliver. Histochemistry95:365-371. 23. Pharoah POD,and Buttfield IH(1971) Neurological damage to the fetus resulting from severe iodinedeficiency during pregnancy. The Lancet13:308-310. 24. Roelfsema F, van der Heide D, and Smeenk D (1980) Orcadian rythms of urinary electrolyteexcretion infreely moving rats.LifeSei27:2303-2309. 25. 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 tissues of the rat. Endocrinology126: 186-191. 26. Schröder-van der Eist JP, and van der Heide D (1990) Thyroxine, 3,5,3'-triiodothyronine, and 3,3',5'-triiodothyronine concentrations in several tissues of the rat: effects of amiodarone and desethylamiodarone on thyroid hormone metabolism.Endocrinology 127: 1656-1664. 27. Silva JE, and Larsen PR (1982) Comparison of iodothyronine 5'-deiodinase and other thyroid-hormone-dependent enzyme activities in the cerebral cortex of hypothyroid neonatalrat. Evidenceforadaptationto hypothyroidism.J. Clin.Invest.70:1110-1123. 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. REFERENCES 1. AdHocCommitteeonStandardforNutritionalStudies(1977) ReportoftheAmerican Institute of Nutrition.JMrfr7:1340-1348. 2. Caivo R, Obregon MJ, RuizdeOna C, Ferreiro B, Escobar delRey F,and Morreale de Escobar G(1990) Thyroid hormone economy inpregnantratsnearterm: a"physiological"animal modelof nonthyroidal illness ?Endocrinology 127:10-16. 3. DiStefano IIIJJ, Jang M, MaloneTK,and Broutman M(1982) Comprehensive kinetics of triiodothyronine production, distribution, and metabolism in blood and tissue pools of theratusingoptimizedblood-sampling protocols. Endocrinology 110:198-213. 4. DoornvanJ,vander HeideD,andRoelfsema F(1983) Sourcesandquantityof 3,5,3triiodothyronine inseveraltissuesoftherat.J.Clin.Invest. 72:1778-1792. 5. Doorn van J, Roelfsema F, and van der Heide D (1985) Concentrations of thyroxine and 3,5,3'-triiodothyronine at 34 different sites in euthyroid rats as determined by an isotopicequilibrium technique.Endocrinology117: 1201-1208. 6. Heide vander D, andEnde-Visser M(1980) T„, T3and reverseT3inthe plasma of rats duringthefirst3monthsof life.ActaEndocrinologica 93:448-454. 7. Hetzel BS (1983) Iodinedeficiency disorders (IDD)andtheir eradications. TheLancet2: 1126-1129. 8. Kjeld JM, KukuSK, Diamant I,Fraser TR,JoplinGF,and Mashiter K(1975) Production andstorageof ["^-triiodothyronine of highspecific activity. Clin.Chim. Acta61:381389. 9. Leonard JL, Kaplan MM,Visser TJ, Silva JE, and Larsen PR (1981) Cerebral cortex responds rapidlytothyroidhormones.Science214:571-573. 10. Leonard JL, and Visser TJ (1986) Biochemistry of deiodination. In: Thyroid hormone metabolism. HennemanG(ed).Marcel Dekker, NewYork,pp189-229. 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 • ; > " " ' _——— y 10 - 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 alues are me lyroid and kid =? < 3 O 3 3 3 3 3 3 <Q C 3 •c (O 3 M 3" D> a •< •a 01 > c P s CA CO 3. 3 3 fl> *s iy* O. O ÇD CA 3 3 5! m 3 Q. m P > j - • 01 3 VS. oluti w ~> 5 ® Q. S£ ? "O (A #••• • ^ b •fc. ^ j (O HO CD HO 'o b O ^ O en H- *. o> •T* -* Kj b CO HO ro b (O b N> HO o co z p o 's ». -«J 01 5' o (O 01 3 o' 5' o. S. 7} o M OD HO b A O O b -4 co co (O HO ti- b *. —* ro Ol o o ba HO o b NJ z p co "0 HO *—s Ijk b a> o 01 b 00 _ i o ^ 8 ^1 ^-J b !_i 3 CD b© =3 II -A to © -4 M © o in -J. 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CD >< CD Tl ai 3 CO CL CL er 3 3 ço * O 5' 01 3 CD H- 73 01 (A O 3 CD 3 0 HCO "O 01 =:• 3 (A c3 HO o O «A 3 o> 5' HO <D 3 Q. O CA CD O f * o ro 0) o S- CT p O 01 a 3 3 if CD 01 3 CL 0 CA CD 3 1 H 01 - 3 a > •o z 5" P2 "O 01 (0 > • CD c 01 > CA < vf' O >• 01 3 4? a 3 O CD l» 01 > «2 a. o' T3 in O S. c >< c ^* 3" >< 30 1 A en <ll m 01 «y 01 co ? HTj CO H * •o S" n en 3 01 Iodideuptake bythematernalandfetalthyroid 2.0 73 100 « 1.5 h 1.0 Ù 0.5 - ô> 80 c 60 40 •5 o 20 I. ••/• É m 0 CM 0 . 0 VWS- maternal thyroid INID, C ülMID, C NID, P MID, P 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. REFERENCES 1. Aboul-KhairSA,CrooksJ,TurnbullAC,andHytten FE(1964)The physiological changes inthyroidfunctionduringpregnancy. ClinSc;27:195-207. 2. AdHocCommitteeonStandardforNutritionalStudies (1977) ReportoftheAmerican InstituteofNutrition.JAM/"7:1340-1348. 3. 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. 4. 5. 6. 7. DeLange F(1994)Thedisordersinduced byiodine deficiency. Thyroid4:107-128. DiStefano IIIJJ, JangM,MaloneTK,andBroutman M(1982)Comprehensive kinetics oftriiodothyronine production,distribution,andmetabolisminbloodandtissuepoolsof theratusingoptimized blood-sampling protocols.Endocrinology\'\0: 198-213. 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. 77 78 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. 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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: 1126-1129. 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 61:381-389. 19. Lindell R, DiStefano III JJ, and Landaw EM (1988) Statistical variability of parameter bounds for n-pool unidentifiable mammillary and catenary compartmental models. MathematicalBiosciences 91: 175-199. 20. Marino A, DiStefano III JJ, and Landaw EM (1992) DIMSUM: an expert system for multiexponential modeldiscrimination.Am.J.Phys.262546-556. 96 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, 1990. 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 ratio of total T4 to T4-binding globulin in serum through pregnancy. Clinic. Endocrinol. 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. REFERENCES 1. Ad Hoc Committee on Standards for Nutritional Studies. Report of the American Institute of Nutrition (1977) JournalofNutrition 7: 1340-1348. 2. Bonet B, and Herrera E (1988) Different response to maternal hypothyroidism during thefirst andsecond half of gestation intherat.Endocrinology 122:450-455. 3. DiStefano IIIJJ,Jang M,MaloneTK, and Broutman M(1982)Comprehensive kinetics of triiodothyronine production, distribution, and metabolism in blood and tissue pools of the ratusingoptimized blood-sampling protocols. Endocrinology 110: 198-213. DiStefano III JJ, Malone TK, and Jang M(1982) Comprehensive kinetics of thyroxine distribution and metabolism in blood and tissue pools of the rat from only six blood samples: dominance of large, slowly exchanging tissue pools. Endocrinology111: 108117. Doom van J, van der Heide D , and Roelfsema F (1983) Sources and quantity of 3,5,3'-triiodothyronine in several tissues of the rat. Journalof Clinical Investigation72: 1778-1792. Escobar-Morreale HF,Escobar del Rey F,Obregon MJ,and Morrealede Escobar G (1996) Onlythe combinedtreatmentwiththyroxine andtri- iodothyronine ensures euthyroidisminalltissuesofthethyroidectomized rat.Endocrinology 137:2490-2502. Garrib A, and McMurrayWC (1984)Asensitive,continuous spectrophotometry method for assaying a-glycerophosphate dehydrogenase: activation by menadione. Analytical Biochemistry139:319-321. 4. 5. 6. 7. 8. Hadjzadeh M, Sinha AK, Pickard MR, and Ekins RP (1990) Effect of maternal hypothyroxinaemia in the rat on brain biochemistry and adult progeny. Journal of Endocrinology124:387-396. T4 andT3kinetics inpregnant hypothyroidrats 9. 111 Heidevander D,andVisser-Ende M(1980) T4,T3andreverse T3inthe plasma of rats duringthefirst 3months of life.ActaEndocrinologies93:448-454. 10. 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. BiologicalTrace ElementResearch40: 237-246. 11. Kjeld JM, Kuku SK, Diamant I, Fraser TR, Joplin GF, and Mashiter K (1975) Production and storage of ["^-triiodothyronine of high specific activity. ClinChimActa 61: 381-389. 12. Lindell R, DiStefano III JJ, and Landaw EM (1988) Statistical variability of parameter bounds for n-pool unidentifiable mammillary and catenary compartmental models. MathematicalBiosciences 91: 175-199. 13. MaiaAL,KiefferJD,HameyJH, andLarsen PR(1995) Effect of 3,5,3'-triiodothyronine (T3) administration on dio! gene expression and T3 metabolism in normal and type I deiodinase-deficient mice.Endocrinology 136:4842-4849. 14. Man EB, and Serunian SA (1976) Thyroid function in human pregnancy. IX. Development or retardation of 7-year-old progeny of hypothyroxineic women.American JournalofObstetricsandGynecology 125:949-957. 15. Marino A, DiStefano III JJ, and Landaw EM (1992) DIMSUM: an expert system for multiexponentialmodeldiscrimination.AmJPhysiol 262:546-556. 16. Morreale de Escobar G, Calvo R, Obregon MJ, and Escobar del Rey F (1990) Contribution of maternal thyroxine to fetal thyroxine pools in normal rats near term. Endocrinology 126:2765-2767. 17. Morreale de Escobar G,Obregon MJ, Ruiz deOnaC,and Escobar del ReyF(1988) Transfer of thyroxine from the mother to the rat fetus near term: effects on brain 3,5,3'-triiodothyroninedeficiency. Endocrinology 122: 1521-1531. 18. Morreale de EscobarG,Obregon MJ,RuizdeOna C,andEscobar delReyF(1989) Comparison of maternal to fetal transfer of 3,5,3'-triiodothyronine versus thyroxine in rats, as assessed from 3,5,3'-triiodothyronine levels in fetal tissues. ActaEndocrinologica(Copenhagen)120:20-30. 19 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 embryonic tissues, before and after onset of fetal thyroid function. Endocrinology117: 1890-1900. 20. Porterfield SP,and HendrichCE (1991) The thyroidectomized pregnant rat-an animal modelto studyfetaleffects of maternal hypothyroidism.AdvancedExperimentalMedical Biology299: 107-132. 21. Roelfsema F, van der Heide D, and Smeenk D (1980) Orcadian rythms of urinary electrolyte excretion infreely movingrats.LifeScience27:2303-2309. 112 Chapter6 22. Ruiz de Ona C, Morreale de Escobar G, Calvo R, Escobar del Rey F, and Obregon MJ (1991) Thyroid hormones and 5'-deiodinase inthe ratfetus late ingestation: effects ofmaternalhypothyroidism.Endocrinology128:422-432. 23. 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.Endocrinology126:186-191. 24. SPSSInc. SPSS/PC * STATISTICS4.0(1990) McGraw-Hill,Chicago, IL:McGraw-Hill. 25. Versloot PM,GerritsenJ,Boogerd L, Schröder-van der EistJP,and van der Heide 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. 125 126 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. 127 128 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. 129 131 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 133 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.