Reverse triiodothyronine in the chicken

Live Sciences, Vol. 21, pp . 205-212, 1977 .

PriaCsd In The U.S.A.

Pergamoa Press

REVERSE TRIIODOTHYRONINE IN THE CHICKEN B . N, Promachandra, S . ~ne, J, A, Andrada and J .H, Kite, Jr, Veterans Administration Hasp Jefferson Barracks and Department of Physiology, Washington University School of Medicine, St . Laois, Mo, Dept, of Microbiology, State University aF New Yak, Buffalo, N, Y . (Received in final form Juae 13, 1977)

SUMMARY

Reverse trüodothyronine (rT3) which is apparontly a product of 5-monodeiodination of thyroxine (T~ at the periphery, was measurod in 199 chickens of carious strains, rT3 was virtually absent in young birds (less tFwn 1 week to 8 weeks of age) in marked contrast to the elevated rT3 levels found in human and other mammalian neonates. At one to two years of age them was a significant incroase in the number of birds with detectable rT3 . However, rT3 concentrations wero low and often close to the detection limits of the assay in contrast to significant rT3 levels found in mammals (man, monlaey, sheep, rat and dog), An apparont sex differonce in rotation to rT3 formation was noted ; 46,4% of 97 females and 9 .396 of 54 males showed detectable rT3 levels . The abserwtiôns described suggest a species differonce in rogard to peripheral T4 monodeiodination between birds and mammals . Recent investigations of thyroxine (T,,~ and triiodothyranine (T3) kinetics in man and in animalshave suggested that T3 generation from T4 at the periphery can account for approximately half of the T4 that undergoes deiodination (1,2,3) . On this basis, it was proposed thatthe initial monadeiodirwtion of T4, from the phenolic (outer) and tyroayl (inner) rings was random in naturo (4,5) . These conceptual advances implied that as a rosult of monodeiodination of T4 from the phenolic (5'~eiodination) and tyrayl (5-deiodination) rings, respectively, approciable amounts of both 3:3' :5 trüodothyranine (T3) and 3,3',5' tri iodothyronine (reverse T3; rT3) must be prosent in ciroulation, While the prosence of T3 in ciroulation is well known, evidence for rT3 in man was only rocently obtained by its specific and diroct measuroment in serum by RIA (6,7,8) and moro rocenfly in the thyroid venous blood (9), Although the existence of rT3 in the thyroid gland (and in circulation) of animals has been noted in the earlier roports (10,11), rT3 is considerod to arise largely at the periphery as a rowlt of T4 monodeiodination . Also, rT3 has been shown to be calaigenically inert (12,13) and to exert anti-T4 effects (14), In several situations to humans and other mammals, e .g, systemic illnesses (15), neonates (15,16), protein~alorie mainutrition (15), starvation (17) and surgery (18), them is an inverse rotation of T3 to rT3, i .e, concentration of T3 decreases and rT3 incroases, These findings cast some doubt on the hypothesis of random peripheral monodeiodirotion of T4. * resent

ross: nst, nvest,

dices, Univ, Buenos Airos, Argentina 205

206

Reverse Trüodothyronine in the (aticken

Vol . 21, No . 2, 1977

We have carried out T3 and rTg meawroments in chickens ranging in age from less 'than 1 week and up to 3 years. We found not only an absence of T3 - rT3 inverse rolation:hip, but a total lack of rT3 in the sera of the majority of chickens tested, tfius demonstrating a species differonce with rasped to peripheral T4 monadeiodination, METH ODS T4 and T3 levels in the sera of chickens wero meawrod by radioimmunoassay (RIA) as proviouslydeacribed (19,20), rT3 antibody was produced in rabbits by immunizing them with 3 weekly injections of rTg~albumin conjugates and the antisera wero harvested 6 to 8 weeks after primary immunization, After proliminary trials, 1/1000 dilution of rT3 antiserum (final dilution = 1/y500) was found witable for rT3-RIA, Ths details of rT3-RIA wero similar to those of Tg-RIA (20) with the exception that antigen~ntibody incubation was carried out overnight at room terr~eraturo prior to froe and bound hormone separation ; 125 1-labeled rTg was obtained from Abbott Laboratories (North Chicago, Illinois) . The croasreaction of T4 and 3,3' diiodothyronine in rT3-RIA was 0.0896 and 1% rospectivsly . T3 and 3 :5 diiodothyronine did not crass react with rT3 antibody . The lowest rTg concentration consistently detectable in the assay was 5 na/100 ml or 5 pg/ assay tube, The range of rTg values in the sera of 50euthyroid normal human wbjects (25 - 60 ng/100 ml) as noted in our assay was similar to that roporied in other rT3-RIAs 6,7,8), The suitability of rT3-RIA in the chicken however was established by serum 251-rTg extractionefficiency (? 9096), rocovery of exogenously added rTg (99-106%), and the comparability aF assay values in undiluted and diluted sera ; the proceduros used wero similar to those described in detail for T4 (19), Hormonal meawroments wero made in several chicken strains ranging in age from 7 days to 3 years . Of several strains noted in Table 1, the Obese strain (OS) is of particular inierost as these chickens develop spontaneous thyroiditis soon after hatching and exhibit phenotypic featuros a~ociated with hypothyroidism such as excess subcutaneous fat, smaller slaeletal structuro, and changes in feather structuro (21,22) . Some of these birds wero bursectomized, or thymectomized, for investigations of autoimmune phenomena, but separation of these experimental groups is not shown in the table as rT3 data did not seem to warrant it, RESULTS AND DISCUSSION The conspicuous finding in these investigations was the absence of rT3 in the majority of chiclaena (Table 1), In only 71 out of a total of 199 birds (or 35%) was rT3 prosent . In those animals whero rT3 was meawrable, it was often close to the detection threshold of the assay . The maximum rT3 concentration of 28 ng/100 ml was noted in only ores out of 199 birds and in another animal it was 23,8 ng ; in 25 birds the range in rT3 concentration was 10-19 ng, and in the romaining instances whero rT3 was detectable it was below long and close to the away detection limits . The ores animal of the OS strain which showed the highest rT3 level (28 ng/100 ml, Table 1) also had high T4 (but not high Tg), but this cannot be invoked to explain the rT3 in this animal since other birrJs with similarly high Tq, values (individual data) showed no rTg in serum (comparo also rT3 values in 52-60 week old random brad chickens which wero equal to, a in some instances higher than in 28-30 week old obese birds despite the fact that the T4 level in the former was half that of the latter) . Because of the unusual finding of absence of rT3 in the majority of chickens, especially in relation to significant rT3 concentrations in the sera of all mammals tested so far (man = 40, sheep = 61, dog = 1®, and rat = 46 ng/100 ml ; rofs . 6,16,23 and urrpub -

Vol . 21, No . 2, 1977

TABLE 1 . whim Legharn Chkioen (Shalra

Reverse Trüodothpronine in the Chicken

20 7

REVERSE T3, T3 AND T4 MEASUREMENTS IN CHICIO:NS OF DEFERENT STRAINS AT VARIOUS AGES Ape

T4 (Ng/100 ml)

T3 (ng/100 ml)

Above DL

(N)

(N)

(12) ( 2) (25) (12)

(12) ( 2) ( 7) ( 2)

( 0) ( 0) (18) (10)

10,0+S,OQ (5-23,8) 8.1 + 3,50 (5 - 16 .2)

0,65 + 0,18 '(10) 1,17 + 0,68 (15)

206 + 20,9 (10) 139 + 23,8 (15)

(10) ( 3)

( 0) (12)

11,9 + 5.29 (5 - 18 .7)

4-8 52-60 72 -76 104-`110 128 - 132

0,53 + 0,20 0,64+0,09 0.71 +0,17 0,58+0,18 1 ,07+ 0,35

(28) ( 5) (10) ( S) ( 3)

208 + 30,8 162+36 .7 182+36,6 137+21,4 166 + 10,7

(30) ( 5) (10) ( 5) ( 4)

(28) ( 1) ( 4) ( 4) ( 2)

( ( ( ( (

1) 4) 6) 1) 2)

6,2 + _ 5,3+0,60 (5-6,2) 8.1 +2,20 (5-11,2) 8 .0+ _ 15 .0 + _ (13 - 17)

OS

3 _6 28 - 30 104-110 168

0,53 + 0,33 1 ,45 +0,79 1,12+0,49 3,50+ -

(10) "" ( 6) (6) ( 1)

155 + 102,9(16) 91 + 33,9 ( 8) 156+25 .1 ( 6) 167+ - ( 1)

(15) ( 7) ( 3) ( 0)

( ( ( (

1) 2) 3) 1)

6,2 + 7 .5 + - (6 - 9) 10,7+4,60 (8-16) 28 .0+ -

KSl1L

2-6 52-60 T2 - 76 104 - 110

0,71+0,31 1 .12+0,49 O,A4 +0,35 0,75 +0,50

(11) ( 4) ( 7) (10)

248+55 .9 (15) 214+112 .8( 4) 196 + 140,2 ( 7) 152 + 4p,3 (10)

(14) ( 1) ( 3) (10)

( ( ( (

1) 3) 6) 0)

6 .2+ 10,8+4,40 (6,2-15) 9,5 + 1 ,86 d.5 - 11 .2) -

(wssla)

mwn + SD

(N)

1 -4 28-30 52-60 104 - 110

0 .87+ 0,26 0,30+ 0 .79+0,30 0,57 + 0 .22

(12) ( 1) (25) (12)

CS

6-8 88 - 92

Llne G

NormaI Randan Brsd

moan ± SD (N)

rT3 (np/100 ml) Below DL "

255 + 32,7 107+2b .9 186+69 .6 113 + 14,8

mean + SD

(Range)

DL - detection Iimlt - 5 rp/100 ml (N) - in Hr T4 - OS group 4 less than in the Tg group os T4 wlues in 6 animals in this group wen deleted beeww tl»y were below DL ( < 0,2 Iq/100 ml) . AltogsH~ar T4 wlua wen below DL in 8 obese animals, one animal each In Llns G and Normal Random Bred ehialmns, and 4 animals in Hb KSU~ chain, T3 was belay DL (10 rg/100 ml) in one animal In tlr obese group, OS - Obsr chain chlelaerr which develop spontaneous autoimmuro thyroidifis, CS - Ca~nell strain, the pwrH strain ehlekere from whkh OS wasderived . These aro esrMTally normal althwgh thyroldkis may occur in less tlwn 196, KSU{ard Lirr G -_ Normal inbred Tins aF whiro Lsgham ehkkero. " ""

lisped data), rT3 rneasuroments wero also made in ethanol extracts to exclude the possibility of unknown protein interFeronce in the assay of unextracted serum . The roprosentative data aro shown in Table 2; rT3 values in chicken serum exfiacts wero essentially the same as those noted in native sera . In addition, in those birds whero rT3 vw: not detectable sera wero pooled and extracted with ethanol ; rT3 eras not detectable even in ethanol extracts of large volumes of sera (5 ml extract tested),

20 8

TABLE 2,

Reverse Trüodothyronine in the Chicken

rT3 CONCENTRATION (ng/100 ml) IN UNEXTRACTED AND ETHANOL EXTRACTED SERA

White Leghorn Chicken (Stra i n) Normal Random Brod KSU-C CS

Vol . 21, No . 2, 1977

Unextracted Sera

Serum Ethanol Exfiach*

18

Not detectable

Not detectable

6624A 7635A

7 15

11 12

5 3

19 12

21 9

Number

* Details of ethanol extraction proceduro have been noted proviously (24), An inspection of the Table 1 also melees it rowdily apparont that the appearance of rT3 .in the circulation of chickens is an age rotated phenomenon . In all 5 strains of chickens, rTg was virtually absent in the first 8 weeks of life, rTg was defected in only 3 out of B2 birdsand the concentration was close to the defection limits of the assay, Approocimately at one year of age, ihero is a sharp incroase in the froquency of birds manifesting rTg, With incroase in age close to 2 years them is further increase in the frequency of birds with rTg in some stratrn (normal random bred, Cornell strain and OS), but not in others (Table 1), This lack oFcomplefe concordance in observations may partly be rotated to differonces in T4 concentration in old animals of carious strains . Overall, them is a significant corrolation (r = 0,75 p < ,05) between age and appearance of rT3 in the ciroulation of chickens (Fig, 1), It is of inierost to note that age -rT3 rolationship has also been described in the human by Nicod et aF (7), After 70 years of age wide fluctuations in serum rT3 wero noMd with a mean ofbT.4 rg/100 ml in comparisonto the mean wlue of 45 ng/100 ml in adult euthyroid subjects (20-60 years of age), It is pasiblethat the age rotated rT3 rolatiorrship as noted in the chicken may have been somewhat muted by an apparont sex differonce in rotation to rT formation (Table 3), In all 5 strairx, the froquency of detectable rT3 was cansistent~y greater in females than in males, Altogether, 46 .496 aF 97 females had detectable rT3 in circulation whereas only 9.396 â 54 males had rT3 . These differonces in circulatir+g rT3 between males and females could not be explained on the basis of differonces in T4, Mean serom T4 conceMTation in 45 females (wifh detectable rTga was 1,1 ONg/100 ml whic h was not si nificantl~higher than the mean T4 concentration of 1 ,03 Ng%100 ml in 5 males with rT3 . The role aF female sex hormones, if any, and other factors on rT3 formation and plasma transport aro not known and the interostirrg rolatio~nship between sex and peripheral T4 monodeiodination merits further study . It should perhaps be emphasized however, that although the appearance of rT3 in the chicken incroases with oge and maro females than males manifest rT3 in the circulation, the 5-manodsiodiration of T4, (that generates rT3) is still a very inefficient and mina pathway in the peripheral metabolism of thyraucine in this species . The absence of rT3 in the majority of chickens of several strains appears to roinforce the concept tFwt the 5 and 5' deiodirotion from the inner and outer rings aro indeed distinct processes. In our studies, rT3 measuromentswero made in sera which theroforo procludes any comment in

Vol . 21, No . 2, 1977

Baverea Trüodothyroniae is the Chicken

20 9

rogord to thyroidal rT in the chicken . It is of inierost, however, that even in the one exceptional mammal dog, ref . 23) whero a large amount of rTg is prosent in circulations thyroidal contribution of rTg is notapprociabls .

AGE IN WEEKS

1ov

~F' ~1 . Relation of age and circulating rovenfe trüodothyronine in the chicken . The numerates aF the ratios at each point roprosents the number of animals with detectable rTg . The denominates indicates the tôtal number of animals in the rospsctive age groups . When the least squaro rogrossion calculations wero made by weighting each data point for the number of animals in the rospective age groups, the slope (b) of the line increased from 0 .45 to 0 .55 ; similarly a higher correlation coefficient rosulted (0 .1~ instead of 0 .75 obtained for the actual data as noted in this figaro) .

An additional speculation resulting from clinkal observations â elevated rT3 (with a concomitarM decrease in Tg) in disease slates and in neofrates (15,16) is that T4 manodeiodination is proferontially diverted from the phenolic to the tyroayl ring in order to afford protection from the calaigenic effects of Tg . In marked contrast, rTg was absent in a number of 1 - 8 week old birds, wheroas T3 concentrations in these some animals wero in the upper normal to mildly hyperthyroid range as judged by values seen in humans suggesting that the peripheral tiques in the chicken can apparonFly tolerate high T3 levels in ciroulation . The rolatively high T3 concentration in birds has also been noted in another study (25) . In light â these observations, it is tempting to speculate that the absence of 5~siodination in a majority of chickens may rosult from the lack of need for such a pathway in view of the tolerance to rolatively high T3 plasma concentrations in birds .

21 0

Reverse Trüodothyroniae is the Chiches

TABLE NM its LsOhan Chicken (St~a In)

Number aF Animals

3.

RELATION OF SEX TO CIRCULATING

Males

Females

No .

of males with rT3

rT3

Vol : 21, No . 2, 1977

IN THE CHICKEN

No, aF females with rT3

% aF males with rT3

% of females with rT3

KSU-C

31

13

18

1

7

7 .7

38 .9

Llne - G

58

22

36

1

13

4 .5

36 .1

CS

15

6

9

3

9

50 .0

100 .0

OS

21

9

12

0

6

0

50 .0

Normal Random Bred

26

4

22

0

10

0

45,4

Summary Data All Moles All Fsnales

54

5 47

9 .3 45

46 .4

At any rate, the observatitxu described in chickens provide yet anotFter example of exceptions in rogard to the physiology and metabolism of thyroid hormone as it is known in mammals, theroby underscairlg that thyroid function and .metabolism in aviarLS should be investigated without a bias towards rosearch done in mammals (26), ACKNOWLEDGEMENTS Grateful acknowledgement is made to Abbott Laboratories, North Chicago, Illinois for gifts of radioactive rnaierial and other supplies, Also, the authors gratefully acknowledge the cooperation of Drs, E,C, Jorgensen and Hare Cahnrnann, rospectively,for gifts of rT g and 3,3' T2 and for other useful comments, Investigatioru also wpported in part byUSPHS Grant CA-02357 from the National Cancer Institute . ADDENDUM After the manuscript was submitted far publication, similar observations aF a lack of reverse T3 in the sera of chickens have been made by Dr . J,D, May, U,S, Dept, Agri, - A,R .S  Mississippi State College, Mississippi (personal communication),

Vol . 21, No . 2, 1977

Reverse Trüodothyroaine in the Chicken

211

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